Coamplification at lower denaturation temperature pcr increase mutation detection selectivity of taq

pcr熔解的反应条件英文回答：PCR (polymerase chain reaction) is a widely used technique in molecular biology for amplifying specific DNA sequences. The reaction conditions for PCR melting, also known as denaturation, are crucial for the success of the reaction.The denaturation step of PCR typically requires high temperatures to separate the DNA strands. The optimal temperature for denaturation is around 94-98°C. This high temperature breaks the hydrogen bonds between the DNA strands, causing them to separate into single strands.The denaturation step is usually carried out for a short period of time, typically 15-30 seconds. This ensures that the DNA strands are fully denatured without causing excessive damage to the DNA template.During the denaturation step, it is important to maintain a consistent and uniform temperature throughout the reaction. This can be achieved by using a thermal cycler, which is a specialized instrument that can rapidly heat and cool the reaction mixture.After denaturation, the reaction temperature is lowered for the annealing step. During annealing, the reaction mixture is cooled to a temperature that allows the primers to bind to the single-stranded DNA template. The annealing temperature is typically around 50-65°C, depending on the melting temperature of the primers.Once the primers have annealed to the template, the temperature is raised again for the extension step. During extension, the DNA polymerase synthesizes new DNA strands using the primers as a starting point. The extension temperature is usually around 68-72°C, which is the optimal temperature for most DNA polymerases.The extension step is typically carried out for a longer period of time, usually 1-2 minutes per kilobase ofDNA being amplified. This allows the DNA polymerase to synthesize enough new DNA strands to generate a significant amount of PCR product.Overall, the reaction conditions for PCR melting involve high temperatures for denaturation, followed by lower temperatures for annealing and extension. These temperature changes are essential for the successful amplification of DNA sequences.中文回答：PCR（聚合酶链反应）是分子生物学中广泛使用的一种技术，用于扩增特定的DNA序列。

从零开始学PCR技术（一）：PCR技术简介展开全文PCR 可能是分子生物学中使用最广泛的技术。

虽然我本科学的是生物科学，提过DNA，也跑过 PCR，但现在都快忘了 PCR 的步骤是三个还是四个了。

赶快网络搜索之，整理成一个从零开始学系列。

聚合酶链式反应（Polymerase Chain Reaction，PCR）是一项利用 DNA 双链复制原理，在生物体外复制特定 DNA 片段的的核酸合成技术。

可在短时间内大量扩增目的 DNA 片段，而不必依赖大肠杆菌或酵母菌等生物体。

DNA 的半保留复制是生物进化和传代的重要途径。

双链 DNA 在多种酶的作用下可以变性解旋成单链，在 DNA 聚合酶的参与下，根据碱基互补配对原则复制成同样的两分子拷贝。

在实验中发现，DNA 在高温时也可以发生变性解链，当温度降低后又可以复性成为双链。

因此，通过温度变化控制 DNA 的变性和复性，加入设计引物，DNA 聚合酶，dNTP 就可以完成特定基因的体外复制，这是 PCR 的理论基础。

一、反应体系PCR 反应是在体外模拟DNA 的复制过程，因此反应体系中必须具有 DNA 复制所需的基本要素：1.模板（template），含有需要扩增的 DNA 片段。

2.引物（primer），一对引物决定了需要扩增的起始和终止位置。

3.聚合酶（polymerase ），DNA 聚合酶复制需要扩增的区域。

4.脱氧核苷三磷酸（dNTP），用于构造新的互补链。

5.缓冲液（buffer），提供适合聚合酶行使功能的化学环境。

二、反应步骤标准 PCR 过程分为三步：1.变性（Denaturation）：利用高温使 DNA 双链分离。

DNA 双链之间的氢键在高温下（93 - 98℃）被打断。

2.退火（Annealing）：在 DNA 双链分离后，降低温度使得引物可以结合于单链 DNA 上。

3.延伸（Extension）：DNA 聚合酶由降温时结合上的引物处开始沿着 DNA 链合成互补链。

测序覆盖率低的原因英文回答：The low sequencing coverage can be attributed to several factors. One possible reason is the presence of repetitive sequences in the genome. Repetitive sequences are regions of DNA that are repeated multiple times, making it difficult for the sequencing technology to accurately determine the number of repeats. As a result, these regions may be poorly covered or completely missed during sequencing. For example, if a repetitive sequence is not fully covered by sequencing reads, it would result in a lower coverage for that particular region.Another reason for low sequencing coverage could be the presence of sequencing errors. Despite advancements in sequencing technologies, errors can still occur during the sequencing process. These errors can lead to incorrect base calls, resulting in low quality reads. Low quality reads are more likely to be discarded during the data processingsteps, leading to a lower overall coverage. For instance,if a sequencing error occurs in a region that is criticalfor the coverage calculation, it could significantly impact the final coverage value.Furthermore, the presence of GC-rich or AT-rich regions in the genome can also contribute to low coverage. These regions have a high percentage of guanine-cytosine (GC) or adenine-thymine (AT) base pairs, respectively. GC-rich regions can be challenging for sequencing technologies asthe high GC content can cause issues with DNA denaturation and amplification. Similarly, AT-rich regions can bedifficult to sequence due to the weaker hydrogen bonding between adenine and thymine. As a result, these regions may have lower coverage compared to other regions in the genome.In addition, low sequencing coverage can be caused by inadequate library preparation. Library preparation is a crucial step in the sequencing workflow, and any errors or inconsistencies during this process can result in low coverage. For example, if the DNA fragments are notproperly fragmented or size-selected, it can lead to biasedrepresentation of certain regions in the library. This bias can then translate into lower coverage for those regions during sequencing.Lastly, technical limitations of the sequencing platform can also contribute to low coverage. Different sequencing platforms have different capabilities and limitations, and some platforms may inherently have lower coverage compared to others. For instance, certain sequencing platforms may have limitations in read length or throughput, which can impact the overall coverage. Additionally, the sequencing depth or number of reads generated per sample can also affect the coverage. If the sequencing depth is not sufficient, it can result in lower coverage for the target regions.中文回答：测序覆盖率低的原因有多种。

Hereditas (Beijing) 2018年3月, 40(3): 227―236 收稿日期: 2017-11-06; 修回日期: 2018-02-02基金项目:深圳市三名工程-出生缺陷防治研究与转化团队(编号：SZSM201406007)，深圳市出生缺陷重点实验室(编号：ZDSYS201504301707152)和深圳市宝安区医疗卫生基础研究项目(编号：2014067，2017JD001)资助[Supported by Sanming Project of Medicine in Shenzhen-Brith Defects Prevention Research and Transformation Team (No. SZSM201406007), Shenzhen Key Laboratory of Birth Defects (No. ZDSYS201504301707152and Science and Technology Plan Project of Baoan District (Nos. 2014067, 2017JD001)]作者简介: 梁卉，硕士，主管技师，研究方向：出生缺陷疾病的遗传学研究。

E-mail: lianghui2016615@陈国杰，博士，主治医师，研究方向：染色质结构和表观遗传调控。

E-mail: chenguojie.hi@梁卉和陈国杰并列第一作者。

通讯作者:熊礼宽，博士，研究员，研究方向：出生缺陷防治与研究。

E-mail: xionglk@DOI: 10.16288/j.yczz.17-369 网络出版时间: 2018/2/8 11:31:42URI: /kcms/detail/11.1913.R.20180208.1131.001.html综 述低温变性下复合PCR 技术及其应用梁卉1,2，陈国杰3，于燕4，熊礼宽1,21. 暨南大学附属深圳市宝安区妇幼保健院中心实验室，深圳 5181022. 深圳市出生缺陷重点实验室，深圳 5181023. 郑州大学附属第一医院消化科，郑州 4500524. 暨南大学附属深圳市宝安区妇幼保健院产科，深圳 518102摘要: 低温变性下复合PCR(co-amplification at lower denaturation temperature-polymerase chain reaction, COLD-PCR)是一种在高丰度野生型序列背景下选择性变性和扩增低丰度突变型序列的方法，可将突变型序列富集10~100倍。

JAPAN CHINA TOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD. Tel (+81)-6-6348-3888 Tel (+86)-21-58794900 www.toyobo.co.jp/e/bio1F1753KGenNext TM NGS Library Prep KitLPK-101 24 reactions LPK-101T 8 reactions LPK-101L 96 reactionsStore at -20°CContents[1] Introduction [2] Components[3] Protocol1. End repair and A-tailing2. Adapter ligation3. Cleanup (after adapter ligation)4. Library amplification5. Cleanup (after library amplification)6. Size selection (optional) [4] Library QC [5] Application data [6] Troubleshooting [7] Related productsC AUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnostic or clinical purposes. Please observe general laboratory safety precautions while using this kit.- illumina ® and MiSeq ® are registered trademarks of illumina, Inc.- Agencourt ® and AMPure ® are registered trademarks of Beckman Coulter, Inc.JAPAN CHINATOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.Tel (+81)-6-6348-3888 Tel (+86)-21-58794900 www.toyobo.co.jp/e/bio********************1[ 1 ] Introduction DescriptionGenNext™ NGS Library Prep Kit comprises the enzymes and buffers for preparinglibraries for illumina® sequencing from fragmented double-stranded DNA and PCRproducts.With this system, it is possible to conveniently and quickly convert a broad range (1ng -1μg) of input amounts of DNA into libraries for illumina® sequencing. Terminal repairand 3' end adenylation of the fragmented DNA can be conducted in the end repair andA-tailing step. Platform-specific adapters are then ligated to both ends of the DNAfragments.If required, a high-fidelity amplification step can be performed using the reagentsincluded in the GenNext™ NGS Library Prep Kit. Library Amplification Master Mixuses a highly-accurate PCR enzyme developed using genetically-modified KOD DNApolymerase. This minimizes the influence of GC bias on amplification and can amplifyvarious regions evenly.Process Workflow* Adapters and beads required for cleanup after adapter ligation and library amplification are notincluded.Features-Simple and quick operation flowThe steps from terminal repair and 3' end adenylation to adapter ligation can beconducted in the same container.Terminal repair and adenylation at the 3' end can beperformed in 15 minutes. Adapter ligation can be done in 15 minutes.Libraryamplification can be performed in cycles of 10 seconds’ annealing and 15 seconds’extension.-A wide range of input amountGenNext™ NGS Library Prep Kit is compatible with various inputs from 1 ng to 1 μg.JAPAN CHINATOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.Tel (+81)-6-6348-3888 Tel (+86)-21-58794900 www.toyobo.co.jp/e/bio********************2[ 2 ] Components -Low bias library amplificationLibrary Amplification Master Mix uses a highly-accurate PCR enzyme developed using genetically-modified KOD DNA polymerase. It minimizes the influence on GC-bias-induced amplification, and it is possible to amplify various regions evenly.The kits include the following reagents, which can be used for 24 (LPK-101), 8 (LPK-101T) and 96 (LPK-101L) reactions. All reagents should be stored at -20°C.GenNext TM NGS Library Prep Kit (Code No. LPK-101, LPK-101T, LPK-101L)LPK-101 LPK-101T LPK-101L End repair and A-tailing buffer*240 µL 80 µL 960 µLEnd repair and A-tailing enzyme*60 µL 20 µL 240 µLLigation solution 1,200 μL 400 µL 1,600 μL × 3 Library amplification master mix 690 µL 230 µL 1,380 μL × 2 Library amplification primer mix 138 µL 46 µL 552 µL* Do not store the mixed solution.End repair and A-tailing bufferEnd repair and A-tailing Buffer is optimized to convert fragmented DNA to repaired DNA having 5´-phosphorylated, 3´-dA-tailed ends. End repair and A-tailing buffer should be used with end repair and A-tailing enzyme.End repair and A-tailing enzymeEnd repair and A-tailing enzyme is optimized to convert fragmented DNA to repaired DNA having 5´-phosphorylated, 3´-dA-tailed ends. End repair and A-tailing enzyme should be used with end repair and A-tailing buffer. Please pipette slowly as this is viscous.Ligation solutionThis solution contains DNA Ligase and optimized reaction buffer. Please pipette slowly as this is viscous.Library amplification master mixThis is a 2×concentrated master mix containing genetically-modified KOD DNA polymerase, dNTPs (dATP, dGTP, dCTP, dTTP) and Mg2+.V arious regions of the genome can be amplified homogeneously even if these regions contain GC bias. The resulting amplicons are suitable for next-generation sequencing analyses.Library amplification primer mixThe primer mix (10×) is optimized to amplify illumina® libraries flanked by the P5 andP7 flow cell sequences.JAPAN CHINATOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.Tel (+81)-6-6348-3888 Tel (+86)-21-58794900 www.toyobo.co.jp/e/bio********************3[ 3 ] Protocol Required materials not included・Thermocycler・Fragmentation equipment or reagentGenNext™ NGS Library Prep Kit is compatible with mechanical and enzymatic fragmentation methods.・Adapters for illumina®GenNext™ NGS Library Prep Kit is compatible with adapters in which both the cluster generation sequences and sequencing are added during the ligation step, such as those routinely used in TruSeq (illumina®) and other similar library preparation workflows.・SPRI (Solid Phase Reversible Immobilization) paramagnetic beadAgencourt®AMPure®XP Beads (Beckman Coulter, cat. no. A63880, A63881) are recommended for use with the GenNext™ NGS Library Prep Kit.・10 mM Tris-HCl, pH 8.0 - 8.5Dilution of adapter stock in water and elution of DNA in water is not recommended.・Magnetic rack/stand for magnetic bead separation・80% ethanol (freshly prepared)1.End repair and A-tailing(1)Prepare the reaction mix in a tube or PCR plate as follows:Component Reaction volume(60 µL)Fragmented dsDNA 50 µLEnd repair and A-tailing buffer* 8 µLEnd repair and A-tailing enzyme mix* 2 µL* Premixes are stable for at least 24 hours at 4°C. Use the mixed solution within 24 hours.(2)V ortex gently or mix well by pipetting. Spin down briefly and proceed immediatelyto the next step.(3)Incubate the mixture as follows:30°C, 10 minutes65°C, 5 minutes4°C, hold(4)Proceed immediately with adapter ligation as described in the next protocol.JAPAN CHINATOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.Tel (+81)-6-6348-3888 Tel (+86)-21-58794900 www.toyobo.co.jp/e/bio********************42.Adapter Ligation(1)Dilute adapter stocks in 10 mM Tris-HCl, pH 8.0 - 8.5, as follows:FragmentedDNA per 50 µL End repair and A-tailing reaction Adapter stock concentrationAdapter :insertmolar ratio1 µg15 µM10:1500 ng15 µM20:1250 ng15 µM40:1100 ng15 µM100:150 ng15 µM200:125 ng7.5 µM200:110 ng 3 µM200:15 ng 1.5 µM200:12.5 ng750 nM200:11 ng300 nM200:1(2)In the same plate/tubes in which end repair and A-tailing was performed, prepare thereaction mix as follows:Component Reaction volume (110 µL)End repair and A-tailing reaction product 60 µLAdapter stock 5 µLLigation solution 45 µL(3)Mix well and centrifuge briefly.(4)Incubate the mixture as follows:20°C, 15 minutes4°C, hold(5)Immediately proceed to the next process (cleanup).3.Cleanup (after adapter ligation)(1)Perform a 0.8× SPRI-bead-based cleanup as follows:Component V olume (198 µL)Adapter ligation reaction product 110 µLAgencourt® AMPure® XP* 88 µL* Ensure that beads are fully resuspended.(2)Mix well by vortexing and/or pipetting up and down multiple times.JAPAN CHINATOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.Tel (+81)-6-6348-3888 Tel (+86)-21-58794900 www.toyobo.co.jp/e/bio********************5(3)Incubate the plate/tubes at room temperature for 5 - 15 minutes.(4)Place the plate/tubes on a magnetic stand to capture the beads. Incubate until thesolution is clear.(5)Carefully remove and discard the supernatant.(6)A dd 200 μl 80% ethanol in the plate/tubes on the magnetic stand.(7)Incubate the plate/tubes on the magnetic stand at room temperature for 30 seconds.(8)Carefully remove and discard the ethanol.(9)A dd 200 μl 80% ethanol in the plate/tubes on the magnetic stand.(10)Incubate the plate/tubes on the magnetic stand at room temperature for 30 seconds.(11)Carefully remove and discard as much ethanol as possible. Be careful not to disturbthe beads.(12)Air dry the beads at room temperature for 3 - 5 minutes while the plate/tubes are onthe magnetic stand with lids open.NotesOverdrying the beads may reduce the yield.(13)Remove the plate/tubes from the magnetic stand.(14)Resuspend the b eads in 25 μL elution buffer (10 mM Tris-HCl, pH 8.0 - 8.5) andincubate the plate/tubes at room temperature for 2 minutes. If proceeding with size selection (optional: see [3] 6), resuspend the beads in 55 μL elution buffer.(15)Place the plate/tubes the magnetic stand to capture the beads. Incubate until thesolution is clear.(16)Transfer the supernatant to a new plate/tubes.Purified libraries can be stored at–20°C.4.Library Amplification(1)Prepare a reaction mix as follows:Component Reaction volume (50 μL)Library amplification master nix (2×) 25 μLLibrary amplification primer mix (10×) 5 μLAdapter-ligated library 20 μLJAPAN CHINA TOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD. Tel (+81)-6-6348-3888 Tel (+86)-21-58794900 www.toyobo.co.jp/e/bio ********************6(2) Mix well and centrifuge briefly.(3) Amplify using the following cycling conditions:(amplified libraries can be stored at –20°C.) StepTemp Duration Cycles Initial denaturation 94°C 2 minutes 1 Denaturation 98°C 10 seconds Minimum number required for optimal amplification (see below)Annealing 60°C 10 seconds Extension 68°C 15 secondsHOLD4°C-1Recommended cycle numbers* The optimal number of amplification cycles may be 1 - 3 cycles higher or lower, depending on the sample type and size distribution of the input DNA.5. Cleanup (after library amplification) (1) Perform a 1× SPRI-bead-based cleanup as follows:ComponentV olume (100 μL)Library amplification reaction product 50 μL Agencourt ® AMPure ® XP*50 μL* Ensure that beads are fully resuspended.(2) Mix well by vortexing and/or pipetting up and down multiple times. (3) Incubate the plate/tubes at room temperature for 5 - 15 minutes.(4) Place the plate/tubes on a magnetic stand to capture the beads. Incubate until thesolution is clear.(5) Carefully remove and discard the supernatant.Input DNA (into end repair and A-tailing reaction) Cycles* 1 μg 0 500 ng 0 250 ng 0 100 ng 0-2 50 ng 3-5 25 ng 5-6 10 ng 7-9 5 ng 9-11 2.5 ng 11-13 1 ng 13-15JAPAN CHINATOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.Tel (+81)-6-6348-3888 Tel (+86)-21-58794900 www.toyobo.co.jp/e/bio********************7(6)A dd 200 μl 80% ethanol in the plate/tubes on the magnetic stand.(7)Incubate the plate/tubes on the magnetic stand at room temperature for 30seconds.(8)Carefully remove and discard the ethanol.(9)A dd 200 μl 80% ethanol in the plate/tubes on the magnetic stand.(10)Incubate the plate/tubes on the magnetic stand at room temperature for 30 seconds.(11)Carefully remove and discard as much ethanol as possible. Be careful not to disturbthe beads.(12)Air dry the beads at room temperature for 3 - 5 minutes.NotesOverdrying the beads may reduce the yield.(13)Remove the plate/tubes from the magnetic stand.(14)Resuspend the beads in an appropriate volume of elution buffer (10 mM Tris-HCl,pH 8.0 - 8.5). Incubate the plate/tubes at room temperature for 2 minutes. If proceeding with size selection (optional: see [3] 6), resuspend the beads in 55 μL elution buffer.(15)Place the plate/tubes on a magnetic stand. Incubate until the solution is clear.(16)Transfer the supernatant to a new plate/tubes. Purified, amplified libraries can bestored at –20°C. Proceed with size selection, library quality control or sequencing, as appropriate.6.Size selection(optional)If required, size selection may be performed at several points in the process workflow, for example, prior to end repair and A-tailing, after the post ligation cleanup or after library amplification. Size selection results in a narrower library size distribution, but at the cost of a much amount of library. Below is an example of selecting a library of 250 - 450 bp.(1)Add 0.6× SPRI beads to the library as follows:Component V olume (80 μL)Library 50 μLAgencourt® AMPure® XP* 30 μL* Ensure that beads are fully resuspended.JAPAN CHINATOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.Tel (+81)-6-6348-3888 Tel (+86)-21-58794900 www.toyobo.co.jp/e/bio********************8(2)Mix well by vortexing and/or pipetting up and down multiple times.(3)Incubate the plate/tubes at room temperature for 5 - 15 minutes.(4)Place the plate/tubes on a magnetic stand. Incubate until the solution is clear.(5)Transfer 75 μl the supernatant to a new plate/tubes.(6)Add 0.13× SPRI beads to the supernatant as follows:Component V olume (85 μL)Library (supernatant) 75 μLAgencourt® AMPure® XP* 10 μL* Ensure that beads are fully resuspended.(7)Mix well by vortexing and/or pipetting up and down multiple times.(8)Incubate the plate/tubes at room temperature for 5 - 15 minutes.(9)Place the plate/tubes on a magnetic stand. Incubate until the solution is clear.(10)Carefully remove and discard the supernatant.(11)A dd 200 μL 80% ethanol in the plate/tubes on the magnetic stand.(12)Incubate the plate/tubes on the magnetic stand at room temperature for 30 seconds.(13)Carefully remove and discard the ethanol.(14)A dd 200 μL 80% ethanol in the plate/tubes on the magnetic stand.(15)Carefully remove and discard as much ethanol as possible. Be careful not to disturbthe beads.(16)Air dry the beads at room temperature for 3 - 5 minutes.NotesOverdrying the beads may reduce the yield.(17)Remove the plate/tubes from the magnetic stand.(18)Add the required amount (e.g., 20 μL) of 10 mM Tris-HCl (pH 8.0 - 8.5) to theplate/tubes and incubate these at room temperature for 2 minutes.(19)Place the plate/tubes on a magnetic stand. Incubate until the solution is clear.(20)Transfer the supernatant to a new plate/tubes. Purified libraries can be stored at–20°C.JAPAN CHINATOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.Tel (+81)-6-6348-3888 Tel (+86)-21-58794900 www.toyobo.co.jp/e/bio********************9[ 4 ] Library QC Library quantificationLibrary quantification can be performed using GenNext™ NGS Library QuantificationKit (Code No. NLQ-101) or equivalent. The kit allows the specific and accuratequantification of libraries bearing P5 and P7 adapters and can be applied to flow cellamplification.Library quality controlAssess the quality of the library using a capillary electrophoresis device such as AgilentBioAnalyzer or equivalent.JAPAN CHINA TOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD. Tel (+81)-6-6348-3888 Tel (+86)-21-58794900 www.toyobo.co.jp/e/bio ********************10[ 5 ] Application data1. Example of library quantification and quality controlLibraries were prepared from fragmented human genomic DNA, E. coli genomic DNA, or 100 bp DNA ladder with the GenNext TM NGS library prep kit or another company’s Kit (Company A).Libraries were amplified using 0-11 cycles of PCR and the size distribution checked using a MultiNA (Shimadzu Corporation). Library quantifications were performed using a GenNext™ NGS library quantification kit (code NLQ-101).There was no difference in the distribution of libraries between the GenNext™ NGS library prep kit and the other company’s kit (Company A). For most illumina ® sequencing platforms, 2 - 4 nM for each library is the preferred starting concentration for denaturation and dilution guidelines. These data illustrate that the GenNext™ NGS library prep kit achieved sufficient adapter-ligated library yields, even with low input amounts of DNA.Adapters of approximately 60 bp are ligated to both ends of the insert DNA fragments, so the total size of DNA fragments will increase by approximately 120 bp.JAPAN CHINATOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD.Tel (+81)-6-6348-3888 Tel (+86)-21-58794900 www.toyobo.co.jp/e/bio********************112.Example of next generation sequencing resultsLibraries were prepared from 1μg or 1ng of E. coli genomic DNA with the GenNext TM NGS library prep kit or another company’s library construction kit (Company A). Libraries prepared from 1ng DNA were amplified using 12 PCR cycles. Sequencing was performed on an illumina® MiSeq® and data analyzed using CLC Genomics Workbench (QIAGEN / CLC bio). Sequencing reads were down-sampled to 1 million per library prior to analysis.These data illustrate that GenNext TM NGS library prep kit enables high quality sequence data.JAPAN CHINA TOYOBO CO., LTD. TOYOBO (SHANGHAI) BIOTECH, CO., LTD. Tel (+81)-6-6348-3888 Tel (+86)-21-58794900 www.toyobo.co.jp/e/bio ********************12[ 6 ] Troubleshooting[ 7 ] Related productsProduct namePackage Code No. NGS Library quantification for illumina ®’s instrumentGenNext™ NGS library quantification kit 500 reactionsNLQ-101Symptom CauseSolutionPresence of adapter dimersLow-quality adapter- Avoid excessive freezing and thawing of adapter stock solutions.- Dilute and store adapters in 10 mM Tris-HCl (pH 8.0 - 8.5).Non-optimal adapter concentration- The optimal concentration of adapter should bedetermined by preliminary tests: try the recommended adapter concentration (see [3] 2).- If the yield of the library is sufficient, repeat thepurification process or perform size selection (see [3] 6). Low yieldIncorrect ratio of added magnetic beads reagent and library solution - Ratio of SPRI magnetic bead reagent and library solution used for purification greatly influences size distribution and yield. Please check whether the liquid volume proportion is accurate.Overdrying the magnetic beads- Overdrying the magnetic beads may reduce the yield. Air dry the beads at room temperature within 5 minutes.。

基于PCR原理富集低丰度DNA突变的检测技术作者：武金霞， 韩凝， 边红武， Wu Jinxia， Han Ning， Bian Hongwu作者单位：浙江大学生命科学学院遗传研究所, 杭州,310058刊名：中华检验医学杂志英文刊名：Chinese Journal of Laboratory Medicine年，卷(期)：2014,37(12)1.沈胤晨,韩晓红外周血肿瘤标志物的筛选策略及临床应用[期刊论文]-中华检验医学杂志 2013(11)2.Sidransky D Emerging molecular markers of cancer 20023.Kobayashi S;Boggon TJ;Dayaram T EGFR mutation and resistance of non-small-cell lung cancer to gefitinib 20054.Engelman JA;Mukohara T;Zejnullahu K Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer 20065.Lo YM;Corbetta N;Chamberlain PF Presence of fetal DNA in maternal plasma and serum 19976.Hoffmann C;Minkah N;Leipzig J DNA bar coding and pyrosequencing to identify rare HIV drug resistance mutations 20077.Castellanos-Rizaldos E;Milbury CA;Guha M COLD-PCR enriches low-level variant DNA sequences and increases the sensitivity of genetic testing 20148.Li J;Wang L;Mamon H Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing 20089.Liew M;Pryor R;Palais R Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons 200410.Lipsky RH;Mazzanti CM;Rudolph JG DNA melting analysis for detection of single nucleotide polymorphisms 200111.Zuo Z;Chen SS;Chandra PK Application of COLD-PCR for improved detection of KRAS mutations in clinical samples 2009bury CA;Li J;Makrigiorgos GM Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations 201113.Castellanos-Rizaldos E;Liu P;Milbury CA Temperature-tolerant COLD-PCR reduces temperature stringency and enables robust mutation enrichment 201214.Egholm M;Buchardt O;Christensen L PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules 199315.Egholm M;Buehardt O;Nielsen PE Peptide nucleic acids (PNA).Oligonucleotide analogs with an achiral peptide backbone 199216.Luo JD;Chan EC;Shih CL Detection of rare mutant K-ras DNA in a single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe 200617.Singh SK;Koshkin AA;Wengel J LNA (locked nucleic acids):synthesis and high-affinity nucleic acid recognition 1998(04)18.Huang Q;Wang GY;Huang JF High sensitive mutation analysis on KRAS gene using LNA/DNA chimeras as PCR amplification blockers of wild-type alleles 201019.Oldenburg RP;Liu MS;Kolodney MS Selective amplification of rare mutations using locked nucleic acid oligonucleotides that competitively inhibit primer binding to wild-type DNA 200820.Vogelstein B;Kinzler KW Digital PCR 199921.Bhat S;Herrmann J;Armishaw P Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number 200922.Sanders R;Mason DJ;Foy CA Evaluation of digital PCR for absolute RNA quantification 201323.Lo YM;Lun FM;Chan KC Digital PCR for the molecular detection of fetal chromosomal aneuploidy 200724.Morrison T;Hurley J;Garcia J Nanoliter high throughput quantitative PCR 200625.Fan HC;Quake SR Detection of aneuploidy with digital polymerase chain reaction 200726.Ottesen EA;Hong JW;Quake SR Microfluidic digital PCR enables multigene analysis of individual environmental bacteria 200627.Diehl F;Li M;He Y BEAMing:single-molecule PCR on microparticles in water-in-oil emulsions 200628.Hindson BJ;Ness KD;Masquelier DA High-throughput droplet digital PCR system for absolute quantitation of DNA copy number 201129.Zimmermann BG;Grill S;Holzgreve W Digital PCR:a powerful new tool for noninvasive prenatal diagnosis 200830.Lun FM;Chiu RW;Allen Chan KC Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma 200831.Yung TK;Chan KC;Mok TS Single-molecule detection of epidermal growth factor receptor mutations in plasma by microfluidics digital PCR in non-small cell lung cancer patients 200932.Wang J;Ramakrishnan R;Tang Z Quantifying EGFR alterations in the lung cancer genome with nanofluidic digital PCR arrays 201033.Taly V;Pekin D;Benhaim L Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients 201334.Qi Z;Ma Y;Deng L Digital analysis of the expression levels of multiple colorectal cancer-related genes by multiplexed digital-PCR coupled with hydrogel bead-array 201135.Galbiati S;Stenirri S;Sbaiz L Identification of an 18 bp deletion in the TWIST1 gene by CO-amplification at lower denaturation temperature-PCR (COLD-PCR) for non-invasive prenatal diagnosis of craniosynostosis:first case report 201436.Pinzani P;Santucei C;Mancini I BRAFV600E detection in melanoma is highly improved by COLD-PCR 201137.Delaney D;Diss TC;Presneau N GNAS1 mutations occur more commonly than previously thought in intramuscular myxoma 200938.Santis G;Angell R;Nickless G Screening for EGFR and KRAS mutations in endobronchial ultrasound derived transbronchial needle aspirates in non-small cell lung cancer using COLD-PCR 201139.Boisselier B;Marie Y;Labussière M COLD PCR HRM:a highly sensitive detection method for IDH1 mutations 201040.Lee D;Sub YL;Kang SY IDH1 Mutations in Oligodendroglial Tumors:Comparative Analysis of DirectSequencing,Pyrosequencing,Immunohistochemistry,Nested PCR and PNA-Mediated Clamping PCR 201341.DU Juan,ZOU Xin,PAN Yi,LI Shuang-fei,LU Guang-xiu Non-invasive prenatal molecular detection of a fetal point mutation for congenital adrenal hyperplasia using co-amplification at lower denaturation temperature PCR[期刊论文]-中华医学杂志（英文版） 2010(22)42.Carotenuto P;Roma C;Cozzolino S Detection of KRAS mutations in colorectal cancer with Fast COLD-PCR 201243.Galbiati S;Brisci A;Lalatta F Full COLD-PCR protocol for noninvasive prenatal diagnosis of genetic diseases 201144.Tsang YT;Deavers MT;Sun CC KRAS (but not BRAF) mutations in ovarian serous borderline tumour are associated with recurrent low-grade serous carcinoma 201345.How Kit A;Mazaleyrat N;Daunay A Sensitive detection of KRAS mutations using enhanced-ice-COLD-PCR mutation enrichment and direct sequence identification 201346.Charbel C;Fontaine RH;Malouf GG NRAS mutation is the sole recurrent somatic mutation in large congenital melanocytic nevi 201447.Kim HS;Sung JS;Yang SJ Predictive efficacy of low burden EGFR mutation detected by next-generation sequencing on response to EGFR tyrosine kinase inhibitors in non-small-cell lung carcinoma 201348.Kim HR;Lee SY;Hyun DS Detection of EGFR mutations in circulating free DNA by PNA-mediated PCR clamping 201349.Kim H J;Lee KY;Kim YC Detection and comparison of peptide nucleic acid-mediated real-time polymerase chain reaction clamping and direct gene sequencing for epidermal growth factor receptor mutations in patients with non-small cell lung cancer 201250.Messa F;Tonissi F;Millo E A PNA-mediated clamping PCR for routine detection of KRAS mutations in colorectal carcinoma 201451.Dono M;Massucco C;Chiara S Low percentage of KRAS mutations revealed by locked nucleic acid polymerase chain reaction:implications for treatment of metastatic colorectal cancer 201352.Kamila WK;Michal S;Pawel K EGFR activating mutations detected by different PCR techniques in Caucasian NSCLC patients with CNS metastases:short report 201353.Guha M;Castellanos-Rizaldos E;Makrigiorgos GM DISSECT Method Using PNA-LNA Clamp Improves Detection of EGFRT790m Mutation 201354.Dominguez PL;Kolodney MS Wild-type blocking polymerase chain reaction for detection of single nucleotideminority mutations from clinical specimens 200555.Krypuy M;Newnham GM;Thomas DM High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples:KRAS codon 12 and 13 mutations in non-small cell lung cancer 200656.Do H;Krypuy M;Mitchell PL High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies 200857.Ihle MA;Fassunke J;K(o)nig K Comparison of high resolution melting analysis,pyrosequencing,next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p.V600E and non-p.V600E BRAF mutations 2014引用本文格式：武金霞.韩凝.边红武.Wu Jinxia.Han Ning.Bian Hongwu基于PCR原理富集低丰度DNA突变的检测技术[期刊论文]-中华检验医学杂志 2014(12)。

Self-Repairing Function in the Carbon-ContainingRefractoryAkira Yamaguchi*Okayama Ceramics Research Foundation,Bizenn,Okayama705-0021,Japan Self-repairing function is observed in carbon-containing refractory such as MgO–C,Al2O3–C,and so forth.Non-oxides such as pure metallic,alloys,carbides,and nitrides are intentionally added to the refractory composition to bring about this function.As a basis of the development of a self-repairing refractory,the self-repairing mechanism in the carbon-containing refractory is described.IntroductionIn a refractory composed only of oxides,one way to increase corrosion resistance is to decrease porosity(in-creasing density).When the porosity becomes too low, however,the thermal shock resistance of the refractory becomes poor.The relationship between the corrosion resistance and the thermal shock resistance of the re-fractory is well known,and is a delicate balance in areas where thermal shock and corrosion are issues.Mixing carbon in oxides solved the thermal shock problem be-cause of carbon’s low thermal expansion and high ther-mal conductance,which increases the refractory’s thermal shock resistance.The corrosion resistance is also increased because the penetrated slag into the re-fractory was suppressed by the characteristics of carbon, which is hardly wetted by slag.In the results,the cor-rosion resistance and the thermal shock resistance of the refractory are both simultaneously improved.Carbon,however,has a large defect—it is easily oxidized in air at a high temperature.One way to de-crease this oxidation is by adding non-oxides such as metal,alloy,and carbide,which react with the air and expand,sealing pores.Behavior and Effect of Nonoxides Added to a Carbon-Containing RefractoryInitially,non-oxides were added to prevent the oxidation of carbon.It was learned,however,that these non-oxides not only acted as antioxidants,but they also had other effects on the refractory including:(1)Decrease of carbon-loss by the reduction ofCO(g)to C(s).(2)Decrease of porosity.(3)Increase of the modulus of rupture(MOR).(4)Crystallization of amorphous carbon precipi-tated from binder.(5)Improvement of the oxidation resistance andcorrosion resistance by forming a surface-protective layer.Int.J.Appl.Ceram.Technol.,4[6]490–495(2007) Ceramic Product Development and Commercialization*akira-yamaguchi@occ.ecwebr2007The American Ceramic SocietyEach of these areas that additives benefit carbon-containing refractories is described in great depth in terms of the mechanisms.Their use can be engineered in a refractory to increase the service life.Decrease of Carbon-Loss by the Reduction of CO(g) to C(s)Graphite is rapidly oxidized under an oxidation at-mosphere over about7001C,and it generates primarily CO2(g)and CO(g).CO(g)is mainly produced in the open pores of the carbon-containing refractory at a high temperature,because the equilibrium partial pressure of CO(g)is remarkably higher than that of CO2(g)under the existence of condensed carbon above about 10001C.1At the atmospheric pressure,CO(g)exists at a partial pressure of0.1Mpa in the pores in the refractory.Carbon antioxidants of pure metals,alloys,nitrides, and carbides become an oxide that is stabilized under a 0.1Mpa CO(g)atmosphere with solid carbon.For ex-ample,Al and AlN become effective antioxidants be-cause Al2O3is stable under0.1Mpa CO(g).The following reactions occur in metal(M)and carbide (MC)in contact with CO(g):x Mþy COðgÞ!M x O yþy Cð1Þx MCþy COðgÞ!M x O yþðxþyÞCð2ÞThe effect of antioxidants is to suppress oxidation loss of carbon by reducing CO(g)to C(s).In addition, the reactions bring about a volume expansion,which can seal pores.Volume changes of149%and289%are obtained from the oxidation of Al and SiC,respectively, 2Alðs;lÞþ3COðgÞ¼Al2O3ðsÞþ3CðsÞð3ÞSiCðsÞþ2COðgÞ¼SiO2ðsÞþ3CðsÞð4ÞThe change was calculated by taking the true den-sity of Al,Al2O3,SiC,SiO2,and amorphous carbon as 2.70,3.98,3.21,2.32,and1.60g/cm3,respectively. Decrease of PorosityWhen the non-oxide added changes to an oxide,a volume expansion is generated as described in Eqs(3) and(4).If this expansion is absorbed by the pores in the refractory,the porosity of the refractory decreases. Figure1shows an example,2namely open porosity de-creasing in a(77.5mass%MgO–17.5%C)refractory with additives at above about8001C,but remaining constant in a refractory without additives.Phenol resin was mixed in powdered raw materials as a binder,and the mixture was pressed to form a shaped refractory using a vacuum friction press,which created a very low porosity(several percent or less). Pores are formed by decomposing binder gases through about8001C.The porosity,however,decreases by the reaction products of the additive above about8001C. The rate of porosity decrease varies depending on the composition,amount of additive,and particle size. Therefore,it is important to choose an additive that performs the best in the service environment of the re-fractory.Increase of the MORFigure23shows the MOR of MgO–C refractories with and without metal additives during heating.The MOR of the refractory without an additive decreases during heating.On the other hand,the MOR of the refractory with non-oxide additives begins to increase from about600to10001C.This is brought about by carbides,nitrides,and/or oxides that are formed by re-actions between the additives and carbon,CO(g)and/or N2(g)from the air.These reaction products deposit as plate crystals and/or whiskers as shown in Fig.3,andinaddition,react with the aggregate oxide to form a new solid phase.These changes are considered to bring about the increase of MOR of the refractory.Crystallization of Amorphous Carbon Precipitated from BinderFree carbon deposits are created from the decom-position of phenol resin added as a binder.Free carbon is also formed by the reaction between the added non-oxide and CO(g )as shown by expressions (1)–(4).These carbons 2are amorphous and oxidized at low-er temperatures more easily than graphite.Their true density is between 1.2and 1.6g/cm 3.However,only with the use of B 4C or MgO does crystallization of the carbons occur.Figure 44shows X-ray diffraction patterns of free carbon from phenol resin and the free carbon with 5mass%B 4C,which were heated in Ar gas at 800–16501C for 3h.When B 4C is added,crystallization of the carbon is promoted.MgO also promotes crystalli-zation of the free carbon,as shown in Fig.5.This figure shows the X-ray diffraction pattern of free carbon heated at 17001C and of a mixture of MgO and the carbon from phenol resin,which is heated at 1100–16001C in Ar gas.Improvement of the Oxidation Resistance and Corrosion Resistance by Forming a Surface-Protective LayerIt is well accepted that a protective layer is formed toward the surface of MgO/carbon refractories during service by the addition of metals,metal alloys,carbides,or nitrides.The following three examples areexplained.Fig.3.Whisker formed in a MgO–C refractory with Al metal additives duringheating.Example1:Formation of a dense protective layer through Al–Mg alloy additions to a MgO–C refractory.When Al–Mg alloy additions are made to a MgO–C refractory used in steelmaking,a dense magnesia layer is formed when the refractory contacts slag containing iron oxide.The formation of this protective layer is shown in Fig.6.When this refractory is heated during use,the Mg volatilizes at a low temperature and Mg(g)is oxidized near the refractory surface,condensing Mg in a layer as MgO.This MgO condensation becomes a driving force for the continual formation of a dense MgO protective layer.It is important for the MgO layer to grow under use,because it is corroded during service.The growth mechanism for MgO can be explained thermodynam-ically by the following methods:MgOðsÞþCðsÞ¼MgðgÞþCOðgÞð5ÞThe partial pressure of Mg(g)and CO(g)in the pores of the refractory becomes significant above about 14001C,acting as a driving force for the reaction.When Al metal coexists with Mg,it reacts with CO(g)as fol-lows,reducing the partial pressure of CO(g)and in-creasing the partial pressure of Mg(g):2Alðl;gÞþ3COðgÞ¼Al2O3ðsÞþ3CðsÞð6ÞIn practice,oxygen diffuses through the MgO dense layer from outside the refractory to inside the re-fractory microstructure.At the refractory surface,in contact with the slag,the dense MgO that is formed reacts with Fe2O3to form MgFe2O3.Iron diffusion to the inside of this layer leads to the formation of (Mg,Fe)O(wustite).The next reaction occurs in the dense(Mg,Fe)O layer,causing Fe metal deposition,and the growth of a magnesia layer:ðMg;FeÞOþMgðgÞ¼MgOþFeð7ÞFe metal from reaction(7)deposits on the inside of the dense layer in the refractoryExample2:Formation of a surface-protective layer in the B4C addition MgO–C refractory.B4C added as an antioxidant to the MgO–C re-fractory reacts with CO(g)as shown in the following equation:B4CðsÞþ6COðgÞ¼2B2O3ðlÞþ7CðsÞð8ÞThe reaction is brought about through various gas phases of the B–C–O system,especially B2O2(g)and B2O3(g),which have high equilibrium partial pressures. Initially,B4C(s)reacts with CO(g)to form B2O2(g),as shown in the following equation:B4CðsÞþ4COðgÞ¼2B2O2ðgÞþ5CðsÞð9ÞWhen B2O2(g)diffuses near the surface of the re-fractory where CO(g)partial pressure is high,it is condensed as B2O3according to the following reaction: B2O2ðgÞþCOðgÞ¼B2O3ðlÞþCðsÞð10ÞFrom Eqs(9)and(10),Eq.(8)is obtained.Once B2O3has condensed on the surface,it reacts with MgO to form Mg3B2O6,which melts above13401C.Figure7 shows the dense layer formed near the surface of the refractory,which is composed of MgO1Mg3B2O6.Fig.6.Dense magnesia layer formed near the surface of theMgO–C refractory with Al–Mg alloy./ACT Carbon-Containing Refractory493This liquid phase suppresses the diffusion of the oxygen to the refractory.Example 3:Formation of a surface-protective layer in the SiC addition Al 2O 3–C refractory.A dense protective layer is formed in Al 2O 3–C refractory by SiC additions as shown in Fig.8.The mechanisms by which the protective layer form is start-ed by SiC,which exists near the refractory surface;it reacts with CO(g )to form SiO(g )and C(s )as shown in the following equation:SiC ðs ÞþCO ðg Þ¼SiO ðg Þþ2C ðs Þð11ÞThe deposited C(s )condenses near the surface of SiC grain.The SiO(g ),on the other hand,diffuses near the surface of the refractory and reacts with CO(g )to form SiO 2and C(s )as indicated in the following equa-tion:SiO ðg ÞþCO ðg Þ¼SiO 2ðs ;l ÞþC ðs Þð12ÞSiO 2,which is deposited near and builds up the refractory surface,reacts with the slag to form a high-viscosity slag,which contains some free carbon.The high-viscosity slag contributes to the corrosion and ox-idation resistance of the refractory by forming a protec-tive layer.In general,the formation of a layer near the surface of a refractory increases the service life of the underling refractory by a number of different mechanisms de-pending on the type of additive in the refractory.An additive of appropriate type,particle size,and dosage should be chosen depending on the refractory type and the service requirements.In case of the nitride additives,the viscosity of the slag melt near the surface of the refractory is increased by dissolution of N 2(g ),which occurs by the decompo-sition of the nitride when in contact with slag.A small amount of nitride addition seems to be very effective at decreasing refractory/slag interactions.The self-repairing function in carbon-containing refractories,which has been described above,is dia-grammatically shown in Fig.9.Fig.7.Dense liquid phase formed near the surface of the MgO–C refractory with B 4C.Fig.8.Dense protective layer formed in an Al 2O 3–C refractory withSiC.Fig.9.Phenomenon of self-repairing function in a carbon-containing refractory through metal,alloy,carbide,or nitride additions.494International Journal of Applied Ceramic Technology—Yamaguchi Vol.4,No.6,2007The Present and Future State of the Additives to Refractories Contributed to the Self-Repairing FunctionCarbon-containing refractories with metal or metal alloys or with carbides or nitrides are representative ex-amples of a refractory with a self-repairing mechanism that prolongs the refractory service life.Table I shows the additives that are most commonly used or under development.Al and SiC are the additives most fre-quently used for a MgO–C and a Al2O3–C refractory, respectively.Carbides are excellent additives because they create deposits as a free carbon in a refractory.Al4O4C,5,9 Al2OC,5Al4SiC4,6Al8B4C7,7,8and so forth,are con-sidered promising future additives because of their potential to form free carbon.SummaryIt has been shown that added non-oxides,additions to carbon-containing refractory,such as metal,alloys,carbides,nitrides,and so on,bring about a self-repairing function.The self-repairing function includes(1)reduction of CO(g)to C(s)by non-oxides and aiding in reducing carbon loss,(2)decrease of the porosity of the refractory,(3)strengthening the modulus of rupture of the refractory,(4)promotion of the crystallization of free carbon from the binder,and(5)formation of the protective layer,the refractory surface,and increase of oxidation resistance and corro-sion resistance.References1. A.Yamaguchi,‘‘Control of Oxidation–Reduction Reactions in MgO–C Re-fractories,’’Taikabutsu(Japanese),37[7]365–370(1983).2. A.Yamaguchi and J.Yu,‘‘Behavior of Carbon from Pitch and Resin Added toCarbon-Containing Refractories,’’J.Ceram.Soc.Japan,102[1]73–74 (1994).3. A.Watanabe and H.Takahashi,et al.,‘‘Behavior of Different MetalsAdded to MgO–C Brick,’’Taikabutsu(Japanese),38[11]740–746(1986).4.S.Zhang and A.Yamaguchi,‘‘Effect of B4C on the Crystallization and Ox-idation Resistance of Carbon from Resin,’’J.Ceram.Soc.Japan,102[9]830–839(1994).5.S.Zhang and A.Yamaguchi,‘‘Hydration Resistances and Reactions withCO(g)of Al4O4C and Al2OC Formed in Carbon-Containing Refractories with Al,’’J.Ceram.Soc.Japan,104[5]393–398(1996).6. A.Yamaguchi,S.Zhang,J.Yu,and S.Hashimoto,‘‘Behavior of AntioxidantsAdded to the Carbon-Containing Refractories,’’Unified international Tech-nical Conference on Refractories,3140–147(1995).7. A.Yamaguchi,Y.Nakano,and T.Wang,‘‘Effect and Behavior of Al–B–CSystem Antioxidants Added to MgO–C Refractories,’’Can.Metall.Q.,39[4] 59–68(2000).8.T.Wang and A.Yamaguchi,‘‘Antioxidation Behavior and Effect of Al8B4C7Added to Carbon Containing Refractories,’’J.Ceram.Soc.Japan,108[9] 818–822(2000).9.J.Zhao,W.Lin,A.Yamaguchi,J.Ommyouji,and J.Sun,‘‘Synthesis ofAl4O4C from Starting Raw Materials of Alumina and Graphite,’’Taikabutsu (Japanese),59[6]288–295(2007).Table I.Additive for the Self-Repairing RefractorySortMetal Al,Si,MgAlloy Al–Si,Al–Mg,Al–Ca–Mg–SiCarbide SiC,B4C,Al4SiC4,Al8B4C7,Al4O4C,Al2OCBoride B4C,ZrB2,CaB2Nitride Si3N4,AlNBold,practical;italic,trial./ACT Carbon-Containing Refractory495。

manual THUNDERBIRD Probe qPCR Mix 0910 A4250K THUNDERBIRD™ Probe qPCR MixQPS-101T 1 mL x 1QPS-101 1.67 mL x 3Store at -20°C, protected from lightContents[1] Introduction[2] Components[3] Primer/Probe design[4] Template DNA[5] Protocol1. Standard reaction set up2. Cycling conditions2-1. Real-time PCR conditions using Applied Biosystems 7900HT2-2. Real-time PCR conditions using Roche LightCycler 1.1[6] Related ProtocolcDNA synthesis[7] Troubleshooting[8] Related productsC AUTIONAll reagents in this kit are intended for research purposes only. Do not use for diagnosis or clinical purposes. Please observe general laboratory precautions and observe safety procedures while using this kit.-LightCycler™ is a trademark of Idaho Technology, Inc. and Roche Molecular Systems, Inc.-TaqMan® is a registered trademark of Roche Molecular Systems, Inc.-SYBR® is a registered trademark of Roche Molecular Systems, Inc.JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio 1JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio********************1[ 1 ] Introduction [ 2 ] Components DescriptionTHUNDERBIRD™ Probe qPCR Mix is a highly efficient 2x Master Mix for real-time PCR using TaqMan® probes. The master mix contains all required components, except for ROX reference dye, probe and primers (50x ROX reference dye is individually supplied with this kit). The master mix facilitates reaction setup, and improves the reproducibility of experiments.This product is an improved version of Realtime PCR Master Mix (Code No. QPK-101). In particular, reaction specificity and PCR efficiency is enhanced.Features-High specificityThe specificity for the detection of low-copy targets is improved.-Homogeneous amplificationThe dispersion of PCR efficiency between targets is reduced by a new PCR enhancer*. (*Patent pending)-Broad dynamic rangeHigh specificity and effective amplification enable the detection of a broad dynamic range.-Compatibility for various real-time cyclers.The reagent is applicable to most real-time cyclers (i.e. Block type and glass capillary type). Because the 50x ROX reference dye is individually supplied with this kit, the kit can be applied to real-time cyclers that require a passive reference dye.-Hot start PCRThe master mix contains anti-Taq DNA polymerase antibodies for hot start technology. The antibodies are easily inactivated in the first denaturation step, thereby activating the DNA polymerase.About the fluorescent probe detection systemThe TaqMan® probe system utilizes fluorescence emission from the probes. The probes hybridize to the target amplicons and then emit fluorescence upon degradation by the 5'-3' exonuclease activity of Taq DNA polymerase. This type of detection system can achieve higher specificity in real-time PCR assays than the SYBR® Green I detection system.This kit includes the following components for 40 reactions (QPS-101T) and 200 reactions (QPS-101), with 50 μl per reaction. All reagents should be stored at -20°C.<QPS-101T>THUNDERBIRD™ Probe qPCR Mix 1 ml x 150x ROX reference dye 50 μl x 1JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio********************2[ 3 ] Primer/Probedesign <QPS-101>THUNDERBIRD™ Probe qPCR Mix 1.67 ml x 350x ROX reference dye 250 μl x 1Notes:-THUNDERBIRD™ Probe qPCR Mix can be stored, protected from light, at 2-8°C for up to 3 months. For longer storage, this reagent should be kept at -20°C and protected from light. No negative effect was detected by 10 freeze-thaw cycles of THUNDERBRID™ Probe qPCR Mix. This reagent does not contain the ROX reference dye.-50x ROX reference dye can be stored, protected from light, at 2-8°C or -20°C. For real-time cyclers that require a passive reference dye, this reagent must be added to the reaction mixture at a concentration of 1x or 0.1x. The master mix solution with the ROX reference dye can be stored, protected from light, at 2-8°C for up to 3 months. For longer storage, this reagent should be kept at -20°C and protected from light. The pre-mixed reagents can be prepared according to the following ratios. [5] Table 1 shows the optimal concentration of the ROX dye.1x solutionTHUNDERBIRD™ Probe qPCR Mix : 50x ROX reference dye = 1.67 ml : 66.8 μl THUNDERBIRD™ Probe qPCR Mix : 50x ROX reference dye = 1 ml : 40 μl0.1x solutionTHUNDERBIRD™ Probe qPCR Mix : 50x ROX reference dye = 1.67 ml : 6.7 μl THUNDERBIRD™ Probe qPCR Mix : 50x ROX reference dye = 1 ml : 4 μlFor real-time cyclers that do not require a passive reference dye, THUNDERBIRD™ Probe qPCR Mix without the ROX reference dye can be used.1. Primer conditionsHighly sensitive and quantitative data depend on primer design. The primer should be designed according to the following suggestions;-Primer length: 20-30 mer-GC content of primer: 40-60%-Target length: ≤ 200 bp (optimally, 80-150 bp)-Melting temperature (Tm) of primers: 60-65°C-Purification grade of primers: Cartridge (OPC) grade or HPLC gradeNotes:-Longer targets (>200 bp) reduce efficiency and specificity of amplification.-Tm of the primers can be flexible, because the Tm value depends on the calculation formula.JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio********************3[ 4 ] Template DNA 2. Fluorescent probeThe probes should be designed according to the guidelines of each probe system. Because insufficiently purified probes may inhibit the reaction, HPLC-grade probes should be used.The following DNA samples can be used as templates.1. cDNANon-purified cDNA, generated by reverse transcription reactions, can be used directly for real-time PCR using THUNDERBIRD™ Probe qPCR Mix. Up to 10% of the volume of a cDNA solution can be used for a real-time PCR reaction. However, excess volume of the cDNA may inhibit the PCR. Up to 20% (v/v) of the cDNA solution from ReverTra Ace® qPCR RT Kit (Code No. FSQ-101) can be used for real-time PCR (see [6]).2. Genomic DNA, Viral RNAGenomic DNA and viral RNA can be used at up to 200 ng in 50 μl reactions.3. Plasmid DNAAlthough super-coiled plasmids can be used, linearized plasmid DNA produces more accurate assays. The copy number of the plasmid DNA can be calculated by the following formula.Copy number of 1μg of plasmid DNA = 9.1 x 1011 / Size of plasmid DNA (kb)JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio********************4[ 5 ] Protocol1. Reaction mixture setupReaction volume FinalReagent50µl20µlConcentrationDWXµlXµlTHUNDERBIRD™ Probe qPCR Mix 25 µl 10 μl 1xForward Primer 15 pmol 6 pmol 0.3 μM*1Reverse Primer 15 pmol 6 pmol 0.3 μM*1TaqMan® Probe 10 pmol 4 pmol 0.2 μM*150x ROX reference dye 1μl / 0.1 μl 0.4μl / 0.04μl 1x / 0.1x*2DNA solution Y µl Y µlTotal50µl20µlNotes:*1 Primer / probe concentration should be determined according to the manufacturer’sinstructions.Higher primer concentration tends to improve the amplification efficiency, and lowerprimer concentration tends to reduce the non-specific amplification. The primerconcentration should be set between 0.2-0.6 μM.*2 50x ROX reference dye must be added when using real-time cyclers that require apassive reference dye, according to Table 1. Table 1 shows the optimum concentrationof the ROX reference dye. This dye is not necessary for real-time cyclers that do notrequire a passive reference dye.Table 1 Recommended ROX dye concentrationReal-time cycler Optimal dye concentration(dilution ratio)Applied Biosystems 7000, 7300, 7700, 7900HT etc. 1x (50:1)Applied Biosystems 7500, 7500Fast,Stratagene cyclers (Optional) etc.0.1x (500:1)Roche’ cyclers, Bio-Rad cyclers, BioFlux cyclers etc. Not requiredNotes:The ROX dye in Realtime PCR Master Mix (Code No. QPK-101) corresponds to 1xconcentration.JAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/bio********************52. PCR cycling conditionsThe following table shows the recommended thermal conditions using primers designed according to the recommended primer and probe conditions described in [3]. Almost all targets can also be amplified using the ongoing conditions with other real-time PCR reagents.*1Due to the anti-Taq antibody hot start PCR system, the pre-denaturation can be completed within 60 sec. The pre-denaturation time should be determined according to the recommendations of each real-time cycler. If the optimal pre-denaturation time cannot be determined, the time should be set at 60 sec.Table 2 The recommended pre-denaturation time for each real-time cycler Real-time cycler Pre-denaturation time High speed cycler (e.g. Applied Biosystems 7500Fast) 20 sec Capillary cycler (e.g. Roche LightCycler™ 1.x, 2.0) 30 secGeneral real-time cyclers (Applied Biosystems 7700, 7500,7900HT (normal block), Stratagene cyclers, BioFlux cyclers 60 sec *2The following table shows the optimal denaturation times for each real-time cycler. If the optimal denaturation time cannot be determined, the time should be set at 15 sec.Table 3 The recommended denaturation time for each real-time cycler Real-time cycler denaturation time High speed cycler (e.g. Applied Biosystems 7500Fast) 3 sec Capillary cycler (e.g. Roche LightCycler™ 1.x, 2.0) 5 secGeneral real-time cyclers (Applied Biosystems 7700, 7500,7900HT (normal block), Stratagene cyclers, BioFlux cyclers 15 sec *3Insufficient amplification may be improved by decreasing the extension temperature, and non-specific amplification (e.g. abnormal shapes of the amplification curve at low template concentrations) may be reduced by increasing the extension temperature. The extension temperature should be set at 56-64°C. *4If the target size is smaller than 300 bp, the extension time can be set at 30 sec on almost all real-time cyclers. Instability of the amplification curve or variation of data from each well may be improved by setting the extension time at 45-60 sec. Some real-time cyclers or software need over 30 sec for the extension step. In these cases, the time should be set according to each instruction manual (e.g. Applied Biosystems 7000/73000: ≥ 31 sec; Applied Biosystems 7500: ≥ 35 sec.).<3-step cycle> Temperature Time Ramp Pre-denaturation:95°C 20-60 sec *1Maximum Denaturation:95°C 1-15 sec *2 MaximumExtension: 60°C *3 30-60 sec *4 Maximum (data collection should be set at the extension step)JAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/bio********************62-1. Real-time PCR conditions using Applied Biosystems 7900HT(Normal block type, software version 2.2.2)The following is an example of a TaqMan ® assay using Applied Biosystems 7900HT.(1) The cycling parameters should be set according to the following “Thermal CyclerProtocol” window under the “Instrument” tab.Notes:- Appropriate sample volumes should be set. - ≥ 45 sec is necessary for the extension step.(2) Click the “Data collection” tab.(3) Insert the PCR plate(4) Start the programJAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio********************72-2. Real-time PCR conditions using Roche LightCycler 1.1(Software version 3.5)The following is an example of a TaqMan® probe assay using Roche LightCycler 1.1. (1)The cycling parameters should be set according to the following window. Analysisand Acquisition mode of the initial denaturation step must be set at “None”. (2)The cycling parameters should be set according to the following window. Analysismode of the PCR step must be set at “Quantification”. Acquisition modes of 95°C and 60°C must be set at “None” and “Single”, respectively.JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio********************8(3)The cycling parameters should be set according to the following window. Analysisand Acquisition mode of the cooling step must be set at “Non”.(4) Insert the capillaries in the carousel, and start the cycling program.JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio********************9[ 6 ] Related Protocol1. cDNA synthesiscDNA synthesized by various cDNA synthesis reagents can be used withTHUNDERBIRD™ Probe qPCR Mix. However, cDNA synthesized by a reagentspecialized for real-time PCR can increase sensitivity.ReverTra Ace® qPCR RT Kit (Code No. FSQ-101) is a cDNA synthesis kit suitable forreal-time PCR. Here, the protocol with ReverTra Ace® qPCR RT Kit is described.However, for the detailed protocol, please refer to the instruction manual of the kit.(1) Denaturation of RNAIncubate the RNA solution at 65°C for 5 min and then chill on ice.Notes:-This step can be omitted. But this step may increase the efficiency of the reversetranscription of RNA, which forms secondary structures.-Do not add 5x RT Buffer and/or enzyme solution at this step.(2) Preparation of the reaction solutionReagent V olume (amount)Nuclease-freeWaterXµl5x RT Buffer 2 µlPrimerMix 0.5µlEnzymeMix 0.5µlRNAsolution 0.5pg-1 µgTotal10µl(3) Reverse transcription reaction-Incubate at 37°C for 15 min. <Reverse transcription>-Heat at 98°C for 2 min. <Inactivation of the reverse transcriptase>-Store at 4°C or -20°C.**This solution can be used in the real-time PCR reaction directly or after dilution.Notes:The above temperature conditions are optimized for ReverTra Ace® qPCR RT Kit.JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio********************10[ 7 ] TroubleshootingSymptom Cause SolutionLoss of linearity in the high cDNA/DNA concentration region. Inhibition by the components in thecDNA/DNA solution.-DNA: The DNA sample may contain PCRinhibitors. The DNA samples should berepurified.-cDNA: The components in the cDNA synthesisreagent may inhibit the PCR reaction. The cDNAsample should be used after dilution.The template DNA is insufficient. When the DNA/cDNA copy number is lower than10 copies per reaction, the linearity of the reactiontends to be lost. The template concentrationshould be increased.Adsorption of the DNA to the tubewall.The diluted DNA templates tend to be absorbedonto the tube wall. Dilution should be performedjust prior to experiments.Lost of linearity or lower signal in the lowDNA/cDNAconcentration region. Competition with primer dimerformation. In the probe assay, primer dimers are not detected. However, dimer formation may reduce the amplification efficiency of the target, especially for reactions at low template concentration. The reaction conditions should be optimized or the primer sequences should be changed.Loss of linearity of the amplification carves. Competition with non-specificamplification.In the probe assay, non-specific amplification isnot detected. However, non-specific amplificationmay reduce the amplification efficiency of thetarget. The reaction conditions should beoptimized or the primer sequences should bechanged.Inappropriate cycling conditions. Optimize the cycling conditions according to [5]. Degradation of the primers. Fresh primer solution should be prepared.The PCR efficiency is lower than 90% (slope:<-3.6) The calculation of the PCRefficiency is inappropriate. The Ct value on the linear region should be used to calculate PCR efficiency.The PCR efficiency is higher than 110%. The calculation of the PCRefficiency is inappropriate.The Ct value on the linear region should be usedto calculate PCR efficiency.Poor purification of the templateDNA.Low-purity DNA may contain PCR inhibitors.Re-purify the DNA samples.Absorption of the template DNA tothe tube wall.Diluted DNA templates tend to be absorbed ontothe tube wall. Dilution of the templateDNA/cDNA should be performed just prior toexperiments.Plasmid DNA or PCR product isused as a template.In general, plasmid DNA or PCR product is usedat low concentration. Diluted DNA templates tendto be absorbed onto the tube wall. Dilution of thetemplate DNA/cDNA should be performed justprior to experiments. Dilution with a carriernucleic acid solution (Yeast RNA) is alsoeffective in improving linearity.Inappropriate thermal conditions. Optimize the thermal conditions according to [5].Reproducibility is notgood.Low purity of the primers or probes.Different lots of primers or probes may showdifferent results. When the lot is changed, priortesting of the primer or probe should beperformed.JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio********************11Symptom Cause SolutionContamination or carry over of the PCR products. Change the contaminated reagent.Amplification from the non-template control(NTC). Inappropriate settings offluorescence measurement, such asin the case of multiplex PCR. In multiplex experiments, inappropriate setting of fluorescence measurement may cause the detection of noise by the cross talk of fluorescent dyes. Settings should be reconfirmed.Excessive amount of ROX reference dye. Excessive amount of ROX reference dye may cause low signal. 50x ROX reference dye should be used according to [5] Table 1.Inappropriate settings of fluorescence measurement. Settings should be confirmed according to the instruction manual of each detector.Low purity of fluorescent probes. Low purity of the probe may increase the baseline. HPLC grade probes should be used.Excessive intensity of the quencher Dye. Certain quenchers (e.g. TAMRA) may cause a higher baseline because of its fluorescence. Use of a non-fluorescent quencher may improve the high baseline.Degradation of the probe. Store the probes according to the manufacture’srecommendations.Insufficient fluorescence measurement time. Certain detection systems require a longer time to detect the fluorescent signal. Longer extension (measurement) time (45-60 sec) may improve the unstable signal.Low amplificationcurve signal /Unstable amplificationcurve signal.Insufficient reaction volume. Low reaction volume may cause an unstablesignal. Increase the reaction volume.JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio********************12[ 8 ] Related productsProduct name Package Code No.High efficiency real-time PCR master mix THUNDERBIRD™ SYBR® qPCR Mix 200reactionsQPS-201High efficiency cDNA synthesis kit for real-time PCR ReverTra Ace® qPCR RT Kit 200reactionsFSQ-101One-step Real-time PCR master mix for probe assayRNA-direct™ Realtime PCR Master Mix 0.5 mL x 20.5 mL x 5QRT-101TQRT-101One-step Real-time PCR master mix for SYBR® Green assayRNA-direct™ SYBR® Realtime PCR Master Mix 0.5 mL x 20.5 mL x 5QRT-201TQRT-201JAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/bio********************NOTICE TO PURCHASER: LIMITED LICENSEA license to perform the patented 5’ Nuclease Process for research is obtained by the purchase of (i) both Authorized 5' Nuclease Core Kit and Licensed Probe, (ii) a Licensed 5’ Nuclease Kit, or (iii) license rights from Applied Biosystems.This product is an Authorized 5’ Nuclease Core Kit. Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155, 5,677,152, 5,773,258, 5,407,800, 5,322,770, 5,310,652, 5,210,015, 5,487,972, and claims outside the US corresponding to US Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. Separate purchase of a Licensed Probe would convey rights under the applicable claims of US Patents Nos. 5,538,848, 5,723,591, 5,876,930, 6,030,787, 6,258,569, 5,804,375 (claims 1-12 only), and 6,214,979, and corresponding claims outside the United States. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained from the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.。

manual Quick Taq HS Dye Mix 1306 F1138K Quick Taq HS DyeMixDTM-101 100 reactionsStore at -20°C Contents[1] Introduction[2] Components[3] Primer design[4] Analysis and Cloning of PCR products[5] Protocol1. Standard reaction setup2. Cycling conditions[6] Examples[7] TroubleshootingCAUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnosis or clinical purposes. Please observe general laboratory precaution and utilize safety while using this kit.JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/bio1JAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/bio/********************[ 1 ] Introduction[ 2 ] Components[ 3 ] Primer Design[ 4 ] Analysis andCloning of PCR productsDescriptionQuick Taq HS DyeMix is a Taq-based 2× master mix PCR reagent that contains anelectrophoresis dye (BPB; bromophenol blue) and anti-Taq antibodies for hot start PCR. This reagent contains all components for PCR except primers and template DNA. This reagent shows specific and efficient amplification. The amplified products can be directly loaded in the wells of agarose or acrylamide gels.Features-As this reagent contains bromophenol blue (BPB) as an electrophoresis dye; the PCR products can be analyzed directly with an agarose or acrylamide gel.-This reagent exhibits greater PCR performance than conventional rTaq DNA polymerase.-This reagent contains anti-Taq antibodies for hot start PCR. Hot start technology realizes highly specific and sensitive PCR.-This reagent is stable for at least three months at 4°C. No decrease in reaction efficiency is observed following 30 freeze-thaw cycles.-This reagent is suitable for a colony-direct PCR (see [6], Example 2).This reagent includes the following components for 100 reactions, 50 μl total reaction volume:2× Quick Taq HS DyeMix1.25ml× 2*In the case of the long-term storage (>3 months), this reagent should be stored at-20°C.Primers should be 22–35 bases long, with a melting temperature (Tm) > 60°C.-As this reagent contains bromophenol blue (BPB) as an electrophoresis dye and by adjusting its relative density, the PCR products can be applied directly to an agarose or acrylamide gel.-The PCR products can be cloned using general TA cloning technology.-The PCR products can be used as templates for sequencing after an appropriate treatment.JAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/bio/********************[ 5 ] Protocol[ 6 ] Examples1. Standard reactionBefore preparing the reaction mixture, the master mix solution should be completely thawed. *For the PCR reaction, thin-wall tubes are recommended. A total reaction volume of 50 μl is also recommended.2. PCR cycle conditionsExample 1. Amplification of the human p53 gene (2.9 kb)The human p53 gene (2.9 kb) was amplified using 50 ng of human genomic DNA.Quick Taq HS DyeMix successfully amplified the targets.Reaction volume 50 μl 20 μl Final concentrationAutoclaved, distilled water X μl X μl 2x Quick Taq HS DyeMix 25 μl 10 μl 1 × 10 pmol /μl Primer #1 1.0 μl 0.4 μl 0.2 μM 10 pmol /μl Primer #2 1.0 μl 0.4 μl 0.2 μM Template DNAY μlY μlGenomic DNA -200 ng / 50 μl Plasmid DNA -50 ng / 50 μl E. coli colonyTotal 50 μl 20 μl3-step cyclePredenaturation: 94°C , 2min. Denaturation: 94°C , 30sec. °C , 30sec. Extension: 68°C , 1min. /kb 2-step cyclePredenaturation: 94°C , 2min. Denaturation: 94°C , 30sec. Extension: 68°C , 1min. /kbJAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/bio/********************[ 7 ] Trouble shootingExample 2. Insert amplification by a colony-direct PCRThe inserts were amplified using Quick Taq HS DyeMix with universal primers from E. coli DH5α colonies bearing pTA2 plasmid (insert size: 500 bp). Quick Taq HS DyeMix successfully and efficiently amplified all targets.SymptomCauseSolutionNo PCR product / low yieldCycling conditions are not suitable.Increase the number of cycles by 2-5 cycles. Primer is not good.Check the quality of the primers. Redesign the primers.Template DNA is ofinsufficient quality and/or quantity.Check the quality of the template DNA. Increase the amount of the template DNA Too much sample Excessive amounts of bacterial cells may inhibit amplification. Decrease the sample volume. Smearing / Extra bandCycling conditions are not suitable.Decrease the number of cycles by 2-5 cycles.Primer concentration is not appropriate Optimize the primer concentration to around 0.1-0.2 μM.Annealing temperature is too low. Optimize the annealing temperature to around 55°C -65°C . Primer is not good. Check the quality of the primers. Redesign the primers. Too much template DNA Reduce the amount of template DNA。

Mono,dual and triple moxi¯oxacin-based therapies for Helicobacter pylori eradicationS.DI CARO*,V.OJETTI*,M.A.ZOCCO*,F.CREMONINI*,F.BARTOLOZZI ,M.CANDELLI*,A.LUPASCU*,E.C.NISTA*,G.CAMMAROTA*&A.GASBARRINI**Internal Medicine Department,Gemelli Hospital,Rome,Italy;and Hygiene and Public Health Department,Catholic University,Rome,ItalyAccepted for publication17September2001INTRODUCTIONMany drug schemes have been proposed for Helicobacterpylori eradication.A standardized therapy,involving a combination of a proton pump inhibitor with twoantibiotics(clarithromycin and amoxicillin or nitroim-idazole)given for1week,has been reported to achieve an eradication rate in the range85±90%.1However,in clinical practice,many factors can affect the results of therapy.Among these factors,resistance to antibiotics and patient compliance are the main determinants of treatment failure in H.pylori infection.2As a conse-quence,the use of new drugs and simpler eradication schemes may improve the ef®cacy of therapy.The use of more recent antibiotics,such as rifabutin,nitazoxamide and¯uoroquinolones,has not been con®rmed com-pletely in the eradication of H.pylori.3±5Previous studies have evaluated the ef®cacy and tolerability of levo¯oxacin-based triple therapies,and have suggested that¯uoroquinolones could provide not only a useful alternative to standard therapy,but also a chance to overcome the increasing primary resistance to currently used antibiotics.6Moxi¯oxacin(BAY12-8039),a new commercially available8-methoxy-¯uoroquinolone(AVALOX,laun-ched by Bayer in1999for the treatment of respirat-ory tract infections),is a broad spectrum antibacterial agent with an improved coverage of Gram-positive (in particular Streptococcus pneumoniae)and anaerobic bacteria.7±13The antibacterial effect of moxi¯oxacin,as for other¯uoroquinolones,is based on the inhibition ofSUMMARYBackground:Moxi¯oxacin is a broad spectrum¯uoroqu-inolone with single daily administration,currently used, above all,for respiratory tract infections.Aim:To compare the ef®cacy of different1-week moxi¯oxacin-based Helicobacter pylori eradication regi-mens.Methods:One hundred and twenty H.pylori-positivesubjects were randomized to receive moxi¯oxacin (400mg/day),moxi¯oxacin(400mg/day)and lansop-razole(30mg/day)or moxi¯oxacin(400mg/day),lan-soprazole(30mg/day)and clarithromycin(500mg b.d.).H.pylori status was reassessed6weeks after the end of therapy,and both intention-to-treat and per protocol analyses were performed.Results:One hundred and nineteen of the120patients completed the study.H.pylori eradication was achieved in22.5%of patients treated with moxi¯oxacin,in 33.3%of subjects treated with moxi¯oxacin and lansoprazole and in90%of patients treated with moxi¯oxacin,clarithromycin and lansoprazole. Conclusions:Mono and dual moxi¯oxacin-based ther-apies are not acceptable for H.pylori eradication; conversely,moxi¯oxacin-based triple therapy may be considered as a new,effective,®rst-line therapy option.Correspondence to:Dr A.Gasbarrini,Associate Professor of InternalMedicine,Catholic University,Gemelli Hospital,Largo Gemelli8,00168Rome,Italy.E-mail:angiologia@rm.unicatt.itAliment Pharmacol Ther2002;16:527±532.Ó2002Blackwell Science Ltd527bacterial DNA topoisomerase II(the enzyme responsiblefor DNA superspiralized rolling up and unrolling). However,in contrast with other¯uoroquinolones,moxi¯oxacin is associated with fewer phototoxic and central nervous system excitatory effects.14±17The most common adverse events are gastrointestinal disturbanc-es,such as nausea and diarrhoea,due to the impact on the human intestinal micro¯ora,mainly on the enterobacteria.18,19Theophylline,probenecid,raniti-dine and warfarin do not seem to interact with moxi¯oxacin.20The absolute bioavailability of oral moxi¯oxacin is89%,and the peak plasma concentra-tion reached1.5h after oral drug administration is2.5mg/L.Total and renal clearances are14.9and3.03L/h,respectively,and the mean elimination half-life is9±16h.Dosage adjustment is not required in patients of advanced age or those with renal or hepatic failure.Co-administration with antacids or iron prepa-rations reduces drug bioavailability.21±23Clinical studies are necessary to de®ne the ef®cacy and the correct use of moxi¯oxacin in H.pylori infection.In this study,we evaluated the ef®cacy of moxi¯oxacin-based mono,dual and triple schemes given for1week.PATIENTS AND METHODSFrom November2000to April2001,120patients(50 males,70females),aged18±65years,attending the Gastroenterology Out-patient Clinic of Gemelli Hospital in Rome(Catholic University Teaching Hospital),who were H.pylori-positive,were enrolled in this prospective, open-label study.All patients underwent upper gastro-intestinal endoscopy,and H.pylori status was deter-mined through histological assessment of gastric body and antral biopsies(two biopsies from each site)and13C urea breath test.The characteristics of the patients,with comparable demographic features in each group, are summarized in Table1.Dyspeptic patients with nopathological®ndings at endoscopy who were enrolled in this study made their own decision to be treated for the infection.Exclusion criteria included a history of hypersensitivity to the study medications,concomitant serious illness, known pregnancy status,recent(past2months)use of antimicrobial agents,bismuth compounds,proton pump inhibitors or H2receptor antagonists,and previous H.pylori eradication therapy.Written informed consent was obtained for each patient.Patients were randomized into three groups to receive moxi¯oxacin in three different1-week schemes:40patients(25females and15males)received moxi¯oxacin,400mg/day;40 patients(25females and15males)received moxi¯ox-acin,400mg/day,and lansoprazole,30mg/day;and 40patients(20females and20males)received moxi¯oxacin,400mg/day,lansoprazole,30mg/day, and clarithromycin,500mg twice daily.24±27Six weeks after the end of the moxi¯oxacin-based regimens,H.pylori status was re-checked by13C urea breath test and the subjects were asked to report all therapy-related side-effects by®lling in a validated questionnaire.28±30In particular,each subject received three copies of a slightly modi®ed deBoer questionnaire, on which,after verbal and written complete explana-tions,they reported the presence of symptoms(taste disturbances,diarrhoea,nausea,bloating,loss of appet-ite,vomiting,stomach pain,constipation,headache and skin rash)at the end of treatment and the second and third week thereafter.Moreover,subjects were asked to judge the severity of each symptom on a four-point scale from mild to pliance was also evaluated byMono therapy Dual therapy Triple therapy No.of patients404040Male/female ratio15/2515/2520/20 Active duodenal ulcer000Gastric ulcer000Erosive duodenitis121Erosive gastritis214Erosive gastroduodenitis102 Previous duodenal ulcer332 Previous gastric ulcer213 Duodenal hyperaemia336Gastric hyperaemia564 Oesophagitis000Table1.Characteristics of randomized groupsÓ2002Blackwell Science Ltd,Aliment Pharmacol Ther16,527±532 528S.DI CARO et al.counting the number of empty drug boxes returned by each patient at 1-week follow-up interviews.Eradication rates were calculated using both per proto-col and intention-to-treat analyses.The chi-squared test was performed to evaluate homogeneity and to compare data differences among the three groups.A P value of less than 0.05was considered to be statistically signi®cant.A con®dence interval (CI)of 95%was given.Statistical analysis was performed using STATA 6.0äsoftware.RESULTSNine of the 40patients treated with moxi¯oxacin were negative to the control 13C urea breath test (per proto-col,22.5%;intention-to-treat,22.5%;CI,10.9±38.4%).Dual therapy was successful in 13of 40subjects (per protocol,33.3%;intention-to-treat,32.5%;CI,18.6±49.1%).Interestingly,triple therapy reached an eradication rate of 90%(per protocol intention-to-treat;CI,76.3±97.2%)(36of 40patients),which was signi®cantly higher than the success rate of the mono and dual regimens (P <0.001)(Table 2).The power of the study is 90%.Patient compliance was almost absolute and all subjects completed therapy,except for one patient in the dual therapy group.In this case,drop out was due to palpitations that persisted for less than 1h.Slight or mild side-effects,such as nausea,diarrhoea and taste disturbances,occurred in all groups.Three patients treated with mono therapy (7.5%),®ve subjects treated with the dual scheme (12.5%)and ®ve patients in the triple therapy group (12.5%)experienced mild to moderate side-effects,including nausea and diarrhoea.Unexpectedly,palpitations occurred in six patients (6/120;5%)(Table 3).These subjects were subse-quently evaluated by a specialist cardiologist and were screened with 24-h electrocardiogram monitoring,but no evidence of arrhythmic events was found.Table 2.Results with different anti-Helicobacter pylori schemesPer protocol analysis Intention-to-treat analysis Regimen Duration (days)No.patients eradicated(eradication rate and 95%CI)No.patients eradicated(eradication rate and 95%CI)Moxi¯oxacin 79/40(22.5%,10.9±38.4%)9/40(22.5%,10.9±38.4%)Moxi¯oxacin 713/40(33.3%,18.6±49.1%)13/39(32.5%,19±50.2%)Lansoprazole Moxi¯oxacin Clarithromycin Lansoprazole736/40(90%,76.3±97.2%)36/40(90%,76.3±97.2%)CI,con®dence interval.Table 3.Side-effects Regimen No.patients Side-effects Grade (duration)Statistical analysis Moxi¯oxacin1Nausea Slight (3±5days)7.5%(3/40patients)2DiarrhoeaModerate (5±6days)CI,1.5±20.3%Taste disturbance Palpitations (<1h)Moxi¯oxacin 1Nausea Slight (3±5days)12.5%(5/40patients)Lansoprazole 2DiarrhoeaModerate (5±6days)CI,4.2±26.8%Taste disturbance 2Palpitations (<1h)Moxi¯oxacin Nausea 12.5%(5/40patients)Clarithromycin 1DiarrhoeaModerate (5±6days)CI,4.2±26.8%Lansoprazole1Taste disturbance Slight (8±10days)3Palpitations(<1h)CI,con®dence interval.Ó2002Blackwell Science Ltd,Aliment Pharmacol Ther 16,527±532MOXIFLOXACIN-BASED THERAPIES FOR H.PYLORI ERADICATION529DISCUSSIONAccording to Maastricht2001Consensus Conference guidelines,H.pylori eradication is strongly recommen-ded in peptic ulcer,mucosa-associated lymphoid tissue lymphoma,atrophic gastritis,after gastric cancer resection,in patients who have a family history of gastric cancer and in patients who wish to be treated. Recommendation for H.pylori eradication is also advisable in functional dyspepsia,gastro-oesophageal re¯ux disease and in subjects using non-steroidal anti-in¯ammatory drugs.31±35Treatment success is related to different factors:patient compliance to the treatment,bacterial resistance to antibiotic agents,treatment duration,scheme of regi-men and antibiotic-related adverse events.The ideal treatment for patients who have failed®rst-line triple therapy is still under debate.The need for and type of`rescue'therapy are matters of discussion among experts,and different approaches have been studied in relation to the geographical prevalence of antibiotic resistance.31,36Current regimens involve a7-day triple therapy based on the use of a proton pump inhibitor and two different antibiotics:clarithromycin and amoxicillin or a nitro-imidazole.37The primary resistance to macrolides in Europe is in the range3±12%,and is2±10%in the USA.In developing countries,most H.pylori strains are resistant to nitro-imidazoles,and in Western countries primary resistance varies from2%to50%.In particular,the prevalence of nitroimidazole resistance in Italy is2±30%.In several European and Asian countries,in which the resistance to antibiotics is currently being monitored,it has been demonstrated that H.pylori resistance to metronidazole and clarithromycin increased throughout the1990s.38 Moreover,repeated eradication attempts may lead to the development of acquired resistance.Increased costs and patient discomfort are also key factors.39,40 Several different strategies can be considered in the case of®rst-line treatment failure:(i)repeat the same regimen with a higher dosage of medications and longer treatment duration;(ii)use different regimens with different antibiotics;(iii)administer a quadruple ther-apy;(iv)employ ranitidine bismuth citrate in triple therapies;(v)individualize the approach after an antibiogram.The eradication success(usually70%) and patient compliance of second-line therapies are lower than those of®rst-line regimens.41In an attempt to solve the problem of a`rescue'scheme,several studies have evaluated new drugs,such as rifabutin,nitazoxamide and¯uoroquinolones,in theeradication of H.pylori.3±5Some approaches to this issue have explored the utility of¯uoroquinolones. Levo¯oxacin,a laevorotatory isomer of o¯oxacin,is a second generation¯uoroquinolone.The main advant-age of second generation relative to®rst generation ¯uoroquinolones,such as cipro¯oxacin,is the broader spectrum of activity and improved coverage against Gram-positive cocci and atypical pathogens,while retaining good activity against Gram-negative bacteria. Recent data have demonstrated that the minimum inhibitory concentration against H.pylori of new generation¯uoroquinolones is less than0.05l g/mL, similar to that of amoxicillin and clarithromycin.42,43 In particular,Cammarota et al.investigated the ef®c-acy of two different7-day triple regimens using levo¯oxacin with a proton pump inhibitor and amox-icillin or tinidazole.6The data indicated an eradication rate of over90%and the occurrence of side-effects in 10%of patients.However,antibiotic susceptibility was not tested before treatment,and the authors suggested that the high eradication rate achieved could be related to the low primary resistance to levo¯oxacin in the study geographical area.6Moxi¯oxacin is another second generation¯uoroqui-nolone,whose antibacterial spectrum covers all major upper and lower respiratory tract pathogens,including penicillin-and macrolide-resistant pneumococci.In in vitro studies,the emergence of bacterial resistance is less common with moxi¯oxacin,but this result requires con®rmation by other research groups.These preliminary observations indicate that moxi-¯oxacin may be a promising alternative in anti-H.pylori schemes.44±46Moxi¯oxacin-based schemes may allow improved patient compliance with a lower occurrence of side-effects,and the administration of only one pill daily in mono therapy and two or four pills daily in dual or triple regimens,respectively.The most common side-effects observed are diarrhoea and nausea.Diarrhoea is probably due to the antibiotic effect on the normal intestinal micro¯ora.Indeed, moxi¯oxacin causes a signi®cant decrease in entero-cocci,enterobacteria,bi®dobacteria and clostridia, while staphylococci,streptococci,bacillus,Candida spp.,peptostreptococci,lactobacilli,Veillonella,bactero-ides and fusobacteria are not affected.In contrast,clar-ithromycin causes a signi®cant reduction in EscherichiaÓ2002Blackwell Science Ltd,Aliment Pharmacol Ther16,527±532530S.DI CARO et al.coli,anaerobic micro¯ora bi®dobacteria,lactobacilli and clostridia.18Only one of the dual therapy patients dropped out with palpitations.The patient had no underlying cardiovas-cular disease and reported herself to be particularly anxious during therapy.In the literature,tachycardia has been indicated as an uncommon side-effect of moxi¯oxacin treatment.In the present study,palpita-tions persisted for less than1h and occurred in six patients(5%).The only possible relative contraindica-tion to the use of moxi¯oxacin is in patients who are being treated at the same time with drugs reducing potassium and magnesium.Moxi¯oxacin,like other ¯uoroquinolones and macrolides,may prolong the QT interval(4ms,1.2%respect to the base value).Mild or slight side-effects,such as nausea,diarrhoea and taste disturbance,occurred in13of the120subjects treated, but none led to treatment discontinuation.Interest-ingly,in the literature,it has been reported that clarithromycin is also characterized by uncommon cardiovascular adverse events,such as a prolonged QT interval,tachycardia and torsade de pointes,as for other macrolides;however,in our study,palpitations also occurred with dual therapy.Patients who experienced palpitations underwent a Holter electrocardiogram and arrhythmias were excluded.According to the ef®cacy and safety pro®les of moxi¯oxacin-based schemes in H.pylori eradication, mono and dual moxi¯oxacin-based regimens should not be recommended for the eradication of H.pylori infection.The eradication rates were22.5%and 33.3%in mono and dual schemes,respectively.These eradication rates are signi®cantly lower than the success rate of moxi¯oxacin-based triple therapy (>85%).Instead,it is very interesting to underline the features of moxi¯oxacin-based triple therapy:a high cure rate of H.pylori infection and good treatment tolerability. Indeed,the triple therapy regimen,including moxi¯ox-acin,clarithromycin and lansoprazole,seems to be as effective as the standard triple schemes currently in use, but involves the administration of a lower number of pills daily.In this study,moxi¯oxacin was used in combination with clarithromycin and a half-dose administration of lansoprazole(30mg/day).Future approaches should test its ef®cacy in combination with amoxicillin,which has a lower primary resistance than clarithromycin in the general population,and with current standard twice daily administration of a proton pump inhibitor for H.pylori eradication.In conclusion,mono and dual moxi¯oxacin-based therapies cannot be recommended for H.pylori eradi-cation.In contrast,the triple therapy used,including moxi¯oxacin,clarithromycin and lansoprazole,seems to be more promising and could even be considered as a ®rst-line therapy option.ACKNOWLEDGEMENTFinancial support for this study was provided by Associazione Ricerca in 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pcr技术工作流程PCR (Polymerase Chain Reaction) is a widely used molecular biology technique that allows for the amplification of a specific DNA sequence. It is a versatile tool with applications in various fields such as medical diagnostics, genetic research, and forensic analysis. The workflow of PCR involves several key steps, including sample preparation, thermal cycling, and analysis of the amplified products.英文回答：The first step in the PCR workflow is sample preparation. This involves obtaining the DNA template that contains the target sequence to be amplified. The DNA can be extracted from various sources, such as blood, tissue, or cells, using different methods like phenol-chloroform extraction or commercial DNA extraction kits. The quality and purity of the DNA template are crucial for the success of PCR.Next, the DNA template is mixed with a pair of primers, which are short DNA sequences that flank the target region. These primers are designed to be complementary to the specific DNA sequence of interest. The primers serve as starting points for DNA synthesis during PCR.The PCR reaction mixture also contains nucleotides (dNTPs), which are the building blocks of DNA, and a heat-stable DNA polymerase enzyme. The DNA polymerase is usually derived from thermophilic bacteria, such as Taq polymerase, that can withstand the high temperatures used in PCR.The thermal cycling step is the core of the PCR process. It involves a series of repeated temperature changes that enable the amplification of the target DNA sequence. The thermal cycling typically consists of three main steps: denaturation, annealing, and extension.During denaturation, the reaction mixture is heated toa high temperature (around 95°C), which causes the double-stranded DNA to separate into two single strands. This stepensures that the DNA template is available for the subsequent steps.The next step is annealing, where the reaction mixtureis cooled to a lower temperature (around 50-65°C). At this temperature, the primers bind to their complementary sequences on the single-stranded DNA template.Finally, during the extension step, the temperature is raised to the optimal range for the DNA polymerase (usually around 72°C). The DNA polymerase synthesizes new DNA strands using the primers as a starting point. This process is repeated for multiple cycles, typically 25-35 cycles, to amplify the target DNA sequence exponentially.After the thermal cycling, the PCR products areanalyzed to determine the success of the amplification. Various methods can be used for this purpose, such as agarose gel electrophoresis, which separates the PCR products based on their size, or real-time PCR, whichallows for quantification of the amplified DNA in real-time.PCR has revolutionized molecular biology and has become an indispensable tool in many areas of research and diagnostics. Its ability to amplify specific DNA sequences quickly and efficiently has opened up new avenues for understanding genetic diseases, identifying pathogens, and studying gene expression.中文回答：PCR（聚合酶链反应）是一种广泛应用于分子生物学的技术，可以扩增特定的DNA序列。

金灵泰，张铭，方双琪，等. 基于斑马鱼模型探究药食同源复方降尿酸作用及成分分析[J]. 食品工业科技，2023，44（19）：410−416.doi: 10.13386/j.issn1002-0306.2022110251JIN Lingtai, ZHANG Ming, FANG Shuangqi, et al. Study on Lowering Uric Acid Effect and Component Analysis of Drug Food Homologous Compound Based on Zebrafish Model[J]. Science and Technology of Food Industry, 2023, 44(19): 410−416. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022110251· 营养与保健 ·基于斑马鱼模型探究药食同源复方降尿酸作用及成分分析金灵泰1，张　铭2, +, *，方双琪2，徐　蔷2（1.中国科学技术大学附属第一医院离子医学中心（合肥离子医学中心），安徽合肥 230088；2.安徽中森生物技术有限公司，安徽桐城 231400）摘　要：目的：探究芹菜籽、蒲公英、菊苣和玉米须四种药食同源中药联用下的降尿酸活性和作用机制，分析这四种中药复合提取物中具备降尿酸作用的主要效应成分。

方法：随机选取5 dpf 野生型AB 品系斑马鱼，通过250 μmol/L 氧嗪酸钾和10 μmol/L 黄嘌呤钠盐诱导建立斑马鱼高尿酸模型，将已建立的斑马鱼模型随机分为对照组、模型组、别嘌醇组（136 μg/mL ）和复方提取物低、中、高（250、500及1000 μg/mL ）剂量组，每组30尾，连续干预24 h 后，测定斑马鱼体内的尿酸荧光值和次黄嘌呤磷酸核糖转移酶1（HPRT1）、葡萄糖转运蛋白9（GLUT9）、有机阴离子转运体（OAT1）基因表达水平；采用超高液相色谱-串联高分辨质谱仪为主要手段，以乙腈-甲酸水为流动相，梯度洗脱，ESI 离子源正、负离子扫描模式，结合软件数据库搜索及相关文献进行成分鉴定。

·综述·《中国产前诊断杂志（电子版）》　２０２１年第１３卷第４期无创单基因病的研究进展胡听听　综述王继成 校审（广州医科大学附属广东省妇儿医院，广东广州　５１００１０）【摘要】　预防出生缺陷一直是困扰临床的一大难题，但胎儿游离ＤＮＡ（ｃｅｌｌ ｆｒｅｅｆｅｔａｌＤＮＡ，ｃｆｆ ＤＮＡ）的发现，给临床检测提供了一条新的途径。

传统的出生缺陷检测一般是侵入性产前诊断，如穿刺抽取羊水、脐血或绒毛。

然而，侵入性产前诊断可能导致１％的流产或胎儿感染。

随着高通量测序技术和方法的进步，可以精准获取孕妇外周血中胎儿游离ＤＮＡ。

现在，对１３、１８、２１号染色体三体综合征的无创产前筛查（ｎｏｎ ｉｎｖａｓｉｖｅｐｒｅｎａｔａｌｓｃａｎｎｉｎｇ，ＮＩＰＳ）已经大规模临床应用，但是对于胎儿单基因病的无创检测依然处于研究阶段，本文综述了近几年无创单基因病检测研究的新进展，探讨临床应用的发展趋势。

【关键词】　无创产前检测；胎儿游离ＤＮＡ；单基因病【中图分类号】　Ｒ７１４．５５ 【文献标识码】　Ａ犇犗犐：１０．１３４７０／ｊ．ｃｎｋｉ．ｃｊｐｄ．２０２１．０４．０１５基金项目：广东省医学科学技术研究基金项目（Ｂ２０１９１５０） 通信作者：王继成，Ｅ ｍａｉｌ：ｊｉｃｈｅｎｇ０９２７＠１２６．ｃｏｍ １９９７年，Ｌｏ等［１］证实了母体外周血胎儿游离ＤＮＡ（ｃｅｌｌ ｆｒｅｅｆｅｔａｌＤＮＡ，ｃｆｆ ＤＮＡ）可以诊断胎儿遗传性疾病。

最近的临床研究也证实，在单胎妊娠中使用无创产前检测（ｎｏｎ ｉｎｖａｓｉｖｅｐｒｅｎａｔａｌｔｅｓｔｉｎｇ，ＮＩＰＴ）检测２１、１８、１３三体比传统的筛查方案具有更低的假阳性和更高的阳性预测值［２］。

随后，各种方法如雨后春笋一样不断出现。

如今，可以在母体外周血中发现全部的胎儿基因组［３，４］，这也是各种胎儿遗传性疾病可以通过母体外周血检测的基础。

产前诊断是通过各种方法检测和监测胎儿生长发育状况，检测方法分为有创和无创。

tpo40三篇托福阅读TOEFL原文译文题目答案译文背景知识阅读-1 (2)原文 (2)译文 (5)题目 (8)答案 (17)背景知识 (17)阅读-2 (20)原文 (20)译文 (23)题目 (25)答案 (35)背景知识 (35)阅读-3 (38)原文 (38)译文 (41)题目 (44)答案 (53)背景知识 (54)阅读-1原文Ancient Athens①One of the most important changes in Greece during the period from 800 B.C. to 500 B.C. was the rise of the polis, or city-state, and each polis developed a system of government that was appropriate to its circumstances. The problems that were faced and solved in Athens were the sharing of political power between the established aristocracy and the emerging other classes, and the adjustment of aristocratic ways of life to the ways of life of the new polis. It was the harmonious blending of all of these elements that was to produce the classical culture of Athens.②Entering the polis age, Athens had the traditional institutions of other Greek protodemocratic states: an assembly of adult males, an aristocratic council, and annually elected officials. Within this traditional framework the Athenians, between 600 B.C. and 450 B.C., evolved what Greeks regarded as a fully fledged democratic constitution, though the right to vote was given to fewer groups of people than is seen in modern times.③The first steps toward change were taken by Solon in 594 B.C., when he broke the aristocracy's stranglehold on elected offices by establishing wealth rather than birth as the basis of office holding, abolishing the economic obligations of ordinary Athenians to the aristocracy, and allowing the assembly (of which all citizens were equal members) to overrule the decisions of local courts in certain cases. The strength of the Athenian aristocracy was further weakened during the rest of the century by the rise of a type of government known as a tyranny, which is a form of interim rule by a popular strongman (not rule by a ruthless dictator as the modern use of the term suggests to us). The Peisistratids, as the succession of tyrants were called (after the founder of the dynasty, Peisistratos), strengthened Athenian central administration at the expense of the aristocracy by appointing judges throughout the region, producing Athens’ first national coinage, and adding and embellishing festivals that tended to focus attention on Athens rather than on local villages of the surrounding region. By the end of the century, the time was ripe for more change: the tyrants were driven out, and in 508 B.C. a new reformer, Cleisthenes, gave final form to the developments reducing aristocratic control already under way.④Cleisthenes' principal contribution to the creation of democracy at Athens was to complete the long process of weakening family and clanstructures, especially among the aristocrats, and to set in their place locality-based corporations called demes, which became the point of entry for all civic and most religious life in Athens. Out of the demes were created 10 artificial tribes of roughly equal population. From the demes, by either election or selection, came 500 members of a new council, 6,000 jurors for the courts, 10 generals, and hundreds of commissioners. The assembly was sovereign in all matters but in practice delegated its power to subordinate bodies such as the council, which prepared the agenda for the meetings of the assembly, and courts, which took care of most judicial matters. Various committees acted as an executive branch, implementing policies of the assembly and supervising, for instance, the food and water supplies and public buildings. This wide-scale participation by the citizenry in the government distinguished the democratic form of the Athenian polis from other less liberal forms.⑤The effect of Cleisthenes’ reforms was to establish the superiority of the Athenian community as a whole over local institutions without destroying them. National politics rather than local or deme politics became the focal point. At the same time, entry into national politics began at the deme level and gave local loyalty a new focus: Athens itself. Over the next two centuries the implications of Cleisthenes’ reforms were fully exploited.⑥During the fifth century B.C. the council of 500 was extremely influential in shaping policy. In the next century, however, it was the mature assembly that took on decision-making responsibility. By any measure other than that of the aristocrats, who had been upstaged by the supposedly inferior "people", the Athenian democracy was a stunning success. Never before, or since, have so many people been involved in the serious business of self-governance. It was precisely this opportunity to participate in public life that provided a stimulus for the brilliant unfolding of classical Greek culture.译文古雅典①在公元前800年到公元前500年期间，希腊最重要的变化之一是城邦的崛起，并且每个城邦都发展了适合其情况的政府体系。

Advanced Topics in STR DNA AnalysisAAFS 2006 Workshop #6Seattle, WAFebruary 20, 2006Dr. John M. ButlerDr. Bruce R. McCord CETroubleshootingmccordb@john.butler@CE TroubleshootingOutline for This Section1. Chemistry/molecular biology problems –stutter, -A,degradation, inhibition, low copy #2.Sample and buffer problems –formamide, urea, water,salt concentration, free dye (“dye blobs”)3.External factors –power supply, room temperature,cleanliness, voltage leaks4.Instrument problems –optical system, capillaryclogging, air bubbles, syringe leaks5.Troubleshooting benchmarks/QC monitoring1. Chemistry/Molecular Biology Problems•PCR amplification issues•Adenylation•Stutter•Non Specific Amplification•Primer dimers•Pipetting small amounts•Degradation/Inhibition•Allele dropout•Over amplification•Ski slope effect•Mitigation Steps for inhibitionOff-ladder allelesFour types 12341. Spike2.OL Allele3. Free Dye4. NoiseExtract of Pistol Grip and Trigger Relatively low amount of amplified DNA? BlueGreenYellow Enhanced stutter or mixture?Truncated peaks give wrong ratios for peak stutter Why else is overloading bad?1. raised baseline2. non specific amplification3. peak height ratios4. -AOverloaded peaks will also show relatively high stutterNon specificamplification-A StutterDegradation and PCR Inhibition •Degradation affects larger alleles more, however there is no published study on the “threshold at which degradation is apparent”-The amplification efficiency of each set of alleles varies independently and differential amplification across loci can occur –Moretti, JFS 2001-Low quality formamide can mimic the degradation effect-Inhibition generally affects certain loci more than others and may or may not produce a slope effect-McCord, unpublished -There are several likely mechanisms for inhibition including DNA aggregation, Protein-DNA binding, chelation of Mg, interferencewith primer binding, etc.DNA DegradationNon-degraded Positive Control20 pg/µL (0.250 ng/12.5 µL)Degraded Bone Sample20 pg/µL(0.250 ng/ 12.5 µL)Degraded DNA and Amplification Allele drop-outAllele drop-out Oversaturation “Pullup”-Apeak Enhanced stutterWith degraded DNA two injections may be necessary to keep data on-scaleNon-DNA Contamination/Inhibition •Anything that is water soluble may co-extract with DNA unless a capture technique is used.•For capture techniques anything with a similar chemical property to DNA may co-extract•Detergents, metal ions, humic substances are all potent contaminant/inhibitors•Can cause all sorts of strange effects including –Spikes, dye blobs, elevated baselines, loss of signal, odd current effectsDegraded and inhibited bone sample Loss of larger alleles due to degradation differential amplification at certain loci (inhibition?)2. Sample Issues •Formamide Conductivity•Excessive salt in sample due to evaporation•Metal ion contamination•Sensitivity issues with Microcon cleanup (salt removal)•Dye “blobs” –artifacts from primer synthesisGolden Gate Effect Attributed to poor formamideSample Problem?.Check ROX, looks OK320 V/cm 47 cm uncoated capillaryPOP4 PolymerAnswer: Incomplete denaturation of standard due to poor quality formamide320 V/cm 47 cm uncoated capillaryPOP4 PolymerPost PCR manipulation •Reprocessing post PCR to concentrate samples can improve signal but be careful–PCR sample is concentrated but:•Spin filtration may result in removal of background salts,•This can greatly enhance sensitivity due to the stackingprocess•Best idea-remake sample up in buffer, not water to avoidreading stochastic effects.Example of an Interpretational Guideline6FAM(blue)VIC (green)NED (Yellow)PET (Red)LIZ (Orange)439389II 438437391389I 426390385 a/b 393392H446019388448447100 bp 139200250*300150160340350YCAII a/b Residual dye artifactsButler, J.M. (2005) Constructing STR multiplex assays. Methods in Molecular Biology: Forensic DNA Typing Protocols (Carracedo, A., ed.), Humana Press: Totowa, New Jersey, in press .NIST Y-STR 20plex assay439389II 438437391389I 426YCAII a/b 390385 a/b393392H446019388448447100 bp 139200250*300150160340350Dye blob removal with Edge columns Dye Blobs and their Removal3. External Factors•Room temperature–Variations in room temperature can cause mobility shifts with band shifts and loss of calibration–Temperature is also important due to effects of high humidity on electrical conductance•Cleanliness–Urea left in sample block can crystallize and catalyze further crystal formation causing spikes, clogs and other problems.–Best bet is to keep polymer in system and not remove or change block until polymer is used up.Temperature effects •Viscosity –mobility shift–μep= q/6πηr•Diffusion –band broadening–DNA•Conformation –DNA size based sievingvs μep= q/6πηr•Current –Power–P= VI = I2R–Increased current →internal temperature rise→diffusion →band broadeningBand shift in the FGA locusLikely the result of temperature or viscosity induced mobility changeFGA Allele 302532562592622652684045505560657075TemperatureS i z e Effect of Temperature on allele sizeSlope is 0.14 bases/degree centigradeTherefore a small change in temperature has a big effect(A 1-2 degree shift in temperature of the heat plate can produce an OL allele)Due to its structure and its non-calibration, the “250”peak can be used to indicate stabilityInitial RunStability losses toTemperatureElectroosmosisSyringe leaksAdsorptionExcess currentBlockagesTemperature Effects: “OL” Alleles“OL Alleles”“OL alleles ” -look at the 250 peak“OL allele re-injected”And the 250 peak...True off-ladder allelesTrue off-ladder allele: the 250 peakMonitoring Room Temperature Over Time ±10 o C spread(over many weeks)Use of Second Allelic Ladder to Monitor Potential MatchCriteria Problems1st Injection (standard for typing)15th Injection (treated as a sample)-0.75 bp-0.54 bpThese alleles have drifted outside of theirgenotyping bins due to temperature shiftingover the course of the sample batchWhat to do if calibration is lost?The 310 only calibrates to the first run ladder this ladder sample may have been run at a different temperature!•If protocol permits–Go to the next ladder–Rerun sample–Check current–Check allelic ladder•Always check the ROX size standard–Look for extra bands–Check peak height–Check parameters and alignmentCleanliness•Urea sublimates and breaks down to ionic components -these find a path to ground•Similarly wet buffer under a vial creates paths to ground •Capillary windows must be clear or matrix effects will occur•Laser will often assist in this process•Vial caps will transfer low levels of DNA to capillaryCarbon TrailsHigh Humidityor wet buffer vialscan create otherpaths to ground Keep Your System Clean!4. Instrumental Factors•Optical System–Sensitivity changes with age, capillary diameter, capillary cleanliness, instrument calibration•Fluidic System–Effects of bubbles, dust, urea crystals, leaks in syringe and capillary ferrule•Matrix Calculations–Changes in buffer, optics, sample dye can alter the software calibrations•Capillary Problems–Chemisorbed materials on capillary surface can produce osmotic flow, DNA bandbroadening and inconsistant resolution(meltdowns)Consider the optical systemLaser (488nm)Charged coupled deviceGratingLaser Capillary CCD OpticsIssues with the Optical System•Pay attention to signal to noise, not absolute peak intensity •Argon Ion lasers outgas and eventually loose intensity; take note of laser current and monitor it over time•Fluorescence expression:I f = IkεbCφ-changes in input intensity, I-changes in capillary diameter, b-cleanliness of capillary, k–All these things directly affect peak RFUs, however, baseline noise is more affected by detector.•Thus by monitoring signal to noise, you can get a better picture of your optical system.The Detection WindowMake sure that the capillary window is lined up (if it is not, then no peaks will be seen)Window may need to be cleaned with ethanol or methanolCapillaryDetection WindowReview Start of Raw Data CollectionLittle spikes indicate need to change buffer… check currentRaised baseline due to dirty windowFluidic Problems•Syringe leaks–At the barrel–At the capillary nut–At the capillary window•Viscosity changes–Water in the block–Bubbles–Temperature•Capillary conditioning–Preelectrophoresis–cloggingBuffer Issues•The buffer and polymer affect the background fluorescence-affecting the matrix•Urea crystals and dust may produce spikes•High salt concentrations may produce reannealing of DNA•High salt concentrations affect current•Low polymer concentrations affect peak resolutionCurrent SpikesGenerally appear in all lanes and are sharper than regular peaksThese are a natural consequence of the application of high voltage in CERemove all bubbles from the channelsBubbles in the channels can prevent flow ofions and are usually exhibited by zero currentwhen the voltage is appliedSeparation problems, bubbles in capillaryThese spikes resulted from buffer dilution with poor water. The problem disappeared when the HPLC grade water was purchased to dilute buffer and samplesBeware of Urea CrystalsUrea crystals haveformed due to a smallleak where the capillarycomes into the pumpblockUrea sublimates and canevaporate to appearelsewhereUse a small balloon tobetter grip the ferrule andkeep it tightPump block should be well cleaned to avoidproblems with urea crystal formationStorage when ABI 310 is not in use•Keep inlet of capillary in water…if it dries out then urea crystals from thepolymer will clog the opening •The waste vial (normally in position 3) can be moved into position • A special device can bepurchased from Suppelco to rinse the capillary off-line •Store in distilled water•Note that the laser is on when the instrument is onRemember that the water in the open tube will evaporate over time…Matrix Problems• A poor matrix can lead to raised baseline and therefore calling of too many peaks•Larger sized alleles will not be identified as peaks because the GeneScan table for a particular dye color has filled upEffect of bad MatrixNote negative peaks and funny downward spiking baseline。

用cdna为模板扩pcr流程英文回答：The process of using cDNA as a template for PCR amplification involves several steps. First, the cDNA is synthesized from RNA using reverse transcription. This is done by adding a reverse transcriptase enzyme and a primer to the RNA sample, which allows the enzyme to synthesize a complementary DNA strand. The resulting cDNA can then be used as a template for PCR amplification.Next, a PCR reaction mixture is prepared. This usually includes the cDNA template, primers specific to the target gene, nucleotides, and a DNA polymerase enzyme. The primers are designed to bind to specific regions of the cDNA template, flanking the target gene sequence. The nucleotides are the building blocks for DNA synthesis, and the DNA polymerase enzyme is responsible for synthesizing new DNA strands.The PCR reaction is then carried out in a thermal cycler. The reaction mixture is subjected to a series of temperature cycles, which allow for DNA denaturation,primer annealing, and DNA synthesis. Typically, the PCR reaction starts with an initial denaturation step at a high temperature to separate the DNA strands. This is followedby a series of cycles consisting of denaturation at a high temperature, primer annealing at a lower temperature, and DNA synthesis at an intermediate temperature. These cycles are repeated multiple times to amplify the target gene.After the PCR amplification, the products are analyzed. This can be done by running the PCR products on an agarose gel and visualizing the DNA bands under UV light. The sizeof the amplified DNA fragments can be determined by comparing them to a DNA ladder of known sizes.Alternatively, the PCR products can be sequenced to confirm the presence of the target gene and to analyze its sequence.中文回答：使用cDNA作为PCR扩增的模板涉及几个步骤。