EnzymoPure™ High-Fidelity DNA Polymerase

EnzymoPure™ 高保真 DNA 聚合酶

EnzymoPure™ High-Fidelity DNA Polymerase

H745689-2000U

2000U

EnzymoPure™

不含DNA内切酶和磷酸酯酶，不含RNA酶。

-20°C储存

超低温冰袋运输

-20℃保存，至少2年有效。

阿拉丁生产的EnzymoPure™ High-Fidelity DNA Polymerase，即EnzymoPure™高保真DNA聚合酶，是阿拉丁自主研发的PerfectProtein™技术平台表达、纯化获得的重组酶。它是一种以嗜热古细菌DNA聚合酶为基础通过突变等改造其“尿嘧啶结合口袋”而获得的能识别和扩增含有尿嘧啶(uracil)和次黄嘌呤(hypoxanthine)的模板DNA的超高性能DNA聚合酶，因此它除了具有扩增速度快、保真度极高、扩增片段可以轻松达到12kb等优点之外，还可以利用dUTP (2'-deoxy-uridine 5'-triphosphate)和dITP (2'-deoxy-inosine-5'-triphosphate)进行基因扩增，其扩增效果请参考图1和图2。阿拉丁的EnzymoPure™ High-Fidelity DNA Polymerase和NEB公司的Q5U™ HotStart High-Fidelity DNA Polymerase、Thermo公司的Phusion U Hot Start DNA Polymerase、Roche公司的KAPA HiFi HotStart Uracil+ Ready Mix (2X)有非常类似的扩增效果和用途。EnzymoPure™ High-Fidelity DNA Polymerase用于二代测序建库相关扩增时，具有扩增效率极高，低GC偏好性(low GC bias)等优良特性，特别适合用于亚硫酸处理的(bisulfite-converted)、脱氨基的(deaminated)或甲醛固定石蜡包埋(formalin fixation and paraffin embedding, FFPE)等导致的损伤的(damaged) DNA样品的扩增，以及PCR扩增时可以掺入dUTP并和尿嘧啶-N-糖基化酶(uracil-N-glycosylase, UNG)处理相结合可以有效避免PCR扩增产物污染导致的假阳性。DNA长期保存会出现随机的Cytocine脱氨基现象，导致普通的DNA聚合酶无法扩增，而本产品可以兼容并扩增脱氨基的DNA1-3]。EnzymoPure™ High-Fidelity DNA Polymerase是一种高保真DNA聚合酶，它不仅可以非常高效地催化5'至3'方向的依赖于DNA模板的聚合酶活性，同时还具有3'至5'的外切酶活性(proofreading activity)，它的错误发生概率比Taq酶低40-50倍，其扩增产物为平端(blunt end)，因此不能直接进行T载体克隆连接。如需用于T载体克隆，需在其扩增结束后加入常规量的普通Taq酶在72℃反应5-10分钟，使每条链的3'端加上一个A以形成粘端。图1. 阿拉丁生产的EnzymoPure™ High-Fidelity DNA Polymerase (简称EnzymoPure™)和EnzymoPure™ DNA Polymerase 使用dUTP或dITP为底物扩增约1.2kbDNA片段的效果对比图。dUTP组的反应体系为50μl：5μl 10X EnzymoPure™ Buffer或10X EnzymoPure™ Buffer，2μl Primers (10μM each)，5μl DdNTP/dUTP Mixture (2.5mM each/5mM) ，1μl模板质粒(20ng/μl)，36μl Nuclease-free Water，冰上混合后加入1μl EnzymoPure™或EnzymoPure™。dITP组的反应体系为50μl：5μl 10X EnzymoPure™ Buffer或10X EnzymoPure™ Buffer，2μl Primers (10μM each)，5μl dNTP/dITP Mixture (2.5mM each + 0.25mM dITP)，1μl模板质粒(20ng/μl)，36μl Nuclease-free Water，冰上混合后加入1μl EnzymoPure™或EnzymoPure™。按照说明书推荐的PCR程序反应完毕后，加入10μl DNA上样缓冲液(6X) ，取5μl使用1%琼脂糖凝胶电泳检测其扩增效果。参考上图，EnzymoPure™ DNA Polymerase可以高效利用dUTP和dITP为底物扩增目的片段，而EnzymoPure™ DNA Polymerase则完全不能。实际结果会因实验条件、检测仪器等的不同而存在差异，图中数据仅供参考。图2. 阿拉丁EnzymoPure™ High-Fidelity DNA Polymerase (简称Q6U)与N公司生产的同类产品(Competitor)利用dUTP和dITP扩增目的片段(约1.2kb)的效果对比图。Q6U使用图1中的反应体系，N公司产品使用其产品说明书推荐的反应体系，酶的用量为图中所标示的用量，U为活力单位。参考上图，阿拉丁EnzymoPure™ High-Fidelity DNA Polymerase能很好地使用dUTP和dITP进行扩增，并且其扩增效果略优于国外N公司的同类产品。实际结果会因实验条件、检测仪器等的不同而存在差异，图中数据仅供参考。 用途 ： 常规的DNA高效高保真扩增，特别适用于扩增亚硫酸处理的(bisulfite-converted)DNA样品用于DNA甲基化测序，特别适用于扩增脱氨基的(deaminated)或甲醛固定石蜡包埋(formalin fixation and paraffin embedding, FFPE)等导致的损伤的(damaged) DNA样品的扩增，以及PCR扩增时可以掺入dUTP并和尿嘧啶-N-糖基化酶(uracil-N-glycosylase, UNG)处理相结合可以有效避免PCR扩增产物污染导致的假阳性。 来源 ： Q6U™ High-Fidelity DNA Polymerase通过大肠杆菌重组、表达、纯化而获得。 酶储存溶液 ： 40mM Tris-HCl (pH7.5), 1mM DTT, 0.1mM EDTA, 100mM KCl, 300µg/ml BSA and 50% (v/v) glycerol。 失活或抑制 ： 酚氯仿抽提可以使Q6U™ High-Fidelity DNA Polymerase失活。 注意事项 ： PCR反应非常灵敏，在进行扩增反应时请注意避免微量待扩增DNA的污染，并尽量考虑设置不加模板的空白对照。本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。为了您的安全和健康，请穿实验服并戴一次性手套操作。 使用说明 ： 1.PCR反应体系的设置：a.溶解并混匀PCR反应所需的各种溶液。将Q6U™ High-Fidelity DNA Polymerase置于冰浴上或冰盒内。b.参考下表在冰浴上设置PCR反应(如果有多个类似的PCR反应，可以根据反应数量先配制底物和引物之外的混合物，然后分装到各PCR反应管内。根据情况，有时混合物中可以包括底物或引物)： To amplify dsDNA ＜ 6kbTo amplify dsDNA ＞ 6kb ReagentFinal concentrationVolumeFinal concentrationVolume Nuclease-free water - (38.5-x)μl - (32.5-y)μl 10X Q6U™ Buffer 1X 5μl 1X 5μl dNTP (2.5mM each) 0.25mM each 5μl 0.5mM each 10μl Template DNA 10pg-1μg* xμl 10pg-1μg* yμl Primer mix (10μM each) 0.2μM each 1μl 0.4μM each 2μl Q6U™ High-Fidelity DNA 1U/50μl 0.5μl 1U/50μl 0.5μl Total volume - 50μl - 50μl *不同类型的DNA模板的推荐用量有所不同。哺乳动物基因组DNA：100ng；大肠杆菌基因组DNA：100ng；质粒DNA：5-30ng。扩增大于6kb的片段时，模板量宜适当加大，但过多的模板DNA也容易导致非特异性的PCR扩增产物。在推荐用量时相对会非常容易扩增出目的片段，实际应用时如果模板DNA的量比较少，完全可以大大少于推荐的用量的。注：参考上表，在扩增大于6kb的片段时dNTP和引物的用量和扩增小于6kb片段时相比需要加倍。c.用移液器轻轻吹打混匀或轻微Vortex混匀，室温离心数秒，使液体积聚集于管底。d.如果所使用的PCR仪有热盖则省略本步骤。如果PCR仪没有热盖，则在管内滴入一滴矿物油。e.设置好的PCR反应管置于PCR仪上，开始PCR反应。2.PCR反应参数的设置可以参考如下表格： Step To amplify dsDNA To amplify dsDNA Description STEP1 94℃ 3min 94℃ 3min Initial denaturation STEP2 94℃ 30sec 94℃ 30sec Denaturation STEP3 55℃ 30sec 55℃ 30sec Annealing STEP4 68℃ 15s/kb 68℃ 1min/kb Extension STEP5 Go To STEP2 for 30 Go To STEP2 for 30 PCR cycles STEP6 68℃ 10min 68℃15min Final extension STEP7 4℃ forever 4℃ forever Hold a.起始变性和变性的温度设置为94℃，延伸温度设置为72℃，其余条件不变时，都可以进行正常的PCR扩增，但扩增效果会稍有下降。对于较难扩增的序列，推荐使用92℃变性和72℃延长。b.PCR反应的设置需根据模板、引物、PCR产物的长度和GC含量等条件的不同设定不同的温度、时间和循环数等。c.STEP4 (Extension)的时间设置需根据PCR产物的长度进行设置，对于本产品扩增小于6kb的DNA片段时，推荐每kb产物的延伸时间为15秒。例如PCR产物的长度为1kb，则延伸时间可以设置为15秒，PCR产物的长度为2kb，则延伸时间可以设置为30秒，以此类推。扩增大于6kb的DNA片段时，推荐每kb产物的延伸时间为1分钟。例如PCR产物的长度为10kb，则延伸时间可以设置为10分钟，PCR产物的长度为12kb，则延伸时间可以设置为12分钟，以此类推。d.对于初次进行的PCR，为尽量确保可以扩增出预期的PCR产物，可以把循环数设置为35。对于需进行半定量或定量的PCR反应，循环数一定要进行适当优化，使PCR反应没有达到平台期。常见问题：1.PCR产物非常少或没有特异性条带。a.引物设计不佳是PCR过程中最常见的问题。请选择适当的引物设计软件进行引物设计，注意引物的GC含量、二级结构、二聚体、退火温度、长度、特异性等方面的问题。在加入酶切位点等的引物中，一定要注意加入酶切位点等后整条引物的GC含量、二级结构、二聚体、退火温度、长度、特异性等方面的问题。在原有引物效果不佳的情况并且阳性对照引物可以正常工作的情况下，可以考虑更换引物。b.待扩增片段GC含量偏高。GC含量较高的情况下PCR会变得相对比较困难，此时可以使用适合扩增高GC含量DNA片段的GC-rich buffer，并相应地根据GC-rich buffer的要求或说明调整PCR反应参数的设置。直接添加1-10% DMSO或5-20%甘油对于扩增高GC含量的片段也有帮助。c.PCR反应设置时在室温进行容易导致非特异性条件。推荐在冰浴上设置PCR反应。d.由于引物存在一定的二级结构或存在一定的引物二聚体，或引物偏短，导致退火效果不佳。此时可以采用Touch down等方法进行退火，通常采用从65℃逐步缓慢降温到55℃或50℃的方法，使退火更加充分。e.退火温度不佳，需要优化。如果有温度梯度PCR仪，则可以设置退火的温度梯度，摸索退火的最佳温度。如果没有温度梯度PCR仪，则可以通过多次PCR反应摸索最佳的退火温度。f.延伸时间不足。可以在推荐的延伸时间基础上把延伸时间延长2-5倍，对于较难扩增的片段可以设置为每1kb延伸5分钟。g.待扩增片段GC含量较高或长度较长，变性不够充分。可以调节起始变性条件至95℃ 1min甚至95℃ 2-4min。h.在不同PCR仪上进行PCR反应，避免有时PCR仪出现问题。i.循环数不足，适当延长PCR的循环数。通常循环数最高不必超过40，常用的循环数范围为25-35。j.模板含量太低，适当加大模板量，或采用巢式PCR (nested PCR)或二次PCR。巢式PCR即为在原先设计的PCR引物内侧再设计一对PCR引物，然后对第一次PCR产物进行稀释后再进行一次PCR扩增，这样一方面可以起到扩增作用，同时也可以从第一次PCR产物中扩增出特异性条带。二次PCR则为比较简单地用原有引物对第一次PCR产物进行稀释后再进行一次PCR扩增，可以起到扩增作用，但不能去除非特异性条带。k.模板中含有抑制PCR反应的物质，可以用适当的DNA纯化方法例如柱纯化等纯化模板DNA。l.对PCR引物进行脱盐纯化。m.使用高质量的dNTP混合物。n.适当增加DNA polymerase的用量。o.当产生较多非特异性条带时，可以适当提高退火温度。p.注意设置适当的阳性对照和阴性对照通常会有很大帮助。2.杂带较多或条带弥散。a.退火温度提高2-5℃。b.减少DNA模板的用量。c.PCR反应设置时在室温进行容易产生非特异性条带。推荐在冰浴上设置PCR反应。d.适当减少Q6U™ High-Fidelity DNA Polymerase用量。e.适当缩短延伸时间。 Aladdin's EnzymoPure™ High-Fidelity DNA Polymerase is a recombinant enzyme expressed in E. coli and purified using Aladdin's PerfectProtein™ technology platform. It is a modified thermostable DNA polymerase with a mutation in the uracil-binding pocket that enables the ability to read and amplify DNA templates containing uracil and hypoxanthine bases. Its strong 3´→5´ exonuclease (proofreading) activity results in superior accuracy during DNA amplification.The EnzymoPure™ High-Fidelity DNA Polymerase generates PCR products with blunt ends due to its 3´→5´ exonuclease activity. For direct cloning of the PCR products, the blunt-end cloning method should be used. When T vector cloning is required, the PCR products should be dA tailed by Taq DNA polymerase for 5-10 minutes at 72℃.n addition to fast amplification speed, high efficiency, high fidelity, and easy amplification of fragments up to 12 kb, EnzymoPure™ High-Fidelity DNA Polymerase can incorporate dUTP (2' -deoxy-uridine 5'-triphosphate) and dITP (2' -deoxy-inosine-5 '-triphosphate) bases into newly synthesized DNA. Please see Figure 1 and Figure 2 for its amplification effects.Figure 1. Amplification of target fragment by Aladdin's EnzymoPure™ High-Fidelity DNA Polymerase (EnzymoPure™) and EnzymoPure™ DNA Polymerase (EnzymoPure™) in PCR reactions containing dUTP or dITP. Reaction system of dUTP group (50μl) Application ： Regular gene amplification with high fidelity; high fidelity NGS library preparation with high yield and low GC bias; amplification of bisulfite-converted DNA for bisulfite sequencing, deaminated DNA, or damaged DNA by formalin fixation and paraffin embedding for instance; preventing carryover contamination in PCR when combined with dUTP and uracil-N-Glycosylase (UNG) treatment. DNA stored for a long time will have random cytocine deamination, resulting in the inability of ordinary DNA polymerase to amplify, while this product can be compatible with and amplify the deaminated DNA[1-3]. Source ： Recombinant protein expressed in E. coli. Inactivation or inhibition ： This enzyme can be removed and inactivated by phenol-chloroform extraction. Precautions ： Because PCR reactions are extremely sensitive, contamination must be avoided during the preparation of PCR reactions. Negative control without templates is recommened for all PCR assays.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation. Instructions for Use ： 1. Prepare PCR reactions: a. Thaw PCR components at room temperature and mix well prior to use. Keep Q6U™ High-Fidelity DNA Polymerase on ice.b. Set up the following reaction on ice. Mix well by gentle vortex or pipetting. Centrifuge briefly to collect liquid at the bottom of microcentrifuge tube.To amplify dsDNA ＜ 6kbTo amplify dsDNA ＞ 6kbReagentFinal concentrationVolumeFinal concentrationVolumeNuclease-free water-(38.5-x)μl-(32.5-y)μl10X Q6U™ Buffer1X5μl1X5μl dNTP (2.5mM each)0.25mM each5μl0.5mM each10μlTemplate DNA10pg-1μg*xμl10pg-1μg*yμlPrimer mix (10μM each)0.2μM each1μl0.4μM each2μlQ6U™ High-Fidelity DNA 1U/50μl0.5μl1U/50μl0.5μlTotal volume-50μl-50μlNote 1: When multiple reactions are required, prepare a master mix including all reagents except template and primer, and then dispense to different nuclease-free PCR tubes. Sometimes, the master mix can also include template and primer.Note 2: The recommended amount of template varies for different types of DNA. In a reaction volume of 50μl, use 100 ng of Mammalian genomic DNA, 100 ng of E. coli genomic DNA, or 5-30 ng of Plasmid DNA. When amplifying fragments larger than 6kb, the amount of template should be increased appropriately, but too much template DNA can also easily lead to non-specific PCR amplification products. c. (Optional) When using a thermal cycler without a heated lid, place a drop of mineral oil onto the top of PCR reaction mixture.2. Transfer PCR reactions to a thermal cycler and run thermocycling conditions as follows:StepTo amplify dsDNA ＜6kbTo amplify dsDNA ＞ 6kbCyclesInitial denaturation94℃3min94℃3min1Denaturation94℃30sec94℃30sec30Annealing55℃30sec55℃30secExtension68℃15s/kb68℃1min/kbFinal extension68℃10min68℃15min1Hold4℃forever4℃forever-Note 1: The extension temperature can also be set to 72ºC, but the amplification will be slightly reduced. For sequences difficult to amplify, denaturation at 92ºC and extension at 72ºC are recommended. Note 2: PCR running conditions should be adjusted based on the template, primer sequence, the length of PCR product or GC content, etc.Note 3: The extension time should be set according to the length of amplicons. For amplification of DNA fragments shorter than 6kb, the recommended extension time is 15 seconds per kb. For example, set 15 seconds to amplify amplicons of 1kb, 30 seconds for amplicons of 2kb, and so on. When amplifying DNA fragments longer than 6kb, the recommended extension time is 1 minute per kb (e.g. use 10 minutes to amplify amplicons of 10kb). Note 4: For initial PCR, the number of cycles can be set to 35 to ensure that the expected PCR product can be amplified. The number of cycles for semi-quantitative or quantitative PCR analysis must be optimized appropriately so that the PCR reaction does not reach a plateau.3. Analyze PCR products by agarose gel electrophoresisFAQ:1. Few PCR products or no specific bands.a. It could be due to poor design of primers. Use primer design tools for primer design to avoid inappropriate GC content, secondary structure, dimer, annealing temperature, length, specificity and other possible problems. When adding restriction enzyme cutting sites in the primer sequence, the same problems need to be considered. In the case that positive control primers work normally but not your primers, redesign primers.b. DNA to be amplified may have a high GC content. High GC genes are relatively difficult to be amplified. In such a case, GC-rich buffer suitable for amplifying DNA with high GC content can be used, and PCR reaction parameters should be adjusted accordingly. Direct addition of 1-10% DMSO or 5-20% glycerol is also helpful for amplifying fragments with high GC content.c. PCR reactions set up at room temperature tend to produce non-specific bands. It is recommended to set up PCR reactions on ice.d. The presence of secondary structure in primers, primer dimers or short primers, may result in poor annealing of primers to the target sequence. In this case, methods such as touch down can be used to anneal, usually by gradually and slowly lowering the temperature from 65ºC to 55ºC or 50ºC to make the annealing more sufficient. e. The annealing temperature needs to be optimized. If necessary, use a temperature gradient to determine the optimal annealing temperature for each template-primer pair combination. f. Insufficient extension time. The extension time can be extended 2-5 times from the recommended extension time, and can be set to 5 minutes per 1kb for fragments difficult to amplify. g. Insufficient denaturation. To amplify long DNA or high GC DNA fragment, the initial denaturation temperature can be adjusted to 95ºC for 1 min or even 95℃ for 2-4 min.h. Perform PCR reactions on a different thermal cycler to avoid possible problems with the instrument.i. Insufficient number of PCR cycles. Try more PCR cycles, but do not exceed 40 cycles. j. Insufficient amount of template. Add more DNA templates or try nested PCR or secondary PCR. Nested PCR is to design another pair of PCR primers inside the original PCR primers, and then conduct PCR amplification again with the diluted first PCR product as template. Instead, secondary PCR uses the same primers for second PCR amplification with the diluted PCR product as template. Nested PCR usually can remove the non-specific DNA amplification, but secondary PCR can not.k. DNA sample contains substances that inhibit the PCR reactions. In such a case, template DNA can be purified using appropriate DNA purification methods such as column purification.l. Use high-purity primers.m. Use high-quality dNTP mixture.n. Increase the amount of DNA polymerase appropriatelyo. When non-specific fragments are produced, the annealing temperature can be increased appropriately.p. Positive and negative controls are always recommended when optimizing PCR reactions.2. Occurence of non-specific bands or DNA smear when examined by agarose gel electrophoresis.Increase the annealing temperature by 2-5℃.Reduce the amount of DNA template.PCR reactions set up at room temperature tend to produce non-specific bands. It is recommended to set up PCR reactions on ice.Reduce the amount of Q6U™ High-Fidelity DNA Polymerase appropriately.Shorten the extension time appropriately.