Cause and solutions to the polymerase chain reaction smear problem in genotyping.
Cause and solutions to the polymerase chain reaction smear problem in genotyping.
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基因分型中聚合酶链反应涂片问题的原因及解决方法
DOI：
10.1016/j.ab.2006.03.041
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Gu,HowardH
中科院分区：
文献类型：
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作者：
Han,DawnD;Chen,Rong;Hill,ErikR;Tilley,MichaelR;Gu,HowardH
转基因动物和植物模型广泛应用于研究和药物开发。在许多实验室中，测定动物和植物个体的基因型是一项必要的常规程序。基因分型最常用的方法是聚合酶链反应（PCR）。因此，PCR过程经常在实验室中使用同一对引物长时间重复。PCR基因分型的常见现象是琼脂糖凝胶上逐渐出现条纹或涂片，特定条带减弱和消失。这有时被称为“基因分型崩溃”，这是一个经常困扰许多实验室的问题，特别是那些空间有限和/或有多人执行基因分型任务的实验室。我们发现，基因分型中的PCR涂片问题是由于所使用的PCR引物特异性的“可扩增DNA污染物”的逐渐积累。因此，一旦涂片开始出现，那些之前被证明工作良好的引物就不再产生可靠的扩增。预防性措施，如分离pcr前和pcr后的实验室空间、试剂和设备，将减缓污染的积累。最快捷和有效的解决方案是更换一对不同序列的新引物，使累积的污染物无害，从而彻底解决涂抹问题。我们的实验室已经培育出了一种敲入小鼠系，它带有一种改良的多巴胺转运体，这种转运体有功能，但对可卡因不敏感。杂合子小鼠的杂交产生纯合子和杂合子突变体小鼠及其野生型仔鼠。大量小鼠的基因型需要在常规基础上确定。在该小鼠系中，突变等位基因在DAT基因的第三个内含子中插入了一个103碱基对（bp）的额外DNA序列。用一对引物（DATMf1， CATTGG， GGTCCACATACAAATG；和DATMr1， AGACACG， TGGCAGATTCATAGG）对插入位点两侧的序列进行退火，利用pcr确定这些小鼠的基因型。pcr扩增的突变等位基因DNA片段比从野生型等位基因扩增的DNA片段大103bp（见图1A和B）。从小鼠尾尖制备基因组DNA的方法有两种，一种是传统的“盐析”方法[1]，另一种是Truett等人描述的Hot Shot方法[2]。两种方法都很有效，但Hot Shot方法更简单、更可靠，是我们首选的方法。简单地说，将约2mm的小鼠尾部尖端浸入75l碱性裂解缓冲液（25mM NaOH， 0.2 mM EDTA二钠，pH 12）中，在95℃下孵育30 min。样品在4℃冰箱中冷却10min或更长时间，并加入75l中和缓冲液（40mM Tris-HCl, pH 5.0）。通过倒置试管将内容物混合均匀，然后准备基因组DNA样本进行PCR分析。配制主PCR混合物，最终浓度为0.2 mM dNTP， 2 mM MgCl2或MgSO4, 1英镑PCR缓冲液（来自商业聚合酶制造商的10英镑缓冲液），每种引物各0.4 M。在96孔PCR板的每孔中加入20微升的1英镑PCR混合物，然后加入2 - 51 l的DNA样本。然后将PCR板置于热循环器（Hybaid TouchDown）中。当PCR块温度达到80℃时，每孔加入10 l含有1单位Taq DNA聚合酶的1英镑PCR混合物。然后将板材密封，循环程序开始，在94℃下30 s变性，30 s退火，72℃下30 s延伸…
Genetically modified animal and plant models are widely used in research and drug development. The determination of genotypes for individual animals and plants is a necessary routine procedure in many laboratories. Genotyping is most frequently accomplished by using the polymerase chain reaction (PCR). 1 Therefore, the PCR procedures are often repeated in a lab for an extended period of time using the same pair of primers. A common phenomenon in PCR genotyping is the gradual appearance of streaks or smears on agarose gels and the weakening and disappearance of specific bands. This is sometimes called “genotyping crash,” a problem frequently troubling many laboratories, especially those labs that have limited spaces, and/or have multiple people performing the genotyping tasks. We found that the PCR smear problem in genotyping was due to the gradual built-up of “amplifiable DNA contaminants” specific to the PCR primers used. Thus, those primers that had been proven to work well before no longer produce reliable amplifications once the smears started to appear. Preventive practices, such as separating lab spaces, reagents, and equipment for pre-PCR from those for post-PCR, would slow down the contamination buildup. The most eYcient and effective solution is to switch to a new pair of primers with different sequences that render the built-up contaminants harmless and therefore completely solve the smear problem. Our lab has generated a knock-in mouse line with a modified dopamine transporter that is functional but insensitive to cocaine. The heterozygous mice were bred to produce the homozygous and heterozygous mutant mice and their wild-type littermates. The genotypes of a large number of mice need to be determined on a routine basis. For this mouse line, the mutant allele had a 103 base pair (bp) additional DNA sequence inserted in the third intron of the DAT gene. The genotypes of these mice were determined using PCRs with a pair of primers (DATMf1, CATTGG GGTCCACATACAAATG; and DATMr1, AGACACG TGGCAGATTCATAGG) that annealed to the sequences flanking the insertion site. The PCR-amplified DNA fragment from the mutant allele was 103bp larger than that amplified from the wild-type allele (see Figs. 1A and B). Two methods were used to prepare genomic DNA from mouse tail tips, a traditional “salting out” procedure [1] and the Hot Shot method as described by Truett et al.[2]. Both methods worked well, but the Hot Shot method was much simpler and more reliable, and was our preferred method. Briefly, about 2mm tips of mouse tails were submerged in 75l alkaline lysis buffer (25mM NaOH, 0.2 mM disodium EDTA, pH 12) and incubated for 30 min at 95 C. The samples were cooled in 4 C refrigerator for 10min or longer and 75l neutralization buffer (40mM Tris–HCl, pH 5.0) was added. The contents were mixed well by inverting the tubes and the genomic DNA samples were then ready for PCR analysis. A master 1£ PCR mixture was prepared with final concentrations of 0.2 mM dNTP, 2 mM MgCl2, or MgSO4, 1£ PCR buffer (from 10£ buffer of commercial polymerase manufacturers), and 0.4 M of each of the primers. Twentymicroliter aliquots of 1£ PCR mixture were added to each well of a 96-well PCR plate followed by 2–5l DNA samples. The PCR plate was then placed in a thermal cycler (Hybaid TouchDown). When the temperature of the PCR block reached 80 C, 10 l of 1£ PCR mixture containing one unit of Taq DNA polymerase was added to each well. The plates were then sealed and the cycling protocol started with 30 s denaturing at 94 C, 30 s annealing, and 30 s extension at 72 C …