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三核苷酸重复序列PCR扩增中影子带的产生及其消除的方法 |
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武辉 张思仲 肖翠英
【摘要】 目的 为了消除PCR扩增三核苷酸重复序列时产生的影子带。方法 以人
类强直性肌营养不良致病基因-肌张力蛋白激酶基因(myotonin protein kinase gene,
MT-PK)3′端非翻译区中的CTG重复序列为例，主要比较了PCR反应中单独使用Taq DNA聚
合酶和使用Taq＋Pwo混合的DNA聚合酶对影子带产生的影响。结果 实验表明PCR反应中
单独使用Taq DNA聚合酶时，其PCR产物经PAGE电泳，银染显色后常有影子带形成，而向
Taq DNA聚合酶中加入具有3′-5′外切酶活性的Pwo DNA聚合酶则可完全消除影子带。
结论 本实验结果表明影子带产生于PCR过程本身，而比Taq DNA聚合酶具有更高加工性
的组合酶则有可能消除或减弱影子带。
【关键词】 聚合酶链反应 影子带 三核苷酸重复序列
SHADOW BANDS IN PCR AMPLIFICATION OF TRINUCLEOTIDE REPEATS AND
THEIR ELIMINATION Wu Hui, Zhang Sizhong*, Xiao Cuiying.*Department of Medical
Genetics,West China University of Medical Sciences, Chengdu, 610041
【Abstract】 Objective To explore a method for eliminating the shadow bands in PCR amplification
of trinucleotide repeats.Methods CTG trinucleotide repeat sequence in the 3′-untranslated region
of the human myotonin protein kinase gene was used as templates DNA in PCR amplification, and
the effectsof Taq DNA polymerase alone and its mixture with Pwo DNA polymerase on occurrence
of theshadow bands were compared. The PCR products containing (CTG)5, both from Taq DNA
polymerase and from Taq＋Pwo polymerase amplification, were cloned into pBluecript KS and
sequenced on ALFexpressTM DNA sequencer respectively. Results The PCR of DNA sequence
containing CTG repeatsfrequently produced a main band (usually darker) and a shadow band (lighter)
that differed from the main band by 3 base pairs when Taq DNA polymerase was used alone. However,
when the mixture of TaqDNA polymerase and Pwo DNA polymerase was used, the shadow bands
usually disappeared. In the 4 clones containing PCR products amplified by Taq DNA polymerase,
one clone contained only 4 CTGrepeats. However, this kind of decrease of repeat copy was not
observed in the 5 clones containing PCRproducts amplified by using mixture of[1] [2] [3] 下一页
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