A STUDY OF THE ORIGIN OF SHADOW BANDS SEEN WHEN TYPING DINUCLEOTIDE REPEAT POLYMORPHISMS BY THE PCR
A STUDY OF THE ORIGIN OF SHADOW BANDS SEEN WHEN TYPING DINUCLEOTIDE REPEAT POLYMORPHISMS BY THE PCR
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DOI：
10.1093/hmg/2.4.411
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发表时间：
1993-04-01
影响因子：
3.5
通讯作者：
LITT, M
中科院分区：
文献类型：
--
作者：
HAUGE, XY;LITT, M
二核苷酸重复多态性（“微卫星”）通常通过在变性丙烯酰胺凝胶上解析PCR扩增产物来分型。使用这种方法，等位基因不是由单个片段组成，而是由片段的梯状结构组成，通常由2nt的间隔分开。已被援引来解释这些“阴影带”的产生的机制包括在PCR期间发生的滑动链错配和由截短的PCR产物的失准退火引起的假象“重组”。D11S527位点含有微卫星序列（GT）n（CTGT）m。通过对D11S527纯合个体的PCR产物进行直接测序，我们发现这些产物的长度变化完全是由于二核苷酸重复序列长度的变化。这些结果排除了PCR重组，并支持滑动链错配作为产生阴影条带的主要机制。
Dinucleotide repeat polymorphisms ('microsatellites') are usually typed by resolving the products of PCR amplification on denaturing acrylamide gels. With this methodology, an allele consists not of a single fragment, but rather of a ladder of fragments, typically separated by intervals of 2nt. Mechanisms that have been invoked to explain the generation of these 'shadow bands' include slipped strand mispairing occurring during the PCR and artefactual 'recombination' caused by out-of-register annealing of truncated PCR products. The D11S527 locus contains the microsatellite sequence (GT)n(CTGT)m. By performing direct sequencing of PCR products derived from individuals homozygous at D11S527, we show that these products vary in length due solely to variations in the length of the dinucleotide repeat tract. These results rule out PCR recombination and support slipped strand mispairing as the major mechanism for generation of shadow bands.