Detailed investigation of factors influencing amplification efficiency and allele drop-out in single cell PCR:: implications for preimplantation genetic diagnosis
Detailed investigation of factors influencing amplification efficiency and allele drop-out in single cell PCR:: implications for preimplantation genetic diagnosis
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DOI：
10.1093/molehr/gag051
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发表时间：
2003-07-01
影响因子：
4
通讯作者：
Wells, D
中科院分区：
文献类型：
--
作者：
Piyamongkol, W;Bermúdez, MG;Wells, D
单基因疾病的着床前遗传学诊断（PGD）依赖于对单细胞（极体或卵裂球）进行的基于pcr的检测。尽管使用了越来越强大的方案，等位基因脱落（ADO；在杂合细胞中无法扩增两个等位基因中的一个）仍然是单细胞PCR诊断的一个重要问题。在极端情况下，ADO可以影响40%的扩增，并且已经导致了一些PGD误诊。我们建议，对ADO起源的更好理解将允许开发更可靠的PCR分析。在这项研究中，我们仔细地改变了bbbb3000单细胞扩增的反应条件，从而确定了影响ADO率的因素。发现ADO受扩增子大小、DNA降解量、冷冻和解冻、PCR程序和同时扩增的细胞数量的影响。局部DNA序列、变性温度（94℃或96℃）和细胞类型对ADO影响不大或没有影响。考虑到本研究中确定的因果因素，应该允许设计较少ADO的PGD方案，从而提高单基因疾病PGD的准确性。
Preimplantation genetic diagnosis (PGD) of single gene disorders relies on PCR-based tests performed on single cells ( polar bodies or blastomeres). Despite the use of increasingly robust protocols, allele drop-out ( ADO; the failure to amplify one of the two alleles in a heterozygous cell) remains a significant problem for diagnosis using single cell PCR. In extreme cases ADO can affect > 40% of amplifications and has already caused several PGD misdiagnoses. We suggest that an improved understanding of the origins of ADO will allow development of more reliable PCR assays. In this study we carefully varied reaction conditions in > 3000 single cell amplifications, allowing factors influencing ADO rates to be identified. ADO was found to be affected by amplicon size, amount of DNA degradation, freezing and thawing, the PCR programme, and the number of cells simultaneously amplified. Factors found to have little or no affect on ADO were local DNA sequence, denaturing temperature ( 94 or 96 degreesC) and cell type. Consideration of the causal factors identified during this study should permit the design of PGD protocols that experience little ADO, thus improving the accuracy of PGD for single gene disorders.