Bevan I S, Rapley R, Walker M R
University Department of Clinical Biochemistry, Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Edgbaston, Birmingham, UK.
PCR Methods Appl. 1992 May;1(4):222-8. doi: 10.1101/gr.1.4.222.
Alternatives for sequencing of PCR products essentially fall into one of two categories; generation of single-stranded DNA for sequencing or the direct sequencing of double-stranded product. Of the two alternatives, sequencing of double-stranded PCR products is likely to be of greatest immediate significance in terms of general applicability and rapidity. Double-stranded sequencing allows the use of the PCR product for other purposes either prior to or subsequent to generation of sequence data. The single-stranded sequencing methods generally require some prior decision regarding sequencing of the product. Assisted by automated workstation development, sequencing of single-stranded DNA PCR products generated either during thermal cycling or following affinity-capture strand separation may have significant future utility, particularly in genome mapping and routine clinical diagnosis. Despite template type and protocol differences, in all situations the purity and concentration of PCR-amplified DNA template used remains the most critical factor determining the efficiency and reliability of nucleotide sequencing methods.
PCR产物测序的方法基本上可分为两类：生成用于测序的单链DNA或对双链产物进行直接测序。在这两种方法中，就普遍适用性和快速性而言，双链PCR产物测序可能具有最直接的重要意义。双链测序允许在生成序列数据之前或之后将PCR产物用于其他目的。单链测序方法通常需要对产物的测序做出一些预先决定。在自动化工作站发展的辅助下，热循环过程中或亲和捕获链分离后生成的单链DNA PCR产物的测序可能在未来具有重要用途，特别是在基因组作图和常规临床诊断中。尽管模板类型和方案存在差异，但在所有情况下，所使用的PCR扩增DNA模板的纯度和浓度仍然是决定核苷酸测序方法效率和可靠性的最关键因素。
