Ruano G, Kidd K K
Department of Human Genetics, Yale University School of Medicine, New Haven, CT 06510.
Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2815-9. doi: 10.1073/pnas.88.7.2815.
Addition of dideoxyribonucleotides during the exponential phase of the PCR should result in the synthesis of two complementary sequence ladders. We have explored this hypothesis to develop coupled amplification and sequencing of genomic DNA. Coupled amplification and sequencing is a biphasic method for sequencing both strands of template as they are amplified. Stage I selects and amplifies a single target from the genomic DNA sample. Stage II accomplishes the sequencing as well as additional amplification of the target using aliquots from the stage I reaction mixed with end-labeled primer and dideoxynucleotides. We have successfully applied coupled amplification and sequencing to a 300-base-pair fragment 4 kilobases upstream from HOX2B directly from human whole genomic DNA.
在PCR指数增长期加入双脱氧核糖核苷酸应能导致合成两个互补序列梯。我们探究了这一假说以开发基因组DNA的耦合扩增与测序技术。耦合扩增与测序是一种在扩增模板两条链的同时对其进行测序的双相方法。第一阶段从基因组DNA样本中选择并扩增单个靶标。第二阶段使用来自第一阶段反应的等分试样与末端标记引物和双脱氧核苷酸混合，完成靶标的测序以及额外扩增。我们已成功地将耦合扩增与测序技术直接应用于来自人类全基因组DNA、位于HOX2B上游4千碱基处的一个300碱基对片段。
