Shumaker J M, Metspalu A, Caskey C T
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.
Hum Mutat. 1996;7(4):346-54. doi: 10.1002/(SICI)1098-1004(1996)7:4<346::AID-HUMU9>3.0.CO;2-6.
A mutation analysis method based upon a wild-type DNA sequence is presented. Oligonucleotides were utilized for primer extension by T7 DNA polymerase to discriminate between wild-type and mutant sequences in two solid phase approaches. 1. Oligonucleotides were annealed to an immobilized template, extended with fluorescent dideoxynucleotides (ddNTPs), and analyzed on an automated fluorescent DNA sequencer. The oligonucleotide length identified the known mutation site, and the fluorescence emission of the ddNTP identified the mutation. 2. Template DNA was annealed to an oligonucleotide array, extended with alpha-32P dNTPs, and analyzed with a Phosphor Imager. The grid position of the oligonucleotide identified the mutation site and the extended base identified the mutation.
本文介绍了一种基于野生型DNA序列的突变分析方法。采用寡核苷酸通过T7 DNA聚合酶进行引物延伸，以两种固相方法区分野生型和突变序列。1. 将寡核苷酸与固定化模板退火，用荧光双脱氧核苷酸（ddNTP）进行延伸，并在自动荧光DNA测序仪上进行分析。寡核苷酸的长度确定已知的突变位点，ddNTP的荧光发射确定突变。2. 将模板DNA与寡核苷酸阵列退火，用α-32P dNTP进行延伸，并用磷成像仪进行分析。寡核苷酸的网格位置确定突变位点，延伸的碱基确定突变。
