Arbitrarily primed PCR fingerprints resolved on SSCP gels.
Arbitrarily primed PCR fingerprints resolved on SSCP gels.
复制标题
任意引发的 PCR 指纹在 SSCP 凝胶上解析。
DOI：
10.1093/nar/22.9.1770
复制
发表时间：
1994
影响因子：
14.9
通讯作者：
Welsh,J
中科院分区：
文献类型：
--
作者：
McClelland,M;Arensdorf,H;Cheng,R;Welsh,J
Arbitrarily primed PCR methods (1), including RAPD (2), generate a reproducible fingerprint of DNA products from complex nucleic acids. Differences detected between fingerprints derived from different genomes have been used extensively as polymorphisms in genetic mapping (eg, 3). Various strategies can be used to maximize the number of polymorphisms that are scored in each fingerprinting experiment. First, parents can be selected for the mapping population that have highly divergent genomes. Second, a large, but manageable, number of fragments in each fingerprinting experiment can be generated by using Stoffel fragment rather than Taq polymerase holoenzyme (4). Third, primers can be directed against sequences such as purine-pyrimidine microsatellite repeats that are intrinsically more polymorphic (5). Fourth, gel systems that detect more polymorphisms can be used, eg, denaturing gradient gel electrophoresis (6).Another extremely simple method to increase the number of scorable polymorphisms would be to include single stranded conformation polymorphisms (SSCP)(7). This method could increase the number of scorable polymorphisms for two reasons. First, the two DNA strands from the same PCR product often run in different places on SSCP gels. This gives two opportunities to score a polymorphism whereas other gel systems afford only one suchpossibility for each polymorphism. Second, some PCR products from identical places in the two parental genomes may have internal sequence polymorphisms that will resolve as mobility differences on an SSCP gel. To test theutility of this approach we mapped polymorphisms in the mouse genome using the C57BL/6Jx DBA and the A/J xC57BL/6J recombinant inbred mapping populations. Figure 1 shows a fingerprint for two parental strains separated on a denaturing polyacrylamide gel (lanes 1 and 2) and on an SSCP gel (lanes 3 and 4). Two conspicuous examples of resolved strands are labeled'A'and'B'. Figure 2 shows a fingerprint generatedby a pair of primers used on genomic DNAs from a set of recombinant inbred lines. Strands derived from the same polymorphic PCR product could be assigned because of their completely concordant segregation patterns. The polymorphism indicated by an'A'is known to be an SSCP polymorphism because it could not be scored on a denaturing gel (not shown). Length polymorphisms, labeled B, C, and D, could be scored at up to four places on the SSCP gel (Figure 2).
