Construction And Research Of Multiplex PCR Amplification System For Establishing Maize DNA Fingerprinting Database

The construction of maize DNA fingerprinting database is of important value in maize purity & identity testing, variety right' protection, variety quality inspection in regional trial, and is of significance in heterotic group, germplasm innovation and variety breeding. This study is to establish the technical platform of maize standard DNA fingerprint database through the key technic of experimental screening of redesigning primer pairs, basic process of establishing common multiplex PCR combination and fluorescence multiplex PCR combination.The main results were summarized as follows:(1) Redesigned and optimized the selected primer locus by software, and experimental screened the redesigned primer pairs, in 216 primer pairs, 76 primer amplified badly, the other 140 primer pairs amplified well enough as candidate primer to combine multiplex PCR.(2) Combined multiplex PCR for selected primer, illustrated the basic process of establishing multiplex PCR, analyzed main influencing factors of constructing multiplex PCR combination, and put forward important principles of establishing multiplex PCR combination.(3) According to homoplastic process, principle of 10 primer (one primer per chromosome) one set, and combined mode of five two-plex or two three-plex and two two-plex, constructed four sets common multiplex PCR combination used 40 core primer pairs.(4) According the results of common multiplex PCR combination, and through evaluating interaction of primer for two three-plex and two two-plex modes by software, confirmed two sets of fluorescence multiplex PCR combination, one set of ten-plex PCR with amplifying one time and electrophoresis detecting one time, and another set of four multiplex PCR amplification with electrophores is detecting one time.