用生物合成的长链开环探针进行SNP分型

摘要

由于化学合成长链开环探针面临许多困难,因此需要开发一种长链开环探针的生物合成方法.该研究利用生物合成的146 bp长链开环探针进行单核苷酸多态性(SNP)分型,以验证长链开环探针生物合成的可行性.结果表明:长链开环探针能够特异地与目的DNA结合,完美配对的长链开环探针能够被DNA连接酶连接,形成环状单链DNA分子,不能完美配对的开环探针不能被DNA连接酶连接,也就不能形成环状单链DNA分子.长链开环探针的连接产物经过DNA外切酶处理后用5%PAGE检测,可以准确进行SNP分型,同时利用RCA反应也能进行SNP分型.该试验的成功为利用不同长度的长链开环探针进行SNP的多元分型提供了基础.%In order to confirm the fact that biosynthesis of long open circle probes was feasible , the long open-circle probe of 146 bp was used to genotype single nucleotide polymorphism ( SNP). After the biosynthesized long open circle probes matched with their target DNA. the probes were ligated by AmpligaseThermostable DNA Ligase to form circularized DNA. Ligation products of long open circle probes were digested with exonuclease firstly, then the mixture was detected with 5％ polyacrylamide gel electrophoresis ( PAGE). In addition, rolling circle amplification ( RCA) reaction was also used for amplification of circularized open circle probes, and the amplification products were detected with 0. 6％ agarose gel. The results showed that the biosynthesized long open circle probes could specifically bind with target DNA perfectly.Owing to the high fidelity of Ampligase Thermostable DNA Ligase at the nick junction, a perfectly matched base-pair would be sealed to form a circular single-stranded DNA. but a mismatched base-pair was left unsealed. Both 5％ PAGE and RCA reaction could correctly detect SNP genotyping using biosynthesized long open circle probe. Taken together, the multiplexed assays could be performed to genotype SNP using biosynthesized long open circle probes with different lengths.