目的 建立一种高效扩增小片段DNA的方法.方法 本方法利用单链DNA连接酶(single strand DNA ligase,ssDNA ligase)可以连接单链DNA的性质将小片段DNA自连成环,随后利用phi29 DNA聚合酶进行恒温的滚环复制,将扩增产物进行酶切,得到了扩增后的小片段DNA.结果 单链DNA连接酶可以有效的将30 bp的小片段DNA自连成环,phi29 DNA聚合酶可以扩增出大于10 kb的DNA片段,扩增产物经酶切后又可进行第二轮扩增,并且证明了其成环方式为自成环.结论 通过单链成环后滚环复制的这种方法可以有效的将小片段 DNA进行扩增,解决了PCR无法扩增小片段DNA的问题,并有着广阔的应用前景.%Objective To establish a method for highly efficient amplification of small fragment DNA. Methods Single-strand DNA intramolecular ligation was made by using single-stranded DNA ligase( ssDNA ligase), which can link small DNA fragments into a ring, and then isothermal rolling circle replication was carried out by using phi29 DNA polymerase. The amplified products were cut by incision enzyme. In this way, the small fragment DNA can be amplified at high efficiency. Results SsDNA ligase can make 30 bp oligonucleotide DNA cyclization, and phi29 DNA polymerase can make amplification products more than 10 kb in length. Amplification products can be amplified second times after being cut by incision enzyme. It was also proved that the way of single strand DNA cyclization was intramolecular. Conclusion This method can effectively amplify the small DNA fragments, and has broad prospects of application.
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