关键词: 滚环复制, phi29 DNA聚合酶, 小片段DNA

Abstract:

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.

Key words: rolling circle replication, phi29 DNA polymerase, small fragment DNA