利用Taq DNA聚合酶直接从双链RNA模板中扩增靶序列

Direct Amplification of Target Sequences by Taq DNA Polymerise Using Double-stranded Rna as Templates

摘要:

利用Taq DNA聚合酶既具有DNA聚合酶活性义具有反转录酶活性的特点,探索了在Taq DNA聚合酶单独作用下以双链RNA为模板进行PCR反应的条件。结果表明靶序列长度为277 bp、369 bp、987 bp时,均可直接进行PCR扩增;短片段序列扩增的退火温度在47．0℃、47．9℃、50．2℃、52．6℃、54．9℃、56．7℃条件下,均可有效扩增,而长片段序列扩增的退火温度在50．2℃、52．6℃、54．9℃、56．7℃条件下,也可扩增出相应的靶序列。这一结果提示利用Taq DNA聚合酶可以dsRNA为模板直接扩增目的片段,尤其是短片段的扩增。

Abstract:

Taq DNA polymerase has the activities of DNA polymerase and RNA reverse transcriptase.This research used the Double-stran- ded RNAs were used as templates for direct PCR,making use of the characteristics of Taq DNA polymerase.PCR for target sequences of 277,369 and 987 bp from dsRNA templates were performed directly with Taq DNA polymerase.When the sizes of target sequences were 277 bp and 369 bp,they were amplified with dsRNA templates on the denaturing temperatures of 47.0℃,47.9℃,50.2℃,52.6℃, 54.9℃,56.7℃ respectively. The results suggested that target sequences can be amplified with dsRNA templates only using Taq DNA polymerase alone and it is more effective for amplification of smaller sequences.