Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Solna, SE-171 65, Sweden.
Nucleic Acids Res. 2018 Apr 20;46(7):3625-3632. doi: 10.1093/nar/gky190.
Phi29 (Φ29) DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real time (SMRT) sequencing. Here, we report the ability of phi29 DNA polymerase to amplify RNA-containing circular substrates during RCA. We found that circular substrates with single RNA substitutions are amplified at a similar amplification rate as non-chimeric DNA substrates, and that consecutive RNA pyrimidines were generally preferred over purines. We observed RCA suppression with higher number of ribonucleotide substitutions, which was partially restored by interspacing RNA bases with DNA. We show that supplementing manganese ions as cofactor supports replication of RNAs during RCA. Sequencing of the RCA products demonstrated accurate base incorporation at the RNA base with both Mn2+ and Mg2+ as cofactors during replication, proving reverse transcriptase activity of the phi29 DNA polymerase. In summary, the ability of phi29 DNA polymerase to accept RNA-containing substrates broadens the spectrum of applications for phi29 DNA polymerase-mediated RCA. These include amplification of chimeric circular probes, such as padlock probes and molecular inversion probes.
Phi29（Φ29）DNA 聚合酶是一种常用于 DNA 扩增方法的酶，如滚环扩增（RCA）和多链置换扩增（MDA），以及 DNA 测序方法，如单分子实时（SMRT）测序。在这里，我们报告了 phi29 DNA 聚合酶在 RCA 过程中扩增含有 RNA 的环形底物的能力。我们发现，具有单个 RNA 取代的环形底物以与非嵌合 DNA 底物相似的扩增速率被扩增，并且连续的 RNA 嘧啶通常比嘌呤更受青睐。我们观察到随着核糖核苷酸取代数量的增加，RCA 受到抑制，而用 DNA 隔开 RNA 碱基部分恢复了这种抑制。我们表明，作为辅助因子补充锰离子支持在 RCA 过程中复制 RNA。对 RCA 产物的测序表明，在复制过程中，Mn2+和 Mg2+作为辅助因子，phi29 DNA 聚合酶在 RNA 碱基处准确掺入碱基，证明了其逆转录酶活性。总之，phi29 DNA 聚合酶接受含 RNA 底物的能力拓宽了 phi29 DNA 聚合酶介导的 RCA 的应用范围。这些包括扩增嵌合环形探针，如锁式探针和分子反转探针。
