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.

中文翻译：

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phi29 DNA聚合酶的有限逆转录酶活性

Phi29（Φ29）DNA聚合酶是一种通常用于DNA扩增方法（例如滚环扩增（RCA）和多链置换扩增（MDA））以及DNA测序方法（例如单分子实时（SMRT）测序）的酶。在这里，我们报告了RCA期间phi29 DNA聚合酶放大含RNA的圆​​形底物的能力。我们发现具有单个RNA取代的圆形底物以与非嵌合DNA底物相似的扩增速率扩增，并且连续的RNA嘧啶通常比嘌呤更可取。我们观察到RCA抑制具有更高数量的核糖核苷酸取代，这通过将RNA碱基与DNA相互间隔而部分恢复。我们显示，补充锰离子作为辅助因子可支持RCA期间RNA的复制。²⁺和Mg ²⁺作为复制过程中的辅助因子，证明了phi29 DNA聚合酶的逆转录酶活性。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。这些包括嵌合圆形探针（例如挂锁探针和分子倒置探针）的扩增。