DNA and RNA Amplification

New application note: An extraction-free method for sensitive detection of viral RNA using direct RT-PCR chemistry

Cytiva direct RT-PCR chemistry, which is a combination of a proprietary AIC and RT-PCR master mix, enables an extraction-free nucleic acid amplification test (NAAT). This provides a fast, accurate and sensitive detection of viral RNA directly from unpurified nasopharyngeal patient samples, reducing the cost of the assay and saving time.

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RT-PCR anti-inhibitor complex (AIC) process

Why is DNA amplification important?

When nucleic acid (DNA or RNA) sample is scarce, amplification can be used to expand the sample quantity for use in multiple experiments.

Selecting the most suitable amplification technology will depend on sample type, the molecular biology application where it will be used, and the need to preserve sample for additional testing. Some common technologies for DNA amplification are Phi29 polymerase technology, polymerase chain reaction (PCR), rolling circle amplification (RCA), and isothermal multiple strand displacement amplification (MDA).

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Phi29 polymerase technology

Phi29 DNA polymerase-based isothermal DNA amplification is a simple, reliable alternative to other DNA amplification procedures such as polymerase chain reaction (PCR). The highly processive Phi29 DNA polymerase elicits strong strand displacement enabling rapid DNA replication from multiple sites. Phi29 also has 3’-5’ exonuclease proofreading activity, resulting in 100-fold higher fidelity compared to Taq DNA polymerase. But which amplification technology is most suitable for different molecular biology applications?

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Whole genome amplification

GenomiPhi kits, powered by Phi29 polymerase technology, provide an easy-to-use amplification method that delivers highly representative and reliable whole genome amplification by isothermal multiple strand displacement amplification (MDA), with minimal hands-on time and low error frequency. MDA uses Phi29 DNA polymerase in combination with random hexamers to initiate amplification at multiple points throughout the genomic DNA to deliver mirogram quantities of representative product from from as little as a single cell. For occasions where the amount of genomic DNA is insufficient to perform the necessary investigations, or for preservation of precious sample for additional exeriments or in case of failures, Genomiphi offers a simple and effective solution.

Rolling circle amplification (RCA)

TempliPhi kits use isothermal rolling circle amplification (RCA) for the accurate, exponential amplification of circular DNA using Phi29 DNA polymerase. Random hexamer primers anneal to the circular template DNA at multiple sites and Phi29 DNA polymerase extends each of these primers. With TempliPhi, amplification reactions can be performed straight from cells, eliminating need for overnight culture. As yields are consistent, reactions can be added directly to cycle sequencing reactions without prior quantitation.

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Comparison of GenomiPhi V3 Ready-To-Go and GenomiPhi V2 DNA amplification kits

The drawback of DNA amplification kits in liquid format is the need to keep them frozen to maintain performance. Apart from the inconvenience of freezer storage, the reagents must be thawed carefully on ice, mixed for use, and returned promptly to storage, with the added risk of degradation over time after repeated use. The ambient temperature stability of GenomiPhi V3 Ready-To-Go removes these requirements and demonstrates robust and reliable performance with improved yield for downstream applications.

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GenomiPhi V2 DNA Amplification Kit