Two important considerations for choosing a cDNA synthesis kit are the reverse transcriptase enzyme and the priming strategy used to initiate the reaction. The choice of enzyme impacts the synthesis rate and reaction fidelity. Priming strategies influence what is transcribed in the reaction and, if not optimal, may result in a biased library that does not accurately represent the complete transcriptome. Most commercial reverse transcription kits utilize either Moloney murine leukemia virus MMLV reverse transcriptase or avian myeloblastosis virus AMV reverse transcriptase.
Pfu DNA polymerase can be used alone to amplify DNA fragments up to 5kb by increasing the extension time to 2 minutes per kilobase. However, the proofreading activity can shorten PCR primers, leading to decreased yield and increased nonspecific amplification.
Some DNA-dependent DNA polymerases also possess a reverse transcriptase activity, which can be favored under certain conditions.
However, for shorter templates with complex secondary structure, AMV reverse transcriptase may be a better choice because it can be used at higher reaction temperatures. As the names suggest, the deletion mutant had a specific sequence in the RNase H domain deleted, and the point mutant has a point mutation introduced in the RNase H domain. The point mutant is often preferred over the deletion mutant because the point mutant has DNA polymerase activity comparable to that of the wildtype M-MLV enzyme, whereas the deletion mutant has a slightly reduced DNA polymerase activity compared to that of the wildtype enzyme Figure 4.
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Buffer Considerations Most reaction buffers consist of a buffering agent, most often a Tris-based buffer, and salt, commonly KCl. Enzyme Concentration We recommend using 1—1. Template Quantity The amount of template required for successful amplification depends upon the complexity of the DNA sample. Reverse Transcription Primer Design Selection of an appropriate primer for reverse transcription depends on target mRNA size and the presence of secondary structure. C Nuclease-Free Water Cat.
Immediately chill in ice water for at least 5 minutes. Centrifuge 10 seconds in a microcentrifuge. Store on ice until reverse transcription mix is added. Combine on ice, in the order listed. Reactions can be stopped at this point for analysis of the cDNA or may be frozen for long-term storage.
Categories PCR. Ahokas, H. PCR Methods Appl. Andre, P. Genome Res. Arakawa, T. DNA Res. Auer, T. Biochemistry 34 , — Baldino, F. Baltimore, D. Nature , — Barnes, W. USA 91 , — Bej, A.
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Brooks, E. Biotechniques 19 , — Carballeira, N. Biotechniques 9 , — Carothers, A. Biotechniques 7 , —9. Cheng, S. USA 91 , —9. Chiocchia, G. Biotechniques 22 , —8. Chumakov, K. Clark, J. Cline, J. Crockett, A. Dang, C. Demeke, T. Biotechniques 12 , —4. Didenko, V. Biotechniques 31 , — Eckert, K. Edwards, J. Nat Methods 6, an12—an13 Download citation. Issue Date : December Anyone you share the following link with will be able to read this content:.
Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Advanced search. Skip to main content Thank you for visiting nature. Download PDF. Genomics 37 , — Central dogma reversed. Crick, F. On protein synthesis. Mullis, K. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods in Enzymology , — Saiki, R. Science , — doi Swaminathan, S.
Milestone Chain reaction. Temin, H. Verma, I. Restriction Enzymes. Genetic Mutation. Functions and Utility of Alu Jumping Genes.
Transposons: The Jumping Genes. DNA Transcription. What is a Gene? Colinearity and Transcription Units. Copy Number Variation. Copy Number Variation and Genetic Disease.
Copy Number Variation and Human Disease. Tandem Repeats and Morphological Variation. Chemical Structure of RNA.
Eukaryotic Genome Complexity. RNA Functions. Pray, Ph. Citation: Pray, L. Nature Education 1 1 How do these technologies work? Aa Aa Aa. That is the heretical but inescapable conclusion stemming from experiments done in the past few months in two laboratories in the United States. Discovering Reverse Transcription. The experiments supporting the existence of this DNA polymerase produced data that revealed the following: The DNA polymerase only incorporated deoxyribonucleotides, not ribonucleotides, into its product.
The product itself "behaved" like DNA--in other words, it was sensitive to treatment by deoxyribonucleases but not ribonucleases.
The RNA itself was the template, as shown by the fact that treatment of virions with ribonucleases destroyed the ability of the polymerase to incorporate radioactively labeled nucleotides. How Reverse Transcriptase Works. So how exactly does reverse transcriptase work? Figure Detail. Making Copies via Polymerase Chain Reaction.
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