RT-PCR Protocols Second Edition
Once a tedious, highly skilled operation, reverse-transcription polymerase chain reaction (RT-PCR) has become a routine and invaluable technique used in most laboratories. In RT-PCR Protocols, Second Edition, expert researchers fully update the technologi
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1. Introduction The process of synthesis of deoxyribonucleic acid (DNA) from a ribonucleic acid (RNA) template is called “reverse transcription” because it reverses the flow of genetic information (from RNA to DNA, rather than from DNA to RNA found in normal transcription). The reverse transcription (RT) reaction is carried out by a retroviral DNA polymerase named reverse transcriptase. The reverse transcriptase is the replication enzyme within the retrovirus that converts its single-stranded genomic RNA into DNA. In molecular biology laboratories, the reverse transcriptase is an RNA-dependent DNA polymerase enzyme used to transcribe single-stranded RNA into single-stranded complementary DNA (cDNA). During the reverse transcription process, oligonucleotide Nicola King (ed.), RT-PCR Protocols: Second Edition, Methods in Molecular Biology, vol. 630, DOI 10.1007/978-1-60761-629-0_17, © Springer Science+Business Media, LLC 2010
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Fig. 1. Schematic representation of reverse transcription of RNA into cDNA using oligo dT, or random primers. Oligonucleotide primers (oligo dT) or random primers are annealed to the RNA template and extended by the reverse transcriptase in the presence of deoxynucleotides (dNTP) to make cDNA. RNAse H treatment can be used to degrade RNA from RNA/DNA hybrids; however, the RNase H treatment is not required before standard PCR. The denaturing step before PCR annealing and extension will make the cDNA available for the PCR primers and Taq polymerase extension
primers complementary to the RNA strand are annealed to the RNA template and extended in the presence of deoxynucleotides (dNTP) and optimal pH and salt conditions to produce cDNA (Fig. 1). The native reverse transcriptase is a multifunctional enzyme. In addition to its RNA-dependent DNA polymerase activity, it also possesses both (1) a weak DNA-dependent DNA polymerase activity that is lacking the 3¢→5¢ exonuclease activity and (2) an RNase H activity that degrades the RNA template from RNA/ DNA hybrid strands as the cDNA synthesis proceeds. Soon after its discovery in 1970 (1, 2), the reverse transcriptase went on to play a vital role in the advancement of molecular biology research, especially in the fields of gene discovery and biotechnology. Reverse transcriptase became an essential molecular tool used in the cloning of genes, the analysis of gene expression, and in the diagnosis of certain microbial diseases. Its influence extends from cloning to the development of microarrays to the characterization of transcriptomes and the annotation of genomes. For molecular biology applica
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