Reverse transcription of RNA

Hypotheticoi scheme for cloning cDNA frogments from reverse transcripts of RNA into M13 vectors for sequencing... 

HTV (the AIDS virus) is shown here attacking a T-4 lymphocyte. HIV is an RNA virus whose genetic material must be translated to DNA before inserting itself into the host cell s DNA. Several of the anti-AIDS drugs are directed toward stopping this reverse transcription of RNA to DNA. (Magnification 1000X)... 

Reverse transcription of RNA is sometimes used, e.g., for clone selection by colony hybridization (Verbeek and Tijssen, 1988). Although both avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MMLV) reverse transcriptase can be used, we prefer the AMV enzyme if there are no strong secondary structures in the RNA (MMLV enzyme can be used at higher temperatures, even up to 55°C). The optimal pH range for the AMV enzyme is very narrow (should be 8.3) whereas the MMLV enzyme is active in a range of 7.6-8.3. Superscript reverse transcriptase from BRL is a cloned MMLV enzyme from which the RNase H activity has been deleted. 

Biological information circulates universally by means of three processes replication of DNA, transcription of DNA into RNA, and translation of RNA into protein. In addition, viruses with an RNA genome possess an enzyme that catalyzes one of two processes that are not carried out by cells rephcation of RNA and reverse transcription of RNA into DNA. These five processes are shown schematically in Figure 10.1. 

Various methods are available for detecting and quantifying gene-expression levels, including northern blots, differential display, polymerase chain reaction after reverse transcription of RNA, and serial analysis of gene expression. These techniques are used primarily to measure the expression levels of specific genes or to screen for significant differences in mRNA abundance. 

The well known central dogma of molecular biology, as hrst discussed by Francis Crick, is that DNA is transcribed into RNA, which is translated into protein (see Chapter 1 and Figure 3.1). The hrst of these steps is occasionally reversible, as in the case of reverse transcription of RNA to DNA in the life cycle of some viruses, but there remains no instance of... 

Reverse transcription polymerase chain reaction (RT-PCR) is an experimental method for exponential amplification of a defined segment of complementary DNA (cDNA) produced by reverse transcription of RNA. 

The principle of PCR, including the different phases of the amplification reaction and the reagents needed, is described in the next section. This is followed by real time PCR, which allows semi-quantitative measurement of the PCR products during the reaction. Then the process of reverse transcription of RNA into cDNA is outlined. 

---