Reverse transcriptase primer requirements

An amplification reaction that is used to amplify target RNA or denatured DNA is called the transcription-based amplification system (TAS). This technique involves using an enzyme called reverse transcriptase and a primer with sequence complementary to the sample target RNA molecule in order to synthesize a complementary DNA (cDNA) copy of the sample target RNA. After denaturation to separate the strands, another primer and additional reverse transcriptase are added to synthesize a double-stranded cDNA molecule. Since the first primer has also an RNA polymerase binding site, it can, in the presence of T7 RNA polymerase, amplify the double-stranded cDNA to produce 10 to 100 copies of RNA. The cycle of denaturation, synthesis of cDNA, and amplification to produce multiple RNA copies is repeated. With as few as four cycles, a 2- to 5-millionfold amplification of the original sample RNA target is possible. However, the time required to achieve a millionfold amplification is approximately 4 hours, which is the same amount of time required by PCR. The TAS requires, however, the addition of two enzymes at each cycle and, as such, can be cumbersome. 

DNA-directed DNA polymerases [EC 2.7.7.7], also called DNA nucleotidyltransferases (DNA-directed), are enzymes that catalyze the DNA template-directed extension of the 3 -end of a nucleic acid strand one nucleotide at a time. Thus, n deoxynucleoside triphosphates produce n pyrophosphate (or, diphosphate) ions and DNA . This enzyme cannot initiate the synthesis of a polymeric chain de novo it requires a primer which may be DNA or RNA. RNA-directed DNA polymerases [EC 2.7.7.49], also referred to as reverse transcriptases, DNA nucleotidyltransferases (RNA-directed), and revertases, are enzymes that catalyze the RNA template-directed extension of the 3 -end of a nucleic acid strand one nucleotide at a time. Thus, n deoxynucleoside triphosphates produce n pyrophosphate (or, diphosphate) ions and DNA . As was the case above, this enzyme cannot initiate the synthesis of a polymeric chain de novo it requires a primer which may be DNA or RNA. 

RNA to initiate cDNA synthesis. All cellular mRNA contains multiple repeats of adenine bases (poly-A tails). Therefore the complementary thymine bases (oligo-dT) can be used as a primer that binds to the mRNA template required for the reverse transcriptase to synthesize the cDNA. In the case of pancreatic mRNAs (Figure 4.2), the signihcantly higher mRNA for insulin compared with other proteins allowed success in isolating the insulin-specihc cDNA. Subsequent insertion of cDNA into a bacterial expression vector allowed the production of functional insulin that is now marketed as a successful therapeutic product (Figure 4.2). 

Although the existence of this enzyme may not be surprising, the mechanism by which it acts is remarkable and unprecedented. Telomerase, like some other enzymes described in this chapter, contains both RNA and protein components. The RNA component is about 150 nucleotides long and contains about 1.5 copies of the appropriate CyKx telomere repeat. This region of the RNA acts as a template for synthesis of the T -G strand of the telomere. Telomerase thereby acts as a cellular reverse transcriptase that provides the active site for RNA-dependent DNA synthesis. Unlike retroviral reverse transcriptases, telomerase copies only a small segment of RNA that it carries within itself. Telomere synthesis requires the 3 end of a chromosome as primer and proceeds in the usual 5 —>3 direction. Having syn-... 

Since eukaryotic chromosomes are linear, the ends of these chromosomes require a special solution to ensure complete replication. This can be seen in figure 26.26. At the very end of a linear duplex a primer is necessary to initiate DNA replication. After RNA primer removal there is bound to be a gap at the 5 end of the newly synthesized DNA chains. Since DNA synthesis always requires a primer the usual way of filling this gap is not going to solve the problem. This dilemma is overcome by a special structure at the ends (telomeres) of eukaryotic chromosomes and a special type of reverse transcriptase (telomerase) that synthesizes telomeric DNA. In many eukaryotes the telomeres contain short sequences (frequently hexamers) that are tan-demly repeated many times. Telomerase contains an RNA that binds to the 3 ends and also serves as a template for the extension of these ends. Prior to replication, the 3 ends of the chromosome are extended with additional tandemly repeated hexamers. The 3 ends are extended sufficiently so that there is room to accommodate an RNA primer. In this way there is no net loss of DNA from the 5 ends as a result of replication. After replication the 3 end is somewhat... 

The mRNAs coding for nearly all mammalian proteins contain a tail of adenylate molecules. This tail is called poly (A). The tail occurs only at one end of the mRNA molecule. Conversion of the mRNA to the mRMA/DNA hybrid takes advantage of poly (A). This conversion is catalyzed by reverse transcriptase. Reverse transcriptase cannot bind to mRNA as is. It requires a primer for activation. The primer can take the form of a short strand of poly(dT). 1 he polyfdT) primer binds to the poly(A) tail of the mRNA, forming a small hybridized region. Formation of the hybrid is possible because A binds to T. 

Hepadnaviral Pol proteins are multifunctional and contain four domains, namely (in order from amino to carboxy terminus) terminal protein, spacer, polymerase, and RNaseH, respectively. Known functions include (1) acting as a primer for first (-) strand DNA synthesis (2) synthesizing first-strand DNA from RNA pregenome (reverse transcriptase or RNA-dependent DNA polymerase [RDDP] activity) (3) degrading viral RNA in resulting RNA-DNA hybrids (RNase H activity) and (4) copying second- (+) strand DNA from the (-) strand DNA template (DNA-dependent DNA polymerase [DDDP] activity). In addition, multiple Pol domains appear to be required for proper assembly of the pregenomic RNA molecule and associated Pol into cytoplasmic core particles (I). 

The answer is b. (Murray, pp 412—434. Scriver, pp 3-45. Sack, pp 3—29. Wilson, pp 99-121.) A special DNA polymerase called telomerase is responsible for replication of the telomeric DNA. Telomerase contains an RNA molecule that guides the synthesis of complementary DNA. Telomerase is therefore an RNA-dependent DNA polymerase in a category with reverse transcriptase. Telomerase does not require an RNA primer, initiating synthesis of the leading strands at 3 ends within the telomeric DNA. Synthesis of the lagging strands uses primase, DNA polymerase III, and DNA polymerase I, as with the replication of other chromosomal regions. 

There are two different methods to identify modified residues and ribonuclease scissions in RNA molecules the reverse transcriptase method or the end-labelling method. The choice of method depends both on the length of the studied RNA and the method of probing (as discussed in Sections 4.4.1 and 4.4.2). The reverse transcription method uses extension of a primer and therefore is independent of the length of the RNA, while the end-labelling method is restricted to analysis of small RNA molecules (n < 300). The latter method requires scission of the RNA. 

Similar assays can be used to study modifications that inhibit the extension of the reverse transcriptase directly and also modifications which render the RNA susceptible to selective strand scission at the modified residue (e.g. m7 guanosines, Section 4.4.2.2). To quantify the extent of modification, one of the nucleotides in the extension mixture should only be present in the dideoxy form (Fig. 5.7). The primer extension assay can also be used for some modifications which do not affect the extension, provided the modification interferes with the reactivity of the nucleotide. The latter approach has been used to study the 2 -0-methylations of guanosine where the modification renders the modified guanosine resistant to RNase T1 digestion. Introduction of a complete RNase T1 digest before the primer extension allows the detection of the modification (Fig. 5.8a). However, the approach requires that there is no nucleotide as the one modified for at least 12 nucleotides 3 of the modified yresidue. If this is not the case alternative approaches can sometimes be employed (see Fig. 5.8b). 

To analyze RNA by PCR an additional step has to be introduced to transcribe RNA into cDNA, which is a more stable single-strand DNA complementary to the targeted RNA. This can be achieved by the reverse transcription (RT) of the template RNA using a reverse transcriptase enzyme. RT and PCR can be carried out either sequentially in the same tube (one-step RT-PCR) or separately (two-step RT-PCR). One-step RT-PCR requires gene-specific primers for the reverse transcription reaction, whereas in two-step RT-PCR random primers can be used. 

Chapter 36 describes a protocol for an isothermal chain reaction that makes use of the presence of DNAprimere, T7 promoters, T7 polymerase, RNaseH, and AMV reverse transcriptase to produce large quantities of nucleic acid when the primers used match an RNA (or DNA) template. This system has the advantage that no thermal cycling block is required, and hence the number of samples that can be processed daily is not limited by the availability of specialized equipment. 

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