Reverse transcriptase cDNA synthesis

Reverse transcriptase j Synthesizes DNA from RNA template. Synthesis of cDNA from mRNA RNA (S end) mapping studies. 

Genomic DNA is much more complex than cDNA. Since cDNAs are synthesized in the laboratory from mRNAs using reverse transcriptase, they are no more diverse than the number of mRNAs present at the time of isolation. However, only about 2% of the mammalian genome codes for the synthesis of proteins and their mRNAs. Thus genomic DNA libraries are at least 50 times more diverse than cDNA libraries. cDNA libraries are not easier to make, however, both because of the inherent instability of mRNA compared to DNA and because reverse transcriptase prematurely terminates when copying long mRNAs. 

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. 

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). 

A third alternative starts with an extract of RNA, not DNA. Mature eukaryotic mRNA contains a long run or tail of adenine residues at its 3 end. The poly(rA) tail can be hybridized with an oligomer of thymine residues, and the oligo(dT) can then be used as a primer for a particular kind of DNA polymerase known as reverse transcriptase. This enzyme, a polymerase associated with retroviruses, will use RNA as a template to make a complementary DNA copy of the RNA, creating a DNA-RNA double-stranded hybrid. In another round of synthesis, the enzyme can replace the RNA strand entirely with DNA, so that the RNA-DNA hybrid is completely converted to double-stranded DNA containing an exact copy of the original RNA sequence. This DNA molecule is known as cDNA because it has a strand that is complementary to (or a copy of) the original RNA. 

Reverse transcriptases have become important reagents in the study of DNA-RNA relationships and in DNA cloning techniques. They make possible the synthesis of DNA complementaiy to an mRNA template, and synthetic DNA prepared in this manner, called complementary DNA (cDNA), can be used to clone cellular genes (see Fig. 9-14). 

Synthesis of cDNA, usually in radiolabeled form is accomplished with reverse transcriptase, the enzyme from retroviruses that synthesize a DNA-RNA hybrid from ssRNA.570 572 A short oligo (dT) primer is usually hybridized to the 3 poly (A) tail to initiate synthesis. Reverse transcriptase also has ribonuclease (RNase H) activity and will digest away the RNA. If desired, synthesis of the second strand can be carried out by a DNA polymerase to give a complete DNA duplex. Many gene sequences have been deduced from cDNA copies. 

Chen, H. and Gold, L. (1994) Selection of high-affinity RNA ligands to reverse transcriptase inhibition of cDNA synthesis and RNase H activity. Biochemistry, 33, 8746-8756. 

Some viruses have secondary structure, which can prevent the production of cDNA detectable in a PCR assay by early termination of the synthesis reaction. To overcome this problem one can raise the temperature of incubation used in first-strand synthesis to 42°C or higher. This will reduce some secondary structures, but will also reduce the half-life of the reverse transcriptase. AMV reverse transcriptase may be used instead, because it has an optimal temperature of 42°C. Unfortunately, AMV RT has more endogenous RNaseH activity than M-MLV RT, thus on average AMV RT produces shorter cDNA fragments. RNaseH deficient RT enzymes are also available (e.g., the Superscript enzymes from Invitrogen), and there is some evidence that these may be the most sensitive type of RT enzymes for PCR assays. The RT conditions required for the efficient detection of individual viruses can only be determined empirically. 

Downstream application [PCR (polymerase chain reaction), cloning, labeling, blotting, RT (reverse transcriptase)-PCR, cDNA synthesis, RNAse protection assays, gene therapy, etc.]... 

Self-primed synthesis of ds cDNA Reverse transcriptase dNTPs... 

Proteins never serve as templates for RNA. However, RNA chains can in rare circumstances reverse the flow of information from RNA to DNA. An example of this is the infecting RNA of retroviruses that serves as a template for the synthesis of a single-stranded complementary DNA (cDNA) chain that functions as a template for a complementary DNA chain. The resulting double-stranded DNA then acts as a template for the synthesis of the original viral RNA chain. The virus-specific enzyme, reverse transcriptase, catalyzes the synthesis of DNA on the RNA template. Little if any reverse transcriptase activity is present in normal cells, so that very little DNA is formed on RNA templates. 

For first-strand cDNA synthesis SUPERSCRIPT II RT reverse transcriptase (200 U/pL) supplemented with 5X RT buffer (250 mM Tris-HCl, pH 8.3, 375 mM KC1, 15mM MgCl2) (Gibco-BRL), random hexamers (50 ng/pL), 0.1 M DTT, 10 mM dNTP mix, and DEPC-treated water. These solutions should be prepared as RNase-free. We utilize solutions supplemented with cDNA synthesis kit (Gibco-BRL)... 

Restriction endonucleases and DNA ligase Cutting, rejoining DNA Reverse transcriptase Conversion of mRNA into cDNA Chemical DNA synthesis Oligonucleotides Polymerase chain reaction (PCR)... 

Reverse transcription PCR (RT-PCR) is a modification of the standard PCR technique that can be used to amplify mRNA. The first step is to convert isolated mRNA to a complementary DNA (cDNA) molecule using an RNA-dependent DNA polymerase (also known as reverse transcriptase) during a process called reverse transcription (RT). The complementary DNA can be used as any other DNA molecule for PCR amplification. The primers used for cDNA synthesis can be either nonsequence-specific primers (a mixture of random hexa-mers or oligo-dT primers) or sequence-specific primers (Fig. 2.4). Random hexamers are a mixture of all possible combinations of six nucleotide sequences that can attach randomly to mRNA and initiate reverse transcription of the entire RNA pool. Oligo-dT primers are complementary to the poly-A tail of mRNA molecules and allow synthesis of cDNA only from mRNA molecules. Sequence-specific primers are the most restricted because they are designed to bind selectively to mRNA molecules of interest, which makes reverse transcription a target-specific process. 

Retroviruses are RNA-DNA viruses that require host-cell genome integration to complete their replication cycle. The retroviral enzyme reverse transcriptase is an RNA-dependent DNA polymerase that has important uses in recombinant DNA methodologies such as the synthesis of complementary DNA (cDNA). The human immunodeficiency virus (HIV) is the etiologic agent that causes acquired immunodeficiency syndrome (AIDS) in humans. HIV infects and destroys human T cells, which are required for immune system functions. 

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