CDNA - copy DNA

Reverse transcription ofmRNA The use of the enzyme reverse transcriptase (RT) transcribes isolated mRNA into cDNA (copy DNA). 

Fig. 5.1 Regulators of pre- and post-integration latency. Pre-integration latency is regulated as the viral RNA is reverse transcribed into the proviral DNA (A). This is controlled by the avaUabdity of the nucleotide pool, half life of the forming proviral cDNA copy, and the interaction of the viral protein Vif with the cellular antiviral protein APOBEC, espedaUy family members 3G and 3R It is also regulated at the step of transport across the nuclear membrane through the availability of ATP as the process requires energy (B). Post-integration, the proviral DNA copy of the viral genome, is regulated maiiily by the avadabdity of host transcription factors, especially NF-kB and NFAT (C)...

A solution to the problem of introns is to isolate mRNA extracted from the human pancreas cells that make insulin. These cells are rich in insulin mRNA from which introns have already been spliced out. Using the enzyme reverse transcriptase it is possible to convert this spliced mRNA into a DNA copy. This copy DNA (cDNA), which carries the uninterrupted genetic information for insulin can be cloned. Although yeast cells (Saccharomyces) can splice out introns it is normal practice to eliminate them anyway by cDNA cloning. 

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. 

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. 

Messenger RNA is isolated from the tissue source selected to contain the Ab-secreting cells. Spleen, bone marrow, tonsil, and lymph node have all been successfully used as a source Copy DNA (cDNA) is then generated by reverse transcription and Fv or Fab regions amplified by PCR. 

Complementary DNAs (cDNA) are DNA copies of mRNAs. Reverse transcriptase is the RNA-directed DNA polymerase that synthesizes DNA strand, using purified mRNA as the template. DNA polymerase is then used to copy the DNA strand forming a double-stranded cDNA, which is cloned into a suitable vector. Once a cDNA derived from a particular gene has been identified, the cDNA becomes an effective probe for screening genomic libraries (Cowell and Austin, 1997). Annotated human cDNA sequences can be accessed from HUNT at http // www.hri.co.jp/HUNT (Yudate, 2001). 

Preparation of cDNA copy from mRNA involves (1) hybridization with an oligo(dT) primer (2) use of reverse transcriptase (3) DNA olymerase I (4) alkali treatment (5) action of nuclease. The correct sequence is... 

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