Oligo dT primer

ImENA template is annealed to synthetic oligonucleotide (oligo dT) primer. 

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. 

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... 

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. 

This can be done by using reverse transcriptase, an enzyme derived from retroviruses, such as the AIDS virus. Reverse transcriptase synthesizes DNA using RNA as a template, producing complementary DNA (cDNA). In the case of eukaryotic mRNA, an oligo-dT primer is used, exploiting the 3 poly A tail on eukaryotic mRNA. 

Fig. 3.2 Gene expression analysis with microarrays. cDNA is obtained by reverse transcription of mRNA using oligo(dT) primers. With high-density commercial arrays (A), biotinylated ribonucleotide analogs are incorporated during in vitro transcription of the cDNA. After fragmentation and hybridization, fluorescence detection occurs by streptavidin-mediated binding of fluorophores to biotin. One hybridization is performed per each sample analyzed. Custom-made cDNA arrays...

Mix 17 xL (20 Xg) of total RNA with 4 xL oligo-dT primer (supplied with the kit) in a clean thin-walled 200- xL PCR tube. 

With poly(A) as the template, the oligo(dT) primer is physically incorporated via a 3 — 5 -phosphodiester linkage into the 5 end of the poly(dT) products. Although both ribo- and deoxyribooligomers can serve as primers, oligodeoxyri-bonucleotides are considerably more efficient as primers on homopolymeric as well as heteropolymeric templates in vitro (22). This is in contrast to the fact that the natural primer is a tRNA tRNA P for AMV RTase (23), tRNA for MoLV RTase (24), and tRNA -> 3 for HIV-1 RTase. 

The cDNAs synthesized from poly(A)-tailed mRNA in the presence of the oligo(dT) primer range widely in size from 200 bases to several kilobases, all depending on reaction conditions. Reverse transcriptases lacking RNase H activities produce longer cDNAs and at substantially higher yields. 

V. Appearance of initial RNase H activity. After forming a stable complex with the DNA-primed RNA template (with a half-life of 30 and 15 sec for AMV RTase and MoLV RTase, respectively), RTase/RNase H activities cleave the hybrid portion of the RNA before dissociating. In the presence of the oligo(dT) primer, RNase H-mediated deadenylation takes place in the mRNA and competes with the RTase-mediated polymerization, resulting in lower yields of cDNA (50). 

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