Synthesis reverse transcriptase assay

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.]... 

The positions that remain unmodified at the end of the in vitro selection procedure (e.g., the purine residnes in a selection carried out with 2 -fluoro-pyrimidine triphosphates) can be modified post-SELEX for further optimization of the aptamers. A systematic stndy of the 64 variants of the six-membered apical loop of an anti-TAR aptamer led to the identification of locked nucleic acid/2 -0-methyl chimeras fully resistant to nncleases that displayed anti-HIV-1 properties in a cell cnlture assay (Di Primo et al., 2007). Identification of the few residues that cannot be modified in an RNA aptamer can be carried ont by chemical interference, a method nsed to identify chemical variants of the aptamer originally selected. Snch an approach led to the synthesis of a modified anti-HIV-1 reverse transcriptase in which all bnt two of the positions of the RNA aptamer were snbstitnted by 2 -0-methyl residnes (Green et al., 1995). This was also the case for the aptamer nsed for age-related macnla degeneration in hnman beings (Ruckman et al., 1998). 

For quantitation of viral and cellular parameters, cell suspensions (in triplicate) were collected, pooled, and centrifuged at 1400 X g for 10 min at 4°C. Supernatant was used for assays of extracellular reverse transcriptase (RT) activity and p24 antigen. Cell pellets were used for the determination of cellular metabolic activity and protein synthesis rates. 

A further important step in quantitative RT-PCR is the production of a single-stranded (ss) complementary DNA copy (cDNA) of the RNA through the reverse transcriptase (RT). Its dynamic range, sensitivity, and specificity are of prime consideration for a successful kinetic RT-PCR assay. For many quantitative applications, moloney murine leukaemia virus (MMLV) RT is the enzyme of choice, as its cDNA synthesis rate is up to 50-fold greater than that of avian myeloblastosis virus (AMV). Newly available thermostable enzymes maintain their activity up to 70°C, thus permitting increased specificity and efficiency of first primer annealing. Fiowever, this enzyme type may be less robust than more conventional ones as it appears to be more sensitive to inhibitors present in RNA preparation. 

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