Phd Essay: CDNA synthesis protocol rnase h free …
Chemical synthesis and enzymatic properties of RNase …
To remove template DNA, add 70 μl nuclease-free water, 10 μl of 10X DNase I Buffer, and 2 μl of DNase I (RNase-free), mix and incubate for 15 minutes at 37°C.
The RNase H activity of reverse transcriptase comprises the C-terminal one-third of the protein and cleaves RNA in an RNA-DNA hybrid (). RNase H effectively degrades the RNA template both during and after minus-strand synthesis to facilitate strand transfer and plus-strand synthesis. Biochemical studies have shown that polymerization-dependent RNase H cleavages take place concomitant with DNA synthesis, are positioned by the polymerase domain, and occur in the RNA template strand 15 to 20 nucleotides (nt) from the DNA 3′ terminus (, , ). However, the polymerization-dependent form of RNase H activity is not sufficient to eliminate all RNA from the minus-strand DNA (, , , ).
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Shortly after entrance of the viral cores into the cytoplasm of a cell, the single-stranded plus-sense RNA genome of a retrovirus is converted into a double-stranded DNA molecule that subsequently integrates into the host cell genome (). This process, termed reverse transcription, requires two distinct RNA primers to synthesize the double-stranded DNA. The first primer is a host cell-derived tRNA that is used for the initiation of minus-strand DNA synthesis. The second is a short RNA derived by RNase H cleavages within a purine-rich sequence in the viral genome called the polypurine tract (PPT). This primer is used to begin plus-strand synthesis and is referred to as the plus-strand primer or the PPT primer (reference and references therein).
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These RNA primers are extended by the viral-encoded reverse transcriptase, a multifunctional enzyme that carries out DNA polymerization, strand displacement synthesis, and the strand transfer reaction and that possesses an RNase H activity required at several steps during genome replication (). The DNA polymerase activity of reverse transcriptase resides in the N-terminal two-thirds of the protein and utilizes either RNA or DNA as a template. Although displacement synthesis is not as efficient as nondisplacement synthesis (, , , ), reverse transcriptase can simultaneously extend the 3′ terminus of a DNA primer and displace a downstream nontemplate RNA or DNA strand, a function vital to complete reverse transcription. Previous studies have demonstrated that reverse transcriptase can access a 3′ primer terminus and initiate DNA and RNA displacement synthesis at a single-strand break or nick (, , ).
When RNase H removes the PPT primer from plus-strand DNA during reverse transcription, the 3′ end of the PPT primer is followed immediately by the 5′ end of the newly synthesized downstream DNA, which presents an interesting substrate for reverse transcriptase. Despite the ability of reverse transcriptase to carry out DNA displacement synthesis at a nick (, , ), the PPT primer is not efficiently reutilized for additional plus-strand synthesis (, ). A similar situation might arise when RNase H creates the PPT primer if RNase H were to make only a single cleavage at the −1/+1 site. This would offer reverse transcriptase a nick with the 3′ end of the PPT primer abutting downstream RNA as a substrate to initiate plus-strand DNA synthesis. Although numerous studies have addressed utilization of the PPT primer (, , , , , , ), none have considered the effect of extension at a nick with downstream RNA. In this report, we have used the reverse transcriptase of Moloney murine leukemia virus (M-MuLV) to ask whether displacement synthesis is sufficient to initiate plus-strand synthesis or whether further RNase H cleavages might facilitate extension of the PPT primer. The results of these experiments offer insights into how M-MuLV reverse transcriptase initiates plus-strand synthesis.
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Synthesis and expression of RNase T1 gene.
The RNA strand of these hybrids is degraded by the RNase H activity to allow DNA-dependent synthesis of (+) strand DNA
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Specific cleavages by RNase H facilitate initiation of plus-strand RNA synthesis by Moloney murine leukemia virus.
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The enzyme is inhibited by diethyl pyrocarbonate (DEPC), guanidinium salts (4 M GuaSCN), β-mercaptoethanol, heavy metals, vanadyl-ribonucleoside-complexes, RNase-inhibitor from human placenta and competitively by DNA, respectively.
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When RTΔH, a form of reverse transcriptase that contains a C-terminal deletion of the RNase H domain, was used to extend 5′ end-labeled PPT62 in the context of nondisplacement synthesis (substrate I), runoff extension products corresponding to primer length plus 15 to 16 nt were observed, representing full-length extension with some nontemplated addition of a single base () (Fig. , lane 10). When a 17-mer DNA corresponding to positions +1 to +17 of M-MuLV plus-strand DNA (MLVnickD) was placed downstream of PPT62 in substrate II, extension from PPT62 dropped sixfold (Fig. ; compare lanes 10 and 12). When an equivalent 17-mer RNA (MLVnick) abutted PPT62 in substrate II, synthesis of full-length extension products decreased threefold (Fig. , lane 11). Similar results were observed in a time course assay with H− RT, an intact form of reverse transcriptase containing specific point mutations in the active site of the RNase H domain (Fig. , lanes 16 to 27). At the 1-min time points, 90-fold more full-length product was synthesized with substrate I than with both forms of substrate II (lanes 17, 21, and 25). By 16 min, the difference in synthesis of full-length extension products was five- to sevenfold (lanes 19, 23, and 27).
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These substrates were also extended with wild-type reverse transcriptase, but a shorter time period was required to visualize extension products due to RNase H cleavage between the primer RNA and the nascent DNA. When either form of substrate II containing PPT62 was extended with wild-type reverse transcriptase for 1 min, the amount of full-length product synthesized was reduced 20-fold or more compared to that resulting from extension with substrate I (Fig. , lanes 13 to 15).
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Extension of 5′ end-labeled PPT62 in the context of substrate III (containing a 2-, 5-, or 12-base gap) was compared to extension in both substrate II (containing a nick) and substrate I (which lacks downstream RNA) (Fig. ). In this experiment, the lengths of downstream duplex through which displacement synthesis had to occur were kept constant; therefore, extension products differed depending on the size of the gap. As described above, both forms of RNase H-deficient reverse transcriptase showed limited extension of PPT62 when downstream RNA was present (Fig. and D; compare lanes 1 to 8 for H− RT and RTΔH). By contrast, introduction of a 2- or 5-base gap downstream of PPT62 significantly improved synthesis for RTΔH and H− RT (Fig. and D; compare lanes 9 to 24). For both enzymes, a 12-base gap permitted synthesis levels approximately equivalent to those of nondisplacement synthesis with substrate I (Fig. and D, lanes 25 to 32).
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