RT is capable of synthesizing a complementary dna strand ( as in HIV life cycle.) Then why is DNA pol used when cDNA (synthesizing the second strand of it ) has to be synthesized from mRNA ( For eg.to construct a cDNA library) ?
$\begingroup$
$\endgroup$
2
-
$\begingroup$ where did you read that DNA pol is used ? $\endgroup$– WYSIWYGCommented Nov 17, 2013 at 17:42
-
$\begingroup$ @WYSIWYG I read that in Campbell. $\endgroup$– biogirlCommented Nov 17, 2013 at 19:43
Add a comment
|
1 Answer
$\begingroup$
$\endgroup$
Reverse transcriptase, as the name suggests, uses an RNA template to create a DNA transcript (i.e., complement). Once the DNA complement has been made, DNA polymerase is used because it uses a DNA template to produce a DNA complementary strand.
In your specific example, HIV contains a positive-stranded, RNA genome. The HIV RT can use either RNA or DNA templates to produce DNA complements, and this is how the single-stranded RNA genome is transcribed into double-stranded DNA, before integration.
To my knowledge, the RT enzymes sold commercially (for applications like RT-PCR) are typically a mix of an RT enzyme and a DNA polymerase, isolated from other retroviruses, such as murine leukemia virus. An important consideration for lab application is the production of cDNA that truly represents the RNA template of interest. Since RT enzymes typically lack a 3'->5' exonuclease activity, they are unable to "proof-read" the complement strand as it is being produced. In contrast, there are very efficient proof-reading DNA polymerase, and this is why a DNA polymerase (with high proof-reading ability) is used after first-strand synthesis by RT.