KCl in RT-PCR
(by aawara from pontiff-playground.org)
Sat Aug 23 09:44:33 EST 2008
In <db4a14ab-daf6-4fcd-adcc-2fe4ae5583b9 from l33g2000pri.googlegroups.com>,
sayeedansarmaricar from gmail.com <sayeedansarmaricar from gmail.com> wrote:
> hi, can anyone tell me why do we use KCl in the reaction buffer for
> cDNA synthesis.
I don't know, but I can wager a guess. Much of the early biochemistry on
reverse transcriptase was done on AMV reverse transcriptase (from avian
myeloblastosis virus - a chicken retrovirus). The MLV enzyme was much
harder to purify because it was harder to make large amounts of murine
leukemia virus - this is in the pre-cloning & expression days.
AMV RT exists in two forms - an alpha/beta heterodimer, and a beta/beta
homodimer. Both these forms have polymerase and RnaseH activity - the
beta/beta form has higher RnaseH activity. Anyway, polymerase processivity
for AMV RT was slightly higher when K was the cation rather than Na,
without affecting the RnaseH processivity. I worked on AMV RT in the
1980s, and it was my observation that this increased processivity with
K was more easily observed with the beta/beta form than the alpha/beta
form - so my guess is that the early preps used by Hurwitz and Spiegelman
had more beta/beta RT than alpha/beta RT.
I don't think this salt preference has any bearing on the recombinant
"monomeric" RTs that are used commercially today - almost all of them
are modified forms of MLV RT.
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