Molec. Bio help (needed)
mspeek at ebc.ee
Wed Mar 3 02:33:52 EST 1993
pickett at kenyon.edu wrote:
: Here is one:
: 1)The view that DNA polymerase moves progressively along the template DNA as it
: adds one nucleotide after another to the growing chain was initially intuitive.
: You want to study whether DNA polymerase indeed exhibits processivity in its
: replication of templates in vitro. You have available to you two synthetic
: DNA's one of which is composed entirely of A's on one strand and T's on the
: other and another with C's on one strand and G's on the other. Also, you have
: your choice of raioactively labelled DNA precursors (alpha-^32P-dNTP's).
: Finally, you have bacterial extracts containing the necessary machinery
: (including polymerase) to replicate DNA. Devise and describe an experiment to
: test whether DNA polymerase acts processively, i.e. does not dissociate from
: template DNA as a new DNA strand is synthesized. Be sure to be clear about
: experimental procedures, about hypotheses being tested, and about expected
: results. We know of course that bacterial DNA polymerase does act
: processively. What features of the Klenow fragment of DNA Polymerase I
: facilitate this type of action?
Just a quick answer (reference) to your first question!
Processivity of DNA PolI (or the Klenow polymerase) "in vitro"
depends on conditions, under which the reaction is carried out
(e.g., see Anal. Biochem. 158, 242-249 ). Briefly, favourable
factors include: high concentration of dNTPs, excess of enzyme,
high pH >8, low ionic strength, etc.
Hope this helps somehow!
Mart (mspeek at ebc.ee) [ESTONIA]
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