Fill in technique
TFITZWATER at somalogic.COM
Fri Feb 22 11:10:09 EST 2002
Both Not I and Xho I can be heat-inactivated by incubation at 65 degrees for
20 minutes. There is a risk: C. Joyce suggests that some restriction
enzymes stay attached to the cut ends of molecules and may require
phenol:chloroform extraction (the most frequent offenders are BamH I, Hind
III and Pst I) prior to Klenow fragment fill-in. (C. Joyce, 1984 BRL Focus 6
(1) 6.) This effect is probably not 100%.
The minimum dNTP concentration is 33 to 44 uM, depending on which source you
reference, but I would recommend 50 uM to account for concentration errors.
Add 1 Unit of Klenow fragment per ug of DNA. Incubate at 37 degrees for 10
Klenows 3 > 5 exonuclease activity removes the final nucleotide after the
fill-in reaction. Alternating polymerase and 3 > 5 exonuclease activity
eventually convert all of the terminal dNTP to dNMP which results in the
loss of the terminal nucleotide. The exo- version of the enzyme is not
recommended for fill-in reactions prior to DNA ligation (unless you are
doing TA cloning), as an extra 1-2 nucleotides added to the 3 end of a
blunt-end DNA will not be deleted by the exonuclease activity of the intact
enzyme. Do NOT heat inactivate the Klenow, as this can increase the 3' > 5'
exo activity, destroying many of your blunt ends.
"The reaction involved is one of excision/incorporation, in which the 3'
deoxynucleotide residue of the primer DNA strand is partitioned into its
5'-mono and 5-tri-phosphate derivatives, respectively. Mechanistic studies
suggest that the 5' monophosphate product is formed in the first step by
simple 3' > 5' exonuclease cleavage. Rapid polymerization follows with the
concomitant release of inorganic pyrophosphate. In the second step, the
5'-triphosphate product is generated by a pyrophosphoralysis reaction,
which, despite the low concentration of pyrophosphate that has accumulated,
occurs at a rate that is comparable with that of the parallel 3' > 5'
hydrolysis." (J. Mizrahi et al.. 1986 PNAS 83: 231-235)
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