PCR fragmentation of DNA.
A method has been developed to prepare random DNA fragments using PCR. First, two cycles are carried out at 16 degrees C with the Klenow's fragment and oligonucleotides (random primers) with random 3'-sequences and the 5'-constant part containing the site for cloning with the site-specific endonuclease. The random primers can link to any DNA site, and random DNA fragments are formed during DNA synthesis. During the second cycle, after denaturation of the DNA and addition of the Klenow's fragment, the random primers can link to newly synthesized DNA strands, and after DNA synthesis single-stranded DNA fragments are produced which have a constant primer sequence at the 5'-end and a complementary to it sequence at the 3'-end. During the third cycle, the constant primer is added and double-stranded fragments with the constant primer sequences at both ends are formed during DNA synthesis. Incubation for 1 h at 37 degrees C degrades the oligonucleotides used at the first stage due to endonuclease activity of the Klenow's fragment. Then routine PCR amplification is carried out using the constant primer. This method is more advantageous than hydrodynamic methods of DNA fragmentation widely used for "shotgun" cloning.
Zheleznaya
L A
LA
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142292, Russia.
Kossykh
V G
VG
Svad'bina
I V
IV
Oshman
T S
TS
Matvienko
N I
NI
D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D053938:DNA Fragmentation; D017931:DNA Primers; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D004247:DNA