PCR fragmentation of DNA

A method has been developed to prepare random DIVA fragments using PCR. Fir st, two cycles are carried out at 16 degrees C with the Klenow's fragment a nd oligonucleotides (random primers) with random 3'-sequences and the 5'-co nstant part containing the site for cloning with the site-specific endonucl ease. The random primers can link to any DNA site, and random DNA fragments are formed during DNA synthesis. During the second cycle, after denaturati on 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-stra nded DNA fragments are produced which have a constant primer sequence at th e 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. In cubation 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 rou tine PCR amplification is carried out using the constant primer. This metho d is more advantageous than hydrodynamic methods of DNA fragmentation widel y used for "shotgun" cloning.