PCR amplification of megabase DNA with tagged random primers (T-PCR).

A very sensitive and specific method for the random amplification of whole DNA molecules and genomes ranging from 400 base pairs (bp) to 40 Megabase (Mb) is described. This simple, two step PCR (1-3) strategy utilizes tagged random primers that consist of a pool of all possible 3' sequences for binding to the target DNA and a constant 5' region for the detection of incorporated primers. As little as 10-12 g of DNA was readily amplified to amounts that could be visualized on ethidium bromide stained gels. The high specificity of this method was demonstrated by hybridization of PCR products to gridded whole genome cosmid libraries. Tagged random primer PCR (T-PCR) is very useful for the amplification of DNA samples purified by various electrophoresis techniques or flow sorting. This random whole DNA and genome amplification should simplify the analysis of other samples with very little DNA, like individual sperm and oocytes. It has the potential for amplifying any DNA molecule. Tagged random primers (4) containing a 9 to 15 bp arbitrary 3' tail that can bind to any DNA sequence and a constant 17 bp 5' head for the subsequent detection of the incorporated primer were synthesized. In the first PCR step, consisting of two or more cycles, the random 3' part of the primer is annealed to the complementary target DNA, and target specific sequences are added by the polymerase. In the second cycle of this step, molecules containing two random primers at their ends are generated. Unbound primer and primer-primer complexes are subsequently removed by spin column gel filtration at 1100 g for 10 minutes with Biogel P100 (5). During the second PCR step, molecules that have incorporated two random primer are amplified ad libitum with a single primer complementary to the constant 5' part of the tagged primer. The annealing conditions of the first step PCR were determined by the length of the random part of the primer. For example, an annealing temperature of 30°C for 1 minute was used for a primer with 9 random bases. The temperature was then raised to 400C for 1 minute and at 72°C for 2 minutes for polymerization. Samples were denatured for 30 seconds at 960C at the beginning of each PCR cycle. For the first step PCR the primer concentration was adjusted to the sample DNA concentration (see below). For the second step PCR the primer was added to …