Two step single primer mediated polymerase chain reaction. Application to cloning of putative mouse, beta-galactoside...

Using the 2 step single primer mediated polymerase chain reaction(PCR), mouse beta-galactoside alpha 2,6-sialyltransferase cDNA was cloned. Single primer mediated PCR is a method to amplify a particular DNA fragment beyond its known sequence region. It employs only one primer for the reaction. Compared to other PCR methods to amplify an adjacent sequence of known DNA fragment, this method requires no enzymatic manipulation on template DNA and is applicable to a template on long DNA fragment. First, a short DNA fragment of the enzyme was obtained from mouse cDNA by the usual PCR method using degenerate primers synthesized according to a relatively conserved region in rat and human beta-galactoside alpha 2,6-sialyltransferase. Four primers were synthesized based on this sequence, then 2 step single primer mediated PCR were performed to obtain 5' and 3' flanking sequences of this short fragment resulting in 1.0 kb and 1.3 kb fragments being amplified respectively. The integrity of the two fragments was confirmed by an additional PCR using primers synthesized according to the joined sequence, which contained 1.2kb complete putative mouse beta-galactoside alpha 2,6-sialyltransferase coding region. The result showed that the specificity and consequently applicability of the single primer mediated PCR for amplifying a particular DNA fragment beyond known sequence region was remarkably improved by the successive 2nd reaction.