Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA

Abstract

A typical PCR cycle includes an extension step at 72°C after denaturation of double-stranded DNA and annealing of oligonucleotide primers. At this temperature the thermostable poly-merase replicates the DNA at an optimal rate that depends on the buffer and nature of the DNA template ( 1 ). Although the sizes of the fragments that can be amplified have been generally limited to Taq + Pfu ) allows replication and amplification of much larger fragments, including a 42 kb sequence from the bacteriophage l genome (long PCR) ( 3 , 4 ). This ability to amplify genomic DNA in vitro is of particular importance to studies of Plasmodium falciparum , as large DNA fragments from this malaria parasite are generally unstable in Escherichia coli ( 5 ). A common finding with P.falciparum DNA, however, is that even small fragments (5 kb).