Reduced extension temperatures required for PCR amplification of extremely AT-rich DNA

A typical PCR cycle includes an extension step at 72C after denaturation of double-stranded DNA and annealing of oligo-nucleotide 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 <5 kb (2), recent reports have shown that a blend of two polymerases (Taq + Pfu) allows replication and amplification of much larger fragments, including a 42 kb sequence from the bacteriophage λ 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 (<2 kb) can be difficult or impossible to amplif