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

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 polymerase 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). Figure 1a shows the effect of extension temperature on the PCR products from four plasmid clones that contain A+T-rich P.falciparum inserts of 1–2 kb (3F3, 6F9, 3E7, 7A6). PCR amplification of each of the inserts was successful using an extension temperature of 60, but not 65 or 72 C. DNA sequences were determined for three of the four inserts, and all were found to have regions of ∼90% A+T-content extending for several hundred bp. By comparison, the P.falciparum pfhsp86 coding region, which supports PCR extension at 70 C (8), has an average A+T-content of 70% with only a single 100 bp region that approaches 80%. We calculated the effect of these different A+T contents on the melting temperatures (Tm) of the DNA sequences. Figure 1b shows the predicted melting curves for representative regions of the pfhsp86 coding region and the 3E7 insert. At a cation concentration of 0.10 M and a DNA concentration of 0.1 pM, values that correspond approximately to conditions in the early stages of PCR, the nucleotides of the pfhsp86 and 3E7 sequences have a 50% probability of being in an unpaired (open) state at ∼73 and 64 C respectively. The difference between these temperatures corresponds to the empirically determined reduction in extension temperature necessary for the amplification of the 3E7 sequence. Figure 1. Effect of temperature on the amplification and melting of A+T-rich DNA sequences. (a) Results from DNA amplifications using PCR extension temperatures of 60 and 65 C. Reactions were performed in 50 μl volumes (0.5 ml tubes) containing 1 ng plasmid DNA, 25 pmol each M13 forward and reverse primer (5′-GTAAAACGACGGCCAGT-3′, 5′-CAGGAAACAGCTATGAC-3′), 1 μl of 10 mM each dNTP, 5 μl 10× buffer (100 mM Tris–HCl/15 mM MgCl2/500 mM KCl pH 8.3, Boehringer Mannheim), and 1.5 U Taq polymerase. After initial heating at 94 C for 120 s, 20 cycles of PCR amplification were performed, each consisting of four steps: denaturation at 94 C for 20 s; annealing at 55 C for 10 s followed by 50 C for 10 s; and extension at 60 or 65 C for 120 s. Five microlitres of each amplification reaction were loaded in the agarose gel (0.8%). (b) Temperatures at which individual nucleotides of the 3E7 and pfhsp86 sequences are calculated to have a 50% probability of the open (melted) state. Computations were performed using 1000 bp sequences from the 3E7 insert (85% avg. A+T content) and from part of the pfhsp86 coding region (70% avg. A+T content); results are shown for bp 80–920 of each sequence. Temperatures were computed by the algorithm of Poland (16) as implemented by Steger (17) (program POLAND available at http://www.biophys.uni-duesseldorf.de/service/polandform.html). An ionic strength of 0.10 M NaCl and a DNA concentration of 1.0 × 10–13 M were used in the computations.