Cycle sequencing protocol using deep VentR (exo-) DNA polymerase and reduced dNTP and [alpha-35S]dATP concentrations.

Direct sequencing of polymerase chain reaction (PCR) products is a very powerful technique, especially for clinical investigations where quantities of biological material are often very limited and results are rapidly needed. However, numerous researchers still prefer to sequence cloned PCR fragments, a very time-consuming strategy, as most published protocols for direct sequencing of PCR products suffer from fairly serious drawbacks. Manual sequencing using exonuclease-deficient bacteriophage T7 derived DNA polymerases gives good results but requires large amounts of material: 0.5–1 pmol (corresponding to ±325 to 650 ng of a 1-kb fragment) for one set of reactions, generally providing no more than 200-bp information. In contrast, protocols using PCR technology allow the sequencing of femtomolar quantities of fragment due to the fact that template molecules are released at the beginning of each cycle and thus are again available for the synthesis of new complementary strands. However, most commercial thermostable DNA polymerases for PCR sequencing are supposed to require relatively high nucleotide concentrations, in the range of 100–200 μM. Therefore, the high concentration of nonradioactive dATP strongly competes with the commonly used [α-35S]dATP nucleotide, which consequently is poorly incorporated. The synthesized strands are thus weakly labeled, imposing long autoradiography exposure times. This is often compensated for by increasing the input of expensive radioactivity (up to 20 μCi), which has an adverse effect on waste disposal. It is, therefore, often preferable to label the sequencing primer, but this dictates the use of high-exposure (32P) or very expensive (33P) radionucleotides, and this may be very tedious if different primers are needed for the sequencing of long fragments. Here, I propose an easily implemented modification that eliminates most drawbacks of current protocols for cycle sequencing with [α-35S]dATP incorporation. This modification is based on the observation that VentR 