Direct Sequencing of PCR Products Using Chemiluminescent Detection

Abstract

The following protocol describes a method that can be used for the direct sequencing of polymerase chain reaction (PCR) products using nonradioactive detection procedures. It is based on our experience with a method we have previously published (1). Direct sequencing of PCR products is generally more difficult than sequencing single-stranded DNA (2) owing to the presence of primers and deoxynucleoside triphosphates (dNTPs) carried over from the PCR into the sequencing phase and by the rapid reannealing of the double-stranded DNA template. These problems are addressed in our method in the following manner:

• 1. Excess of primers: The same primers used in the PCR are used in the sequencing of the DNA. Speck sequencing products are then detected with biotinylated probes using multiplexing and chemiluminescent procedures. This overcomes the need to use PCR products free of primers (3) or complicated procedures such as those using nested primers (4).

• 2. Excess of dNTPs: Excess dNTPs carried over from the PCR into the sequencing reaction alter the dideoxynucleoside triphosphate (ddNTPs) to dNTP ratio and hence the optimum conditions for sequencing PCR products (5). This can be overcome by using limited amounts of dNTPs in the PCR or, if necessary, a simple gel purification procedure. The latter procedure is necessary if the PCR is relatively inefficient at low dNTP concentrations or is difficult to optimize to produce a specific product.

• 3. Reannealing of double-stranded DNA templates. Reannealing of template DNA reduces the efficiency of sequencing of DNA products. This is overcome by cycle sequencing using Taq polymerase.