Utility of MultiNA in PCR Direct Sequencing

Introduction

PCR direct sequencing is a technique in which, rather than cloning the amplification products obtained as a result of the PCR reaction, they are used directly as a template for determining the base sequence. This is a very effective method because it allows base sequence information to be obtained quickly and reduces the influence of base take-up mistakes following cloning. However, those results may vary widely depending on the amplification situation following PCR. In order to increase that success rate, it is necessary to verify the purity (quality and quantity) of the PCR products prior to the sequencing reaction. That verification has traditionally been conducted using agarose gel electrophoresis. However, there are cases in which the sequence data obtained in the sequence analysis indicates the possibility of closely related substrates for reaction, even when it appears that there is only a single target PCR product. One possible cause for this is the limited sensitivity of agarose gel electrophoresis. With this method, it is difficult to elucidate the existence of trace amounts of PCR side reaction products among the principal reaction products. Here, we introduce a PCR product purity assay in a PCR direct sequencing process conducted in the search for the housekeeping genes of cruciferae plant family by the Research Institute for Biological Sciences, Okayama Prefectural Technology Center for Agriculture, Forestry, and Fisheries.

November 22, 2011 GMT