Direct sequencing with highly degenerate and inosine-containing primers.

Abstract

Among the many techniques of cloning new genes, one approach involves degenerate primers (, , , , , , ). The approach usually requires the following three steps: 1. Using degenerate primers to amplify part of the gene of interest by PCR: The degenerate primers’ sequences may be designed from known protein sequences or conserved regions of a gene family (e.g., ref. ,). Because deoxyinosine can base pair with all of the four deoxyribonucleotides, it has been substituted for specific nucleic acids in degenerate primers to reduce the number of different primer sequences that would otherwise be needed in the reaction (,,). 2. A determination of which amplified PCR product(s) is from the gene of interest: If the target gene and the primers are only partially homologous, a moderate annealing stringency in PCR is usually necessary to obtain amplification. Moderate stringency may result in multiple PCR products. Although from the size of the PCR products it may be possible to predict which is from the gene of interest, sequencing analysis of the PCR products may be required. 3. The screening of a cDNA library using the correct PCR product as a probe and cloning the gene of interest.