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Inosine-Containing Primers 367
54
Direct Sequencing with Highly Degenerate
and Inosine-Containing Primers
Zhiyuan Shen, Jingmei Liu, Robert L. Wells, and Mortimer M. Elkind
1. Introduction
Among the many techniques of cloning new genes, one approach involves degenerate
primers (1–7). 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. 2,4). 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 (2,7,8).
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.
Sequencing the amplified PCR product is one of the most important steps in this
approach to gene cloning. To sequence the PCR fragment amplified by degenerate
inosine-containing primers, the PCR fragment may be cloned into a sequencing vector,
such as M13 bacteriophage. Sequencing is straightforward if primers specific to the
vector are used. Theoretically, this method allows any unknown cloned DNA fragment
to be sequenced. However, the Taq polymerase, which is used to amplify the target
fragment, is thought to have relatively high misincorporation rates for dNTPs, or ~10 –4 .
Hence, it is possible that a copy of the product may contain one or more incorrect
nucleotides. If such a copy has been cloned into the sequencing vector, the resulting
sequencing data would be incorrect for that particular clone. Direct sequencing of PCR
products can circumvent this problem because most of the fragments are exact replicas
of the target molecule. Thus, the majority of the products used for sequencing would
have the right nucleotide at a specified position and result in the correct sequencing
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
367