Title: Improved efficiency in amplification of Escherichia coli o-antigen gene clusters using genome-wide sequence comparison Authors
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 1, 2010
Publication Date: N/A
Technical Abstract: Background: In many bacteria including E. coli, genes encoding O-antigens are clustered in the chromosome, with a 39-bp JUMPstart sequence and gnd gene located upstream and downstream of the cluster, respectively. For determining the DNA sequence of the E. coli O-antigen gene cluster, one set of PCR primers targeting the JUMPstart region and gnd gene has traditionally been used in long PCR assays. However, due to variability in the sequences among different strains, a PCR product can not always be obtained. Therefore, following sequence analyses, a more efficient primer set was designed for long PCR. Methods: Available sequences from GenBank, as well as sequences from our in-house database were compared, and new JUMPstart (NEWJump, 33 bp) and gnd (NEWgnd, 40 bp) primers were designed. Results: A comparison of the previously and newly designed primer sets in long PCR assays for amplification of the O-antigen gene clusters of E. coli O6, O11, O12, O20, O25, O44, O69, O70, O74, O75, O78, and O119 showed that there was a notable improvement in the PCR assays using the new primer set. All of the strains tested using the new primers produced a PCR product, whereas, amplification was inconsistent using the old set of primers. Conclusion: The NEWJump and NEWgnd primers were very efficient for amplification of long PCR products for O-antigen sequencing in E. coli, and potentially can also be used for other bacteria.