基因组步移法

A specific and versatile genome walking technique

Haitao Guo,Jin Xiong ⁎

Department of Biology,Texas A and M University,College Station,TX 77843,USA Received 2March 2006;received in revised form 2June 2006;accepted 15June 2006

Available online 21June 2006Received by A.Bernardi

Abstract

We describe here a nested PCR-based strategy for genome walking to extend a known sequence region to its uncharacterized flanking regions.This technique involves the use of a partially degenerate primer as a walker primer and a set of nested specific primers to perform two to three successive rounds of nested PCR.To increase the success rate of genome walking,four different walker primers were designed to allow the setup of parallel reactions.This technique was applied to amplify flanking sequences of known genomic loci of two highly divergent photosynthetic organisms,Rhodobacter capsulatus and Heliophilum fasciatum .Specific products were preferentially amplified using this strategy,which were verified using DNA sequencing.The extremely high success rate of extension of genomic regions in these two organisms suggests that this technique can be applied to a wide range of genomes.©2006Elsevier B.V .All rights reserved.

Keywords:Heliophilum fasciatum ;Nested PCR;Partial degenerate primers;Rhodobacter capsulatus ;Touchdown PCR

1.Introduction

Genome walking is a basic molecular biology technique.It involves a stepwise determination of uncharacterized DNA sequence flanking a known sequence region.This technique has many applications such as closing genome sequence gaps in the finishing phase of whole genome sequencing (Carraro et al.,2003;Rogers et al.,2005)and identification of insertion sites of transposons in gene disruption analysis (Huang et al.,2000;Levano-Garcia et al.,2005).

A number of PCR-based methods have been developed to define flanking sequences from known genomic loci.The methods essentially fall into two categories:preprocessing-dependent and preprocessing-independent.The first category requires restriction digestion and ligation of genomic DNA,which includes inverse PCR (Ochman et al.,1988;Triglia et al.,1988),ligation-mediated PCR (Pfeifer et al.,1989),vectorette PCR (Arnold and Hodgson,1991),and panhandle PCR (Jones and Winistorfer,1993).The procedures rely on successful DNA self circularization or ligation to specifically designed adaptors.

Their main limitations are the requirement of a large amount of starting material (2–10μg of DNA)and the dependence of the availability of restriction sites.

The second category is nearly entirely PCR-based and does not require restriction and ligation.These methods include universal fast walking (Myrick and Gelbart,2002),targeted gene-walking (Parker et al.,1991),and interlaced PCR (Liu and Whittier,1995).These methods rely on the binding of degenerate primers near the ends of a known sequence,which is amplified by PCR with specific primers from the known region.They have been shown to be effective under certain circumstances.Though no preproces-sing is needed,some methods in this category still require a significant level of sample handling during or after the amplification,such as purification of biotinylated intermediate products and exonuclease treatment.In many cases,high rates of non-specific PCR products are encountered.

Recently,Levano-Garcia et al.(2005)reported using a touchdown PCR protocol to amplify gene flanking regions with the use of partially degenerate primers (hybrid consensus degenerate primers)and a single specific primer.This method,belonging to the second category,requires simple manipulations and only a limited amount of starting DNA.The authors showed an example of using this strategy by mapping transposon insertion sites of Xanthomonas citri .This technique,however,based on

our

Gene 381(2006)18–

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Abbreviations:nt,nucleotide;Tm,melting temperatures of oligonucleotides.⁎Corresponding author.Tel.:+9794583462;fax:+9798452891.E-mail address:jxiong@ (J.Xiong).

0378-1119/$-see front matter ©2006Elsevier B.V .All rights reserved.doi:10.1016/j.gene.2006.06.002