Gene 253 (2000) 145–150 www.elsevier.com/locate/gene Walking into the unknown: a ‘step down’ PCR-based technique leading to the direct sequence analysis of flanking genomic DNA Ziguo Zhang, Sarah Jane Gurr * Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK Received 23 May 2000; accepted 22 June 2000 Received by S. Salzberg Abstract We describe a novel and efficient PCR-based technique for walking into unknown flanking genomic DNA without recourse to protracted laborious library screening for overlapping sequences. This two component ‘hot start’ and ‘step down’ PCR method uses 6×1 mg of genomic DNA (ca 20 kb in length) restricted with six different endonucleases and ligated to adaptors with the inclusion of two further restriction enzymes to prevent self-ligation. This allowed us to walk, in a single step, up to 6 kb into flanking DNA and gave sufficient PCR products for up to 200 different walking experiments. This technology enabled us to clone and characterize the previously elusive 5∞ sequence of the barley powdery mildew chitin synthase gene, BgChs2, which includes a myosin motor-like sequence fused to a type V chitin synthase gene. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Blumeria graminis; Chitin synthase; Myosin motor 1. Introduction restriction site PCR, and in 1999, Cormack and Somssich (1997) described a method for the rapid amplification of genomic ends, and Rudi et al. (1999) Several PCR-based methodologies are available for described a technique to amplify and clone genomic walking from a known region into cloned or uncloned DNA without restriction digestion. However, some of genomic DNA, including inverse PCR (Ochman et al., these PCR-based methods have proved to be compli- 1988, Triglia et al., 1988); randomly primed PCR cated or inefficient, or to demand genomic DNA of ca (Parker et al., 1991) and adaptor ligation (Riley et al., 50 kb as the starting material. We are not able to 1990; Rosenthal and Jones, 1990; Lagerstrom et al., routinely prepare genomic DNA from Blumeria graminis 1991; Jones and Winistorfer, 1993). Siebert et al. (1995) DC f.sp. hordei Em Marchal (syn. Erysiphe graminis DC improved upon the adaptor ligation method by combin- Speer f.sp. hordei Em. Marchal ) greater than 20 kb in ing ‘vectorette PCR’ (Lagerstrom et al., 1991) with length. Indeed, the collection of tissue from this fungus ‘suppression PCR’ (Lukyanov et al., 1994), and is troublesome, and the difficulty in obtaining adequate Padegimas and Reichert (1998) described an adaptor- amounts of nucleic acids has been documented (Delye ligation PCR technique using blocked adaptors and et al., 1998). exonuclease digestion to remove unligated products. In B. graminis is a highly specialised, obligately biot- Weber et al. (1998) recorded a technique for the rapid rophic, ectopic fungal plant pathogen that causes pow- acquisition of unknown DNA sequences by multiplex dery mildew disease of barley ( Hordeum vulgare L.). It is a devastating crop pathogen that spreads by the production of millions of asexual spores or conidia from Abbreviations: aa, amino acid(s); AGT, appressorial germ tube; CHS, chitin synthase; CTAB, cetyltrimethylammoniumbromide; EST, colonial hyphae on barley. Upon initial contact with its expressed sequence tag; f.sp., formae speciales; kb, kilobase(s) or host, the conidium produces a short primary germ tube 1000 bp; ORF, open reading frame; PCR, polymerase chain reaction; (PGT ), and subsequently, a second-formed germ tube PGT, primary germ tube. emerges and becomes the appressorial germ tube * Corresponding author. Tel.: +44-1865-275813; (AGT ). Fungal penetration occurs when the AGT pro- fax +44-1865-275074. E-mail address: sarah.gurr@plant-sciences.ox.ac.uk (S.Jane Gurr) duces a peg that breaches the host epidermal wall, and 0378-1119/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0378-1119(00)00289-4