Use of the polymerase chain reaction for physical mapping of Escherichia coli genes.

Several investigators are currently positioning published Escherichia coli DNA sequence data on the Kohara physical map (9) by computer-aided alignment of restriction patterns (13, 14, 17). To facilitate direct correlation of the E. coli K-12 physical and genetic maps, we have used a rapid method based on the amplification of specific gene products by the polymerase chain reaction (PCR). PCR products obtained from amplification of total E. coli genomic DNA were utilized as probes in hybridization experiments with a gene mapping membrane. In an alternative strategy, the primers were used directly for the PCR amplification of phage and cosmid DNAs to detect specific sequences at defined positions in ordered E. coli genomic libraries. To physically map genes of interest, we screened a mapping panel constructed from the Kohara phage collection (version 9010) that covers greater than 99% of the E. coli genome. A single filter, or gene mapping membrane, which contains a "miniset" of 476 overlapping phage clones (16), was hybridized with each specific probe. This membrane was prepared by Takara Shuzo Co., Ltd., Kyoto, Japan, and the National Institute of Genetics, Mishima, Japan, and was provided by Akihiro Noda (16). The glpK and cca coding sequences were amplified from total E. coli K-12 W3110 genomic DNA (100 ng) by PCR using the conditions described below for the phage lysates (Table 1), gel purified (6), oligolabeled (5), and hybridized to the ordered phage filter. A small amount (0.1 ng) of 32P-labeled lambda DNA was mixed with the labeled fragment at a ratio of 1:1,000 to allow one to distinguish individual background lambda phage clones. Specific phage clones from the ordered set demonstrated a hybridization signal (data not shown) which is consistent with the physical map position published previously (1) (Table 1). This technique allows one to map genes whose locations are completely unknown when minimal DNA sequence information is available for synthesis of PCR primers. Direct PCR amplification of a selected sample from the ordered phage lysate series was performed to demonstrate the feasibility and rapidity of PCR-based screening of phage lysates. For this procedure some information about the approximate chromosomal location of a specific gene is necessary. The entire phage library (version 9010) of the E. coli W3110 chromosome was obtained from Kohara as an ordered series of phage lysates (9). One microliter of each of a selected sample of lysates from the region of interest was added to the PCR tubes in a buffer containing 10% dimethyl sulfoxide (8). Reaction mixtures contained 50 pmol of each primer, 1.25 mM each of four deoxynucleoside triphosphates, and 2 U of AmpliTaq (Cetus) DNA polymerase. The PCR primers were 20to 30-mers which annealed to opposite ends of the gene of interest. The amplification conditions entailed an initial denaturation at 94°C (9 min) followed by a