JOURNAL OF BACTERIOLOGY, Sept. 1991, p. 5253-5255 0021-9193/91/175253-03$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 173, No. 17

Use of the Polymerase Chain Reaction for Physical Mapping of Escherichia coli Genes JAMES VERSALOVIC,' THEARITH KOEUTH,l EDWARD R. B. McCABE,"2 AND JAMES R. LUPSKIl 2* Institute for Molecular Genetics' and Department of Pediatrics,2 Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030-3498

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 20- to 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

TABLE 1. Gene-specific primer sets and conditions used to identify genomic clones for physical mapping Gene (reference)

cca (4)

(+) 5'-TGCTGTTCGGGATGCATTGTTAGGG-3'

dnaG (18)

(-)5'-TTTGGGCAACGTTGTTCCTTCCAGC-3' (+) 5'-GAATTGCTAAAAATCGGGGCCT-3'

glpK (11) parC (7) parE (7)

rpoD (2) rpsU (3, 10)

toiC (15)

Phage PCR (anneal; extend; no. of cycles)

PCR primersa

(-)5'-GCCAACGATAATTACGAGGGCG-3' (+) 5'-AAGCTTCGCTGTAATATGACTA-3' (-) 5'-TTATTCGTCGTGTTCTTCCCAC-3' (+) 5'-GACCGTGCGTTGCCGTTTATTGGTG-3' (-)5'-TATCACCGCTGCTGGCACGGCGAGG-3' (+) 5'-GCTGATGCCATTGAGGTACTCACCG-3' (-)5'-ACTGCACCAGACGGCGAGTGTTCGG-3' (+) 5'-ACITAIGCIACITGGTGGATCIGICAGGC-3' (-) (+) (-) (+) (-)

5'-TTIGCTTCGATITGICGIATACG-3' 5'-ACGAGCCGTTCGACGTAGCTCTGCG-3' 5'-CACGCGTGGAAATTCTTGCC-3'

5'-CTCCCCATTCTTATCGGCCTGAGCC-3' 5'-AAAGGGTTATGACCGTTACTGGTGG-3'

56°C, 1 min; 65°C, 8 min; 28 55°C, 1 min; 65°C, 8 min; 30

3258

19F2 (509)

4H12b (539)

E329, E405&

3588

E4541, E1204

3216-3221

E4541, E1204

3227-3233

ND

3268-3270

70°C,

6H4 (505), 17B2 (506) 17B2 (506), 6B12 (507) 19F2 (509), 17E4 (510) 19F2 (509)

ND

3265

65°C,

6B12 (507)

E1204

3233-3240

65°C, 65°C,

70'C,

a(+), sense strand; (-), antisense strand. Also identified by hybridization of PCR fragments amplified from total W3110 chromosomal DNA.

b

c *

ND, not determined.

Corresponding author. 5253

9F9b (508)

E1204, E716, E4177 NDc

59°C, 1 min; 68°C, 5 min; 24 56"C, 1 min; 8 min; 42 56°C, 1 min; 8 min; 42 55°C, 1 min; 5 min; 30 50°C, 1 min; 5 min; 28 56°C, 1 min; 8 min; 28

Physical map KhrTata position cosmid phage phg omd(kb)

3266-3267

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FIG. 1. The Kohara lambda phages used are listed by clone numbers (9), and miniset serial numbers are shown in parentheses. In the positive control lane the template is W3110 genomic DNA; no template DNA was added to the negative control lanes. The DNA molecular weight marker is a 1-kb ladder (BRL). The figure shows PCR amplification products of the expected sizes from the lambda phages containing cca (A), dnaG (B), glpK (C), and tolC (D), while no amplification product is seen from adjacent phages. Similar results were obtained for parC, parE, rpsU, and rpoD (data not shown). The gels were 1% agarose-1 x Tris-acetate-EDTA and contained 0.5 ,ug of ethidium bromide per ml.

variable number of cycles of 90°C denaturation (1 min) and variable annealing and extension conditions (Table 1), depending on the primer set used, in an automated thermal cycler (Perkin-Elmer/Cetus). As shown in Fig. 1, only the expected phage lysates amplified a single fragment of the predicted size. It is important to emphasize that the lysates were not pretreated in any way. The time required to proceed from lysate to PCR products visible on a gel spanned only several hours, and the technique bypassed the need for radioisotope. An ordered cosmid library has been recently established in E. coli W3110 and was obtained from Tabata et al. (19). Relevant cosmid clones from each region of interest were selected and specific genes were amplified by using purified cosmid DNA and gene-specific primers. PCR conditions consisted of an initial denaturation at 94°C (9 min) followed by 30 cycles of 90°C denaturation (1 min), 60°C annealing (1 min), and 70°C extension (5 min) in an automated thermal cycler (Perkin-Elmer/Cetus). Specific cosmids corresponding to the genes of interest were amplified and are shown in Table 1. The cosmids in this genomic library were originally positioned relative to each other on the genetic map by hybridization and complementation (19). PCR data obtained by the amplification of the cosmid library are consistent with the physical map positions on the Kohara map (9). The cosmid library, partially due to its larger insert size, provides more potential for overlap and hence a greater probability of multiple positive clones. A careful examination of these data is warranted since the PCR may lead to the amplification of

sequences present on contaminating E. coli chromosomal DNA that remains after the cosmid preparation procedure. These results establish the feasibility of using PCR technology in direct physical mapping approaches of known genes on the E. coli chromosome when DNA sequence information is available. The utility of this approach was demonstrated with both the phage and cosmid ordered libraries of E. coli W3110.

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