PRODUCT REVIEW

Genomic footprinting with Taq

H. P. Sa/uz and J. P. Jost

The linear amplification of chemically sequenced DNA with DNA polymerase from aquaticus permits the direct detection of DNA methylation and protein-DNA interaction sites in vivo.

DNA methylation and protein-DNA Total genomic DNA fragments to produce many radioactively interactions play a crucial role in gene Restriction' digestion labelled copies. The resulting increase in regulation''. Genomic sequencing - Chemical sequencing' reactions sensitivity allows the more rapid detection pioneered by Church and Gilbert', and A B of single-copy mammalian genes, and further refined in our laboratory' - Linear amplification~-- Sequencing gel should permit the investigation of such enables the study of the methylation state with I complex genomes as those of higher and labeled primer Electrotransfer of any gene in a strand- and sequence­ to a nylon plants. The technique retains all of the specific manner. membrane benefits of the original genomic sequenc­ I When used to investigate areas of Fixing DNA onto ing procedure. The unique I membrane protein/DNA interaction, the technique Sequencing gel of Taq polymerase permits the optimal is referred to as genomic footprinting. In Hybridisation' with cloned annealing of the primer to the denatured this technique, cells in suspension are single-stranded probe target template, and its faithful elonga­ treated with dimethylsulphate (DMS) or l tion. But so far, procedures for studying Fix and dry gel Processing of membrane ultraviolet light to modify specific DNA ' DNA methylation are still based on the bases, which undergo a quantitative Autoradiography detection of chemical cleavage products. cleavage reaction (f)-eliminationf'. The FIG. 1 Flow diagram for performing genomic 5-Methylcytosine reacts poorly with modified sites arc then identified by --- hydrazine, resulting in a lack of cleavage experiments using the new expedited pro• genomic sequencing. Proteins bound to cedure (a), and the classical method of upon alkaline treatment. The position of DNA protect these areas from modifica­ genomic sequencing (b). the 5-methylcytosine is then revealed by a tion by DMS and subsequent chemical gap in the sequence ladder. cleavage, leaving their "footprint" on the DNA in the reaction mixture, less expen­ Because chemical cleavage of the DNA DNA. In contrast, ultraviolet light cleaves sive cndonucleases can be used, as long as results in fragments of different lengths, at sites of protein- DNA interaction. they do not cleave within the target it is only possible to amplify each frag­ Despite the potential of in vivo foot­ sequence. ment linearly. But if procedures were printing for revealing protein-DNA But the success of our technique is available quantitatively to deaminate interactions and genomic sequencing for highly dependent upon the purity of the 5-methylcytosine residues to produce detecting modifications to the DNA's Taq polymerase used, which can vary thymidine, the modified DNA could be native state, the current procedure is from batch to batch. The recent commer­ amplified exponentially by the popularly rather time-consuming and complex. We cial availability of recombinant Taq poly­ used symmetrical amplification procedure sought therefore to simplify the technique merase should eliminate this problem. of the polymerase chain reaction (PCR)" ". and make it more accessible to less experi­ [Editor's note: Recombinantly produced Following dideoxy sequencing, the posi­ enced users'. A flow diagram of our new Taq polymerase can be purchased from tion of the original 5-methylcytosine method is shown in Fig. la. Perkin-Elmer Cetus and United States would be detected by the presence of an Biochemical Corporation. Purified natural A-T pair instead of a G-C pair in the Abbreviated strategy Taq is sold by Perkin-Elmer Cetus, Strata­ undeaminated control DNA. We are The first steps of both the old method and gene, BRL, Beckman Instruments, and now investigating this approach, because our new technique are the same: total Promega.] it would speed up the procedure genomic DNA is digested with a suitable Our simplified procedure for studying enormously. 0 restriction , and the resulting the native state of genomic DNA relies on DNA fragments are chemically sequenced the linear amplification of cleaved DNA Hanspeter Saluz and Jean-Pierre Jost are at as described by Maxam and Gilbert", the Friedrich Miescher-lnstitut, PO Box 2543, CH-4002, Basel, Switzerland. For more infor• Fritzsche eta/."', or Rubin and Schmid". c c c 5' ---+---+-+---- 3' mation, fill in reader service number 100. But in our technique, sequencing is fol­ C-specific ~ reaction lowed by selective, linear amplification 5'--- 1. Raz1n, A. Cedar, H. & R1ggs, A.D. DNA Methylation. 392 with the thermostable DNA polymerase 5'------(Springer. New York. 1984). from Thermus aquatic us, using a synthetic 2. Adams, R.L.P. & Burdon. R.H. of DNA 5'------Methylation. 247 (Springer, NewYork.1985). primer labelled to a very high specific ---3' 3. Church. G.M. & Gilbert, W. Proc. natn. Acad. Sc1. U.S.A radioactivity. The products of the linear -----3' 81, 1991-1995 (1984). 4. Saluz. H.P. & Jost. J.P. Gene 42. 151-157 (1986); A amplification (Fig. 2) are then run on a ------3' Laboratory Guide to Genomic Sequencing. 163 (Birk• sequencing gel, and the sequence infor­ Linear amplification l with labeled primer hauser. Boston, 1987). and Taq polymerase 5. Becker, P.B., Ruppert. S. & Schutz. G. Ce/151. 435-443 mation is directly obtained by exposing ...... 3' (1987). the dried gel to an X-ray film...... 3' 6. Saluz. H.P .. Feavers. I.M .. Jiricny. J. & Jost. J.P. Proc ...... 3' natn. Acad. Sc1. U.S.A. 85.6697-6700 (1988) . 7. Becker, M.M. &Wang. J.C. Nature309. 795-798 (1984). A step forward FIG. 2 A diagram illustrating the linear amplifi• 8. Saluz, H.P. & Jost. J.P. Proc. natn. Acad. Sci. U.S.A. (in The new technique permits sequence cation of the sequencing reaction products. the press). -digested DNA is first sub• 9. Maxam. A.M. & Gilbert. W. Meth. Enzym. 65. 499- 560 information to be gathered for any gene, (1980) jected to chemical sequencing reactions. The regardless of its copy number and the size 10. Fritzsche, E , Hayatsu. H., lgloi. G.L .. I ida. S. & Kosse I. of the total genome, without electroblot­ fragments generated by ['\-elimination are H. Nucleic Ac1ds Res. 15, 5517-5528 (1987) then annealed to labelled 11. Rubin. C.M. & Schmid. C.W. Nucleic Acids Res. 8. 4613- ting or hybridization steps (Fig. 1 b). primers (black squares), and the complemen• 4619 (1980). Moreover, as restriction are used 12 Saiki. R.K. eta/. Science230.1350-1354 (1985). tary strand (dotted line) is synthesized by Taq 13. Erlich. H.A , Gelfand. D.H. & Saiki. R K Nature331. 461- only to reduce the viscosity of the genomic polymerase. 462 (1988) NATURE ·VOL 338 · 16 MARCH 1989 277