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. 1997 Oct 15;25(20):4169–4171. doi: 10.1093/nar/25.20.4169

A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP.

K K Wong 1, L M Markillie 1, J D Saffer 1
PMCID: PMC147012  PMID: 9321677

Abstract

Partial digestion of DNA fragments is a standard procedure for subcloning analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-methyl-dCTP into the PCR product to protect most of the restriction sites. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reaction done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditional methods. Furthermore, using fluorescein-labeled primers in the reaction, we were able to detect the fluorescein-labeled end fragments resulting from the enzyme digestion using a fluorimager or anti-fluorescein-AP antibody and thus determine the restriction maps.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Cook G. P., Tomilinson I. M. High resolution restriction mapping of YACs using chromosome fragmentation. Nucleic Acids Res. 1996 Apr 15;24(8):1585–1586. doi: 10.1093/nar/24.8.1585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Danna K. J. Determination of fragment order through partial digests and multiple enzyme digests. Methods Enzymol. 1980;65(1):449–467. doi: 10.1016/s0076-6879(80)65055-1. [DOI] [PubMed] [Google Scholar]
  3. Hoheisel J. D., Nizetic D., Lehrach H. Control of partial digestion combining the enzymes dam methylase and MboI. Nucleic Acids Res. 1989 Dec 11;17(23):9571–9582. doi: 10.1093/nar/17.23.9571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Löbrich M., Rydberg B., Cooper P. K. Random-breakage mapping method applied to human DNA sequences. Nucleic Acids Res. 1996 May 15;24(10):1802–1808. doi: 10.1093/nar/24.10.1802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. McClelland M., Nelson M., Raschke E. Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases. Nucleic Acids Res. 1994 Sep;22(17):3640–3659. doi: 10.1093/nar/22.17.3640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Nobile C., Romeo G. Partial digestion with restriction enzymes of ultraviolet-irradiated human genomic DNA: a method for identifying restriction site polymorphisms. Genomics. 1988 Oct;3(3):272–274. doi: 10.1016/0888-7543(88)90088-2. [DOI] [PubMed] [Google Scholar]
  7. Shields D. C., Butler A., Mosurski K. R., Walsh M. T., Whitehead A. S. Mapping genes within a YAC by computer-assisted interpretation of partial restriction digestions. Nucleic Acids Res. 1996 Nov 15;24(22):4495–4500. doi: 10.1093/nar/24.22.4495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Tartof K. D., Hobbs C. A. New cloning vectors and techniques for easy and rapid restriction mapping. Gene. 1988 Jul 30;67(2):169–182. doi: 10.1016/0378-1119(88)90394-0. [DOI] [PubMed] [Google Scholar]
  9. Watts G. S., Futscher B. W. Detecting differences in 5-methylcytosine using restriction enzyme isoschizomers: an endogenous control for complete digestion. Nucleic Acids Res. 1995 Nov 25;23(22):4740–4741. doi: 10.1093/nar/23.22.4740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Wong K. K., McClelland M. PCR with 5-methyl-dCTP replacing dCTP. Nucleic Acids Res. 1991 Mar 11;19(5):1081–1085. doi: 10.1093/nar/19.5.1081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Wong K. K., Stillwell L. C., Dockery C. A., Saffer J. D. Use of tagged random hexamer amplification (TRHA) to clone and sequence minute quantities of DNA--application to a 180 kb plasmid isolated from Sphingomonas F199. Nucleic Acids Res. 1996 Oct 1;24(19):3778–3783. doi: 10.1093/nar/24.19.3778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Yokota H., Johnson F., Lu H., Robinson R. M., Belu A. M., Garrison M. D., Ratner B. D., Trask B. J., Miller D. L. A new method for straightening DNA molecules for optical restriction mapping. Nucleic Acids Res. 1997 Mar 1;25(5):1064–1070. doi: 10.1093/nar/25.5.1064. [DOI] [PMC free article] [PubMed] [Google Scholar]

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