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. 1992 Feb;50(2):331–346.

Detection of DNA sequence polymorphisms in human genomic DNA by using denaturing gradient gel blots.

M R Gray 1
PMCID: PMC1682447  PMID: 1310376

Abstract

Denaturing gradient gel electrophoresis can detect sequence differences outside restriction-enzyme recognition sites. DNA sequence polymorphisms can be detected as restriction-fragment melting polymorphisms (RFMPs) in genomic DNA by using blots made from denaturing gradient gels. In contrast to the use of Southern blots to find sequence differences, denaturing gradient gel blots can detect differences almost anywhere, not just at 4-6-bp restriction-enzyme recognition sites. Human genomic DNA was digested with one of several randomly selected 4-bp recognition-site restriction enzymes, electrophoresed in denaturing gradient gels, and transferred to nylon membranes. The blots were hybridized with radioactive probes prepared from the factor VIII, type II collagen, insulin receptor, beta 2-adrenergic receptor, and 21-hydroxylase genes; in unrelated individuals, several RFMPs were found in fragments from every locus tested. No restriction map or sequence information was used to detect RFMPs. RFMPs can be used as genetic markers, because their alleles segregate in a Mendelian manner. Unlike most other methods for detecting DNA sequence polymorphisms, a genomic DNA blot made from one gel can be hybridized consecutively with many (30 or more) different probes.

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Selected References

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

  1. Abrams E. S., Murdaugh S. E., Lerman L. S. Comprehensive detection of single base changes in human genomic DNA using denaturing gradient gel electrophoresis and a GC clamp. Genomics. 1990 Aug;7(4):463–475. doi: 10.1016/0888-7543(90)90188-z. [DOI] [PubMed] [Google Scholar]
  2. Ala-Kokko L., Prockop D. J. Completion of the intron-exon structure of the gene for human type II procollagen (COL2A1): variations in the nucleotide sequences of the alleles from three chromosomes. Genomics. 1990 Nov;8(3):454–460. doi: 10.1016/0888-7543(90)90031-o. [DOI] [PubMed] [Google Scholar]
  3. Antonarakis S. E., Youssoufian H., Kazazian H. H., Jr Molecular genetics of hemophilia A in man (factor VIII deficiency). Mol Biol Med. 1987 Apr;4(2):81–94. [PubMed] [Google Scholar]
  4. Bell G. I., Karam J. H., Rutter W. J. Polymorphic DNA region adjacent to the 5' end of the human insulin gene. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5759–5763. doi: 10.1073/pnas.78.9.5759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bernatzky R., Tanksley S. D. Toward a saturated linkage map in tomato based on isozymes and random cDNA sequences. Genetics. 1986 Apr;112(4):887–898. doi: 10.1093/genetics/112.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cheah K. S., Stoker N. G., Griffin J. R., Grosveld F. G., Solomon E. Identification and characterization of the human type II collagen gene (COL2A1). Proc Natl Acad Sci U S A. 1985 May;82(9):2555–2559. doi: 10.1073/pnas.82.9.2555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cotton R. G., Rodrigues N. R., Campbell R. D. Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4397–4401. doi: 10.1073/pnas.85.12.4397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cox N. J., Spielman R. S., Kahn C. R., Müller-Wieland D., Kriauciunas K. M., Taub R. Four RFLPs of the human insulin receptor gene: PstI, KpnI, RsaI (2 RFLPs). Nucleic Acids Res. 1988 Aug 25;16(16):8204–8204. doi: 10.1093/nar/16.16.8204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Donis-Keller H., Green P., Helms C., Cartinhour S., Weiffenbach B., Stephens K., Keith T. P., Bowden D. W., Smith D. R., Lander E. S. A genetic linkage map of the human genome. Cell. 1987 Oct 23;51(2):319–337. doi: 10.1016/0092-8674(87)90158-9. [DOI] [PubMed] [Google Scholar]
  12. Drayna D., White R. The genetic linkage map of the human X chromosome. Science. 1985 Nov 15;230(4727):753–758. doi: 10.1126/science.4059909. [DOI] [PubMed] [Google Scholar]
  13. Elbein S. C., Corsetti L., Ullrich A., Permutt M. A. Multiple restriction fragment length polymorphisms at the insulin receptor locus: a highly informative marker for linkage analysis. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5223–5227. doi: 10.1073/pnas.83.14.5223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  15. Fischer S. G., Lerman L. S. Two-dimensional electrophoretic separation of restriction enzyme fragments of DNA. Methods Enzymol. 1979;68:183–191. doi: 10.1016/0076-6879(79)68013-8. [DOI] [PubMed] [Google Scholar]
  16. Gitschier J., Drayna D., Tuddenham E. G., White R. L., Lawn R. M. Genetic mapping and diagnosis of haemophilia A achieved through a BclI polymorphism in the factor VIII gene. 1985 Apr 25-May 1Nature. 314(6013):738–740. doi: 10.1038/314738a0. [DOI] [PubMed] [Google Scholar]
  17. Gitschier J., Wood W. I., Goralka T. M., Wion K. L., Chen E. Y., Eaton D. H., Vehar G. A., Capon D. J., Lawn R. M. Characterization of the human factor VIII gene. Nature. 1984 Nov 22;312(5992):326–330. doi: 10.1038/312326a0. [DOI] [PubMed] [Google Scholar]
  18. Gray M., Charpentier A., Walsh K., Wu P., Bender W. Mapping point mutations in the Drosophila rosy locus using denaturing gradient gel blots. Genetics. 1991 Jan;127(1):139–149. doi: 10.1093/genetics/127.1.139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hodgson C. P., Fisk R. Z. Hybridization probe size control: optimized 'oligolabelling'. Nucleic Acids Res. 1987 Aug 11;15(15):6295–6295. doi: 10.1093/nar/15.15.6295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hofker M. H., Skraastad M. I., Bergen A. A., Wapenaar M. C., Bakker E., Millington-Ward A., van Ommen G. J., Pearson P. L. The X chromosome shows less genetic variation at restriction sites than the autosomes. Am J Hum Genet. 1986 Oct;39(4):438–451. [PMC free article] [PubMed] [Google Scholar]
  21. Kobilka B. K., Dixon R. A., Frielle T., Dohlman H. G., Bolanowski M. A., Sigal I. S., Yang-Feng T. L., Francke U., Caron M. G., Lefkowitz R. J. cDNA for the human beta 2-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor. Proc Natl Acad Sci U S A. 1987 Jan;84(1):46–50. doi: 10.1073/pnas.84.1.46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kobilka B. K., Frielle T., Dohlman H. G., Bolanowski M. A., Dixon R. A., Keller P., Caron M. G., Lefkowitz R. J. Delineation of the intronless nature of the genes for the human and hamster beta 2-adrenergic receptor and their putative promoter regions. J Biol Chem. 1987 May 25;262(15):7321–7327. [PubMed] [Google Scholar]
  23. Lerman L. S., Silverstein K., Grinfeld E. Searching for gene defects by denaturing gradient gel electrophoresis. Cold Spring Harb Symp Quant Biol. 1986;51(Pt 1):285–297. doi: 10.1101/sqb.1986.051.01.034. [DOI] [PubMed] [Google Scholar]
  24. Maniatis T., Jeffrey A., van deSande H. Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry. 1975 Aug 26;14(17):3787–3794. doi: 10.1021/bi00688a010. [DOI] [PubMed] [Google Scholar]
  25. Miller W. L., Morel Y. The molecular genetics of 21-hydroxylase deficiency. Annu Rev Genet. 1989;23:371–393. doi: 10.1146/annurev.ge.23.120189.002103. [DOI] [PubMed] [Google Scholar]
  26. Myers R. M., Fischer S. G., Maniatis T., Lerman L. S. Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis. Nucleic Acids Res. 1985 May 10;13(9):3111–3129. doi: 10.1093/nar/13.9.3111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nakamura Y., Leppert M., O'Connell P., Wolff R., Holm T., Culver M., Martin C., Fujimoto E., Hoff M., Kumlin E. Variable number of tandem repeat (VNTR) markers for human gene mapping. Science. 1987 Mar 27;235(4796):1616–1622. doi: 10.1126/science.3029872. [DOI] [PubMed] [Google Scholar]
  28. Orita M., Iwahana H., Kanazawa H., Hayashi K., Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2766–2770. doi: 10.1073/pnas.86.8.2766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sangiorgi F. O., Benson-Chanda V., de Wet W. J., Sobel M. E., Tsipouras P., Ramirez F. Isolation and partial characterization of the entire human pro alpha 1(II) collagen gene. Nucleic Acids Res. 1985 Apr 11;13(7):2207–2225. doi: 10.1093/nar/13.7.2207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Seino S., Seino M., Nishi S., Bell G. I. Structure of the human insulin receptor gene and characterization of its promoter. Proc Natl Acad Sci U S A. 1989 Jan;86(1):114–118. doi: 10.1073/pnas.86.1.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sheffield V. C., Cox D. R., Lerman L. S., Myers R. M. Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci U S A. 1989 Jan;86(1):232–236. doi: 10.1073/pnas.86.1.232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  33. Strom C. M. A three allele restriction fragment length polymorphism within the human Col2A1 gene. Nucleic Acids Res. 1988 Sep 26;16(18):9077–9077. doi: 10.1093/nar/16.18.9077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sykes B., Smith R., Vipond S., Paterson C., Cheah K., Solomon E. Exclusion of the alpha 1(II) cartilage collagen gene as the mutant locus in type IA osteogenesis imperfecta. J Med Genet. 1985 Jun;22(3):187–191. doi: 10.1136/jmg.22.3.187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Traystman M. D., Higuchi M., Kasper C. K., Antonarakis S. E., Kazazian H. H., Jr Use of denaturing gradient gel electrophoresis to detect point mutations in the factor VIII gene. Genomics. 1990 Feb;6(2):293–301. doi: 10.1016/0888-7543(90)90569-g. [DOI] [PubMed] [Google Scholar]
  36. Ullrich A., Bell J. R., Chen E. Y., Herrera R., Petruzzelli L. M., Dull T. J., Gray A., Coussens L., Liao Y. C., Tsubokawa M. Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. 1985 Feb 28-Mar 6Nature. 313(6005):756–761. doi: 10.1038/313756a0. [DOI] [PubMed] [Google Scholar]
  37. Urabe K., Kimura A., Harada F., Iwanaga T., Sasazuki T. Gene conversion in steroid 21-hydroxylase genes. Am J Hum Genet. 1990 Jun;46(6):1178–1186. [PMC free article] [PubMed] [Google Scholar]
  38. Weber J. L., May P. E. Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am J Hum Genet. 1989 Mar;44(3):388–396. [PMC free article] [PubMed] [Google Scholar]
  39. White P. C., New M. I., Dupont B. Structure of human steroid 21-hydroxylase genes. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5111–5115. doi: 10.1073/pnas.83.14.5111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wood W. I., Capon D. J., Simonsen C. C., Eaton D. L., Gitschier J., Keyt B., Seeburg P. H., Smith D. H., Hollingshead P., Wion K. L. Expression of active human factor VIII from recombinant DNA clones. Nature. 1984 Nov 22;312(5992):330–337. doi: 10.1038/312330a0. [DOI] [PubMed] [Google Scholar]

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