Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1994 Oct 11;22(20):4321–4325. doi: 10.1093/nar/22.20.4321

A program for selecting DNA fragments to detect mutations by denaturing gel electrophoresis methods.

S Brossette 1, R M Wartell 1
PMCID: PMC331956  PMID: 7937161

Abstract

A computer program was developed to automate the selection of DNA fragments for detecting mutations within a long DNA sequence by denaturing gel electrophoresis methods. The program, MELTSCAN, scans through a user specified DNA sequence calculating the melting behavior of overlapping DNA fragments covering the sequence. Melting characteristics of the fragments are analyzed to determine the best fragment for detecting mutations at each base pair position in the sequence. The calculation also determines the optimal fragment for detecting mutations within a user specified mutational hot spot region. The program is built around the statistical mechanical model of the DNA melting transition. The optimal fragment for a given position is selected using the criteria that its melting curve has at least two steps, the base pair position is in the fragment's lowest melting domain, and the melting domain has the smallest number of base pairs among fragments that meet the first two criteria. The program predicted fragments for detecting mutations in the cDNA and genomic DNA of the human p53 gene.

Full text

PDF
4321

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. Børresen A. L., Hovig E., Smith-Sørensen B., Malkin D., Lystad S., Andersen T. I., Nesland J. M., Isselbacher K. J., Friend S. H. Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8405–8409. doi: 10.1073/pnas.88.19.8405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cheng J., Haas M. Frequent mutations in the p53 tumor suppressor gene in human leukemia T-cell lines. Mol Cell Biol. 1990 Oct;10(10):5502–5509. doi: 10.1128/mcb.10.10.5502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Costes B., Girodon E., Ghanem N., Chassignol M., Thuong N. T., Dupret D., Goossens M. Psoralen-modified oligonucleotide primers improve detection of mutations by denaturing gradient gel electrophoresis and provide an alternative to GC-clamping. Hum Mol Genet. 1993 Apr;2(4):393–397. doi: 10.1093/hmg/2.4.393. [DOI] [PubMed] [Google Scholar]
  5. Delcourt S. G., Blake R. D. Stacking energies in DNA. J Biol Chem. 1991 Aug 15;266(23):15160–15169. [PubMed] [Google Scholar]
  6. Fixman M., Freire J. J. Theory of DNA melting curves. Biopolymers. 1977 Dec;16(12):2693–2704. doi: 10.1002/bip.1977.360161209. [DOI] [PubMed] [Google Scholar]
  7. Ke S. H., Kelly P. J., Wartell R. M., Hunter S., Varma V. A. Selecting DNA fragments for mutation detection by temperature gradient gel electrophoresis: application to the p53 gene cDNA. Electrophoresis. 1993 Jul;14(7):561–565. doi: 10.1002/elps.1150140188. [DOI] [PubMed] [Google Scholar]
  8. Lerman L. S., Fischer S. G., Hurley I., Silverstein K., Lumelsky N. Sequence-determined DNA separations. Annu Rev Biophys Bioeng. 1984;13:399–423. doi: 10.1146/annurev.bb.13.060184.002151. [DOI] [PubMed] [Google Scholar]
  9. Lerman L. S., Silverstein K. Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis. Methods Enzymol. 1987;155:482–501. doi: 10.1016/0076-6879(87)55032-7. [DOI] [PubMed] [Google Scholar]
  10. McCampbell C. R., Wartell R. M., Plaskon R. R. Inverted repeat sequences can influence the melting transitions of linear DNAs. Biopolymers. 1989 Oct;28(10):1745–1758. doi: 10.1002/bip.360281008. [DOI] [PubMed] [Google Scholar]
  11. Myers R. M., Lerman L. S., Maniatis T. A general method for saturation mutagenesis of cloned DNA fragments. Science. 1985 Jul 19;229(4710):242–247. doi: 10.1126/science.2990046. [DOI] [PubMed] [Google Scholar]
  12. Nigro J. M., Baker S. J., Preisinger A. C., Jessup J. M., Hostetter R., Cleary K., Bigner S. H., Davidson N., Baylin S., Devilee P. Mutations in the p53 gene occur in diverse human tumour types. Nature. 1989 Dec 7;342(6250):705–708. doi: 10.1038/342705a0. [DOI] [PubMed] [Google Scholar]
  13. Rosenbaum V., Riesner D. Temperature-gradient gel electrophoresis. Thermodynamic analysis of nucleic acids and proteins in purified form and in cellular extracts. Biophys Chem. 1987 May 9;26(2-3):235–246. doi: 10.1016/0301-4622(87)80026-1. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Wartell R. M., Hosseini S. H., Moran C. P., Jr Detecting base pair substitutions in DNA fragments by temperature-gradient gel electrophoresis. Nucleic Acids Res. 1990 May 11;18(9):2699–2705. doi: 10.1093/nar/18.9.2699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Yoshino K., Nishigaki K., Husimi Y. Temperature sweep gel electrophoresis: a simple method to detect point mutations. Nucleic Acids Res. 1991 Jun 11;19(11):3153–3153. doi: 10.1093/nar/19.11.3153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Zakut-Houri R., Bienz-Tadmor B., Givol D., Oren M. Human p53 cellular tumor antigen: cDNA sequence and expression in COS cells. EMBO J. 1985 May;4(5):1251–1255. doi: 10.1002/j.1460-2075.1985.tb03768.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES