Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1991 Jul 25;19(14):3929–3933. doi: 10.1093/nar/19.14.3929

Single-base mutational analysis of cancer and genetic diseases using membrane bound modified oligonucleotides.

Y Zhang 1, M Y Coyne 1, S G Will 1, C H Levenson 1, E S Kawasaki 1
PMCID: PMC328485  PMID: 1861985

Abstract

A convenient format for the detection of PCR amplified sequences is the hybridization of the PCR products to oligonucleotide probes which are immobilized on a solid phase. We describe a new method for site-specific attachment of such probe oligonucleotides to nylon membranes. The method is based on the formation of an amide bond between carboxyl groups present on the membranes and amino-linkers situated on the 5' end of the oligonucleotides. The covalent attachment is via a carbodiimide mediated condensation. The single, 5' end attachment of the oligonucleotides to the membrane surface leaves the probe free to interact with complementary sequences, thus increasing the hybridization efficiency relative to methods where heat or ultraviolet light is used for non-specific fixation. Using biotinylated PCR products in hybridization reactions along with a non-radioactive chemiluminescent detection system, high efficiency hybridization is obtained as well as a very good signal to noise ratio. The method has been applied successfully to the detection of RAS point mutations, cystic fibrosis deletion and point mutations and others. The sensitivity, simplicity and reproducibility of this method make it an ideal tool for the diagnosis of infectious and genetic diseases, as well as analysis of mutations in neoplasias, HLA typing and other areas.

Full text

PDF
3929

Images in this article

Selected References

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

  1. Bos J. L. ras oncogenes in human cancer: a review. Cancer Res. 1989 Sep 1;49(17):4682–4689. [PubMed] [Google Scholar]
  2. Farr C. J., Saiki R. K., Erlich H. A., McCormick F., Marshall C. J. Analysis of RAS gene mutations in acute myeloid leukemia by polymerase chain reaction and oligonucleotide probes. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1629–1633. doi: 10.1073/pnas.85.5.1629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ghosh S. S., Musso G. F. Covalent attachment of oligonucleotides to solid supports. Nucleic Acids Res. 1987 Jul 10;15(13):5353–5372. doi: 10.1093/nar/15.13.5353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Li H. H., Gyllensten U. B., Cui X. F., Saiki R. K., Erlich H. A., Arnheim N. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature. 1988 Sep 29;335(6189):414–417. doi: 10.1038/335414a0. [DOI] [PubMed] [Google Scholar]
  5. Miller S. A., Dykes D. D., Polesky H. F. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988 Feb 11;16(3):1215–1215. doi: 10.1093/nar/16.3.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Roberts L. Cystic fibrosis pilot projects go begging. Science. 1990 Nov 23;250(4984):1076–1077. doi: 10.1126/science.2251497. [DOI] [PubMed] [Google Scholar]
  7. Saiki R. K., Walsh P. S., Levenson C. H., Erlich H. A. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6230–6234. doi: 10.1073/pnas.86.16.6230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Sinha N. D., Biernat J., McManus J., Köster H. Polymer support oligonucleotide synthesis XVIII: use of beta-cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucleic Acids Res. 1984 Jun 11;12(11):4539–4557. doi: 10.1093/nar/12.11.4539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Tainsky M. A., Cooper C. S., Giovanella B. C., Vande Woude G. F. An activated rasN gene: detected in late but not early passage human PA1 teratocarcinoma cells. Science. 1984 Aug 10;225(4662):643–645. doi: 10.1126/science.6740333. [DOI] [PubMed] [Google Scholar]
  10. Wood W. I., Gitschier J., Lasky L. A., Lawn R. M. Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1585–1588. doi: 10.1073/pnas.82.6.1585. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES