Multiplex detection of single-nucleotide variations using molecular beacons - PubMed (original) (raw)
Comparative Study
Multiplex detection of single-nucleotide variations using molecular beacons
S A Marras et al. Genet Anal. 1999 Feb.
Abstract
We demonstrate that single-nucleotide differences in a DNA sequence can be detected in homogeneous assays using molecular beacons. In this method, the region surrounding the site of a sequence variation is amplified in a polymerase chain reaction and the identity of the variant nucleotide is determined by observing which of four differently colored molecular beacons binds to the amplification product. Each of the molecular beacons is perfectly complementary to one variant of the target sequence and each is labeled with a different fluorophore. To demonstrate the specificity of these assays, we prepared four template DNAs that only differed from one another by the identity of the nucleotide at one position. Four amplification reactions were prepared, each containing all four molecular beacons, but each initiated with only one of the four template DNAs. The results show that in each reaction a fluorogenic response was elicited from the molecular beacon that was perfectly complementary to the amplified DNA, but not from the three molecular beacons whose probe sequence mismatched the target sequence. The color of the fluorescence that appeared in each tube during the course of the amplification indicated which nucleotide was present at the site of variation. These results demonstrate the extraordinary specificity of molecular beacons. Furthermore, the results illustrate how the ability to label molecular beacons with differently colored fluorophores enables simple multiplex assays to be carried out for genetic analysis.
Similar articles
- Real Time PCR Using Molecular Beacons : A New Tool to Identify Point Mutations and to Analyze Gene Expression in Mycobacterium tuberculosis.
Manganelli R, Tyagi S, Smith I. Manganelli R, et al. Methods Mol Med. 2001;54:295-310. doi: 10.1385/1-59259-147-7:295. Methods Mol Med. 2001. PMID: 21341083 - Multicolor molecular beacons for allele discrimination.
Tyagi S, Bratu DP, Kramer FR. Tyagi S, et al. Nat Biotechnol. 1998 Jan;16(1):49-53. doi: 10.1038/nbt0198-49. Nat Biotechnol. 1998. PMID: 9447593 - Using molecular beacons to detect single-nucleotide polymorphisms with real-time PCR.
Mhlanga MM, Malmberg L. Mhlanga MM, et al. Methods. 2001 Dec;25(4):463-71. doi: 10.1006/meth.2001.1269. Methods. 2001. PMID: 11846616 - Molecular beacons: an optimal multifunctional biological probe.
Li Y, Zhou X, Ye D. Li Y, et al. Biochem Biophys Res Commun. 2008 Sep 5;373(4):457-61. doi: 10.1016/j.bbrc.2008.05.038. Epub 2008 May 19. Biochem Biophys Res Commun. 2008. PMID: 18489905 Review. - Molecular beacons: colorful analysis of nucleic acids.
Vet JA, Van der Rijt BJ, Blom HJ. Vet JA, et al. Expert Rev Mol Diagn. 2002 Jan;2(1):77-86. doi: 10.1586/14737159.2.1.77. Expert Rev Mol Diagn. 2002. PMID: 11963813 Review.
Cited by
- A highly fluorescent DNA toolkit: synthesis and properties of oligonucleotides containing new Cy3, Cy5 and Cy3B monomers.
Hall LM, Gerowska M, Brown T. Hall LM, et al. Nucleic Acids Res. 2012 Aug;40(14):e108. doi: 10.1093/nar/gks303. Epub 2012 Apr 11. Nucleic Acids Res. 2012. PMID: 22495935 Free PMC article. - Genotyping of single nucleotide polymorphisms by 5' nuclease allelic discrimination.
Malkki M, Petersdorf EW. Malkki M, et al. Methods Mol Biol. 2012;882:173-82. doi: 10.1007/978-1-61779-842-9_10. Methods Mol Biol. 2012. PMID: 22665234 Free PMC article. - Use of molecular beacons for rapid, real-time, quantitative monitoring of cytotoxic T-lymphocyte epitope mutations in simian immunodeficiency virus.
Peyerl FW, Barouch DH, Bazick HS, Manuel E, Letvin NL. Peyerl FW, et al. J Clin Microbiol. 2005 Sep;43(9):4773-9. doi: 10.1128/JCM.43.9.4773-4779.2005. J Clin Microbiol. 2005. PMID: 16145140 Free PMC article. - In vitro selection of molecular beacons.
Rajendran M, Ellington AD. Rajendran M, et al. Nucleic Acids Res. 2003 Oct 1;31(19):5700-13. doi: 10.1093/nar/gkg764. Nucleic Acids Res. 2003. PMID: 14500834 Free PMC article. - A new class of homogeneous nucleic acid probes based on specific displacement hybridization.
Li Q, Luan G, Guo Q, Liang J. Li Q, et al. Nucleic Acids Res. 2002 Jan 15;30(2):E5. doi: 10.1093/nar/30.2.e5. Nucleic Acids Res. 2002. PMID: 11788731 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Other Literature Sources