Abstract
A rapid in-solution method for the detection of all 12 single-base mismatches is described. The technique is based on the hybridization protection assay (HPA) format that utilizes oligonucleotide probes labeled with a highly chemiluminescent acridinium ester (AE). Hydrolysis by weak base renders AE permanently non-chemiluminescent. When an AE-labeled probe hybridizes to an exactly complementary target, AE is protected from hydrolysis relative to the unhybridized conformation. Single-base mutations in the duplex adjacent to the site of AE attachment disrupt this protection resulting in rapid AE hydrolysis and loss of chemiluminescence. The discrimination effect was seen in both DNA and RNA. Studies of Tm values revealed that this effect is not due to a decrease in the overall stability of the duplex, suggesting the AE is responding to local structural changes in the double helix induced by mismatches. Using this principle all 12 single mismatches were clearly discriminated from the corresponding matched sequences. The assay is homogeneous, simple, sensitive, applicable to both amplified and non-amplified targets, and is completed in 30-60 min. An example showing discrimination between wild-type and mutant sequences corresponding to the reverse transcriptase coding region of HIV-1 is given.
Full Text
The Full Text of this article is available as a PDF (154.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Babon J. J., Youil R., Cotton R. G. Improved strategy for mutation detection--a modification to the enzyme mismatch cleavage method. Nucleic Acids Res. 1995 Dec 25;23(24):5082–5084. doi: 10.1093/nar/23.24.5082. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bhattacharyya A., Lilley D. M. Single base mismatches in DNA. Long- and short-range structure probed by analysis of axis trajectory and local chemical reactivity. J Mol Biol. 1989 Oct 20;209(4):583–597. doi: 10.1016/0022-2836(89)90596-2. [DOI] [PubMed] [Google Scholar]
- Cebula T. A., Payne W. L., Feng P. Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their Shiga-like toxin type by mismatch amplification mutation assay-multiplex PCR. J Clin Microbiol. 1995 Jan;33(1):248–250. doi: 10.1128/jcm.33.1.248-250.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen X., Baumstark T., Steger G., Riesner D. High resolution SSCP by optimization of the temperature by transverse TGGE. Nucleic Acids Res. 1995 Nov 11;23(21):4524–4525. doi: 10.1093/nar/23.21.4524. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hale Y. M., Melton M. E., Lewis J. S., Willis D. E. Evaluation of the PACE 2 Neisseria gonorrhoeae assay by three public health laboratories. J Clin Microbiol. 1993 Feb;31(2):451–453. doi: 10.1128/jcm.31.2.451-453.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jonas V., Alden M. J., Curry J. I., Kamisango K., Knott C. A., Lankford R., Wolfe J. M., Moore D. F. Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by amplification of rRNA. J Clin Microbiol. 1993 Sep;31(9):2410–2416. doi: 10.1128/jcm.31.9.2410-2416.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ke S. H., Wartell R. M. Influence of nearest neighbor sequence on the stability of base pair mismatches in long DNA; determination by temperature-gradient gel electrophoresis. Nucleic Acids Res. 1993 Nov 11;21(22):5137–5143. doi: 10.1093/nar/21.22.5137. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Khrapko K., Hanekamp J. S., Thilly W. G., Belenkii A., Foret F., Karger B. L. Constant denaturant capillary electrophoresis (CDCE): a high resolution approach to mutational analysis. Nucleic Acids Res. 1994 Feb 11;22(3):364–369. doi: 10.1093/nar/22.3.364. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kovach J. S., Hartmann A., Blaszyk H., Cunningham J., Schaid D., Sommer S. S. Mutation detection by highly sensitive methods indicates that p53 gene mutations in breast cancer can have important prognostic value. Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):1093–1096. doi: 10.1073/pnas.93.3.1093. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miret J. J., Parker B. O., Lahua R. S. Recognition of DNA insertion/deletion mismatches by an activity in Saccharomyces cerevisiae. Nucleic Acids Res. 1996 Feb 15;24(4):721–729. doi: 10.1093/nar/24.4.721. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nelson N. C., Cheikh A. B., Matsuda E., Becker M. M. Simultaneous detection of multiple nucleic acid targets in a homogeneous format. Biochemistry. 1996 Jun 25;35(25):8429–8438. doi: 10.1021/bi960085+. [DOI] [PubMed] [Google Scholar]
- Newton C. R., Graham A., Heptinstall L. E., Powell S. J., Summers C., Kalsheker N., Smith J. C., Markham A. F. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 1989 Apr 11;17(7):2503–2516. doi: 10.1093/nar/17.7.2503. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Petersen K. B., Kølvraa S., Bolund L., Petersen G. B., Koch J., Gregersen N. Detection of alpha 1-antitrypsin genotypes by analysis of amplified DNA sequences. Nucleic Acids Res. 1988 Jan 11;16(1):352–352. doi: 10.1093/nar/16.1.352. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Phelps R. S., Chadwick R. B., Conrad M. P., Kronick M. N., Kamb A. Efficient, automatic detection of heterozygous bases during large-scale DNA sequence screening. Biotechniques. 1995 Dec;19(6):984–989. [PubMed] [Google Scholar]
- Richman D., Shih C. K., Lowy I., Rose J., Prodanovich P., Goff S., Griffin J. Human immunodeficiency virus type 1 mutants resistant to nonnucleoside inhibitors of reverse transcriptase arise in tissue culture. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11241–11245. doi: 10.1073/pnas.88.24.11241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rojas J. M., Sánchez-Palomino S., Santana M., López-Galíndez C., Tabarés E. Genetic analysis of herpes simplex virus type 1 isolates from recurrent lesions and clinical reinfections. J Infect Dis. 1995 Dec;172(6):1602–1605. doi: 10.1093/infdis/172.6.1602. [DOI] [PubMed] [Google Scholar]
- Rust S., Funke H., Assmann G. Mutagenically separated PCR (MS-PCR): a highly specific one step procedure for easy mutation detection. Nucleic Acids Res. 1993 Aug 11;21(16):3623–3629. doi: 10.1093/nar/21.16.3623. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sarkar G., Yoon H. S., Sommer S. S. Screening for mutations by RNA single-strand conformation polymorphism (rSSCP): comparison with DNA-SSCP. Nucleic Acids Res. 1992 Feb 25;20(4):871–878. doi: 10.1093/nar/20.4.871. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shuber A. P., Skoletsky J., Stern R., Handelin B. L. Efficient 12-mutation testing in the CFTR gene: a general model for complex mutation analysis. Hum Mol Genet. 1993 Feb;2(2):153–158. doi: 10.1093/hmg/2.2.153. [DOI] [PubMed] [Google Scholar]
- Takamatsu S., Kato R., Kuramitsu S. Mismatch DNA recognition protein from an extremely thermophilic bacterium, Thermus thermophilus HB8. Nucleic Acids Res. 1996 Feb 15;24(4):640–647. doi: 10.1093/nar/24.4.640. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wagner R., Debbie P., Radman M. Mutation detection using immobilized mismatch binding protein (MutS). Nucleic Acids Res. 1995 Oct 11;23(19):3944–3948. doi: 10.1093/nar/23.19.3944. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Walsh T. J., Francesconi A., Kasai M., Chanock S. J. PCR and single-strand conformational polymorphism for recognition of medically important opportunistic fungi. J Clin Microbiol. 1995 Dec;33(12):3216–3220. doi: 10.1128/jcm.33.12.3216-3220.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Youil R., Kemper B. W., Cotton R. G. Screening for mutations by enzyme mismatch cleavage with T4 endonuclease VII. Proc Natl Acad Sci U S A. 1995 Jan 3;92(1):87–91. doi: 10.1073/pnas.92.1.87. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zimmerman P. A., Carrington M. N., Nutman T. B. Exploiting structural differences among heteroduplex molecules to simplify genotyping the DQA1 and DQB1 alleles in human lymphocyte typing. Nucleic Acids Res. 1993 Sep 25;21(19):4541–4547. doi: 10.1093/nar/21.19.4541. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zimmerman P. A., Shapiro M., Tang J., Nutman T. B., Unnasch T. R. Technical report. Optimizing probe selection in directed heteroduplex analysis using HDprobe 1.1. Biotechniques. 1995 Dec;19(6):972–977. [PubMed] [Google Scholar]