Nucleic Acids Research, 1989, Vol. 17, No. 20 8093-8099
© 1989
MOLECULAR BIOLOGY |
Detection of minority point mutations by modified PCR technique: a new approach for a sensitive diagnosis of tumor-progression markers
Institut de Pathologie Moléculaire CHU Cochin, 24 rue du Faubourg St Jacques, F-75014 Paris, France 1Service de Gastroentérologie CHU Cochin, 24 rue du Faubourg St Jacques, F-75014 Paris, France
*To whom correspondence should be addressed
Received July 28, 1989. Revised September 15, 1989. Accepted September 15, 1989.
The detection of point mutations correlated with diseases, in enzymatically amplified DNA sequences (Polymerase Chain Reaction), is currently performed by digestion of PCR products when an existing restriction site disappears at least in one allele of the amplified mutated sequence or by allele specific radiolabeled probes in all other cases. These methods are the most sensitive but they cannot detect a mutation if it is present in less than 5% of the studied cells. We describe here a method based on the introduction of an artificial restriction site, using a modified primer during the PCR, which creates a RFLP indicative of the studied mutation. This RFLP is detected by a radiolabeled oligonucleotide probe which is not related to the mutation. Our approach multiplies the sensitivity by a factor of 1000 and it is practical for use in screening purposes and the detection, after treatment, of the residual disease in human malignancies. Using this method we detected 20% more mutations at codon 12 in the Ki ras oncogene in DNA from colorectal cancers that were undetectable with all the previous methods.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  G. Amicarelli, D. Adlerstein, E. Shehi, F. Wang, and G. M. Makrigiorgos Genotype-Specific Signal Generation Based on Digestion of 3-Way DNA Junctions: Application to KRAS Variation Detection Clin. Chem., October 1, 2006; 52(10): 1855 - 1863. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Zhang, Z. Zhu, H. Zhu, T. Nguyen, F. Yao, K. Xia, D. Liang, and C. Liu SNP Cutter: a comprehensive tool for SNP PCR-RFLP assay design Nucleic Acids Res., July 1, 2005; 33(suppl_2): W489 - W492. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Jeffreys and C. A. May DNA Enrichment by Allele-Specific Hybridization (DEASH): A Novel Method for Haplotyping and for Detecting Low-Frequency Base Substitutional Variants and Recombinant DNA Molecules Genome Res., October 1, 2003; 13(10): 2316 - 2324. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Mannen, M. Morimoto, K. Oyama, F. Mukai, and S. Tsuji Identification of mitochondrial DNA substitutions related to meat quality in Japanese Black cattle J Anim Sci, January 1, 2003; 81(1): 68 - 73. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kaur, Y. Zhang, W.-H. Liu, S. Tetradis, B. D. Price, and G. M. Makrigiorgos Ligation of a primer at a mutation: a method to detect low level mutations in DNA Mutagenesis, September 1, 2002; 17(5): 365 - 374. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Solakoglu, C. Maierhofer, G. Lahr, E. Breit, P. Scheunemann, I. Heumos, U. Pichlmeier, G. Schlimok, R. Oberneder, M. W. Kollermann, et al. Heterogeneous proliferative potential of occult metastatic cells in bone marrow of patients with solid epithelial tumors PNAS, February 19, 2002; 99(4): 2246 - 2251. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Lichtenstein, O.'g. I. Serdjuk, T. I. Sukhova, H. S. Melkonyan, and S. R. Umansky Selective 'stencil'-aided pre-PCR cleavage of wild-type sequences as a novel approach to detection of mutant K-RAS Nucleic Acids Res., September 1, 2001; 29(17): e90 - e90. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. D. Sorenson Detection of Mutated KRAS2 Sequences as Tumor Markers in Plasma/Serum of Patients with Gastrointestinal Cancer Clin. Cancer Res., June 1, 2000; 6(6): 2129 - 2137. [Abstract] [Full Text]
|
|
|
|
 |
|
 C. D. GOCKE, F. A. BENKO, M. S. KOPRESKI, and D. B. EVANS Enrichment Methods for Mutation Detection Ann. N.Y. Acad. Sci., April 1, 2000; 906(1): 31 - 38. [Full Text] [PDF]
|
|
|
|
|
|
B. Vogelstein and K. W. Kinzler Digital PCR PNAS, August 3, 1999; 96(16): 9236 - 9241. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Morgan, J.-p. Xiao, and S.-y. Xu Characterization of an Extremely Thermostable Restriction Enzyme, PspGI, from a Pyrococcus Strain and Cloning of the PspGI Restriction-Modification System in Escherichia coli Appl. Envir. Microbiol., October 1, 1998; 64(10): 3669 - 3673. [Abstract] [Full Text]
|
|
|
|
|
|
A D van Mansfeld and J L Bos PCR-based approaches for detection of mutated ras genes. Genome Res., May 1, 1992; 1(4): 211 - 216. [PDF]
|
|
|
|
|
|
D R Jacobson and T Moskovits Rapid, nonradioactive screening for activating ras oncogene mutations using PCR-primer introduced restriction analysis (PCR-PIRA) Genome Res., November 1, 1991; 1(2): 146 - 148. [PDF]
|
|