Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-05-30
2000-03-07
Jones, W. Gary
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
536 231, 536 243, 435 912, C12Q 168, C07H 2102, C07H 2104, C12P 1934
Patent
active
060338512
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of Invention
The present invention relates to a method for suppressing nonspecific extension of primers in the primer extension method.
2. Background Art
The primer extension reaction, which forms a nucleic acid complementary to a nucleic acid template, not only has an important role in an organism but is also used as an essential technique in genetic engineering. In the reaction, a template and a primer which is complementary to the template form a double strand, and then polymerase adds the mononucleotides complementary to the template at the 3' end of the primer. Vary et al. proposed a method of detecting nucleic acids using the above template-dependent primer extension reaction (U.S. Pat. No. 4,851,331).
Various gene amplification methods utilizing the above primer extension method (for example, the PCR method, SDA method, RCR method, NASB method, 3SR method: Keller, G. H. et al., DNA Probes, pp. 255-297, Stockton Press (1993); Persing, D. H. et al., Diagnostic Molecular Microbiology, pp. 51-87, American Society for Microbiology (1993)) have been developed, which have significantly contributed to the advancement of genetic engineering.
However, in these gene amplification methods using the primer extension method, excessive amounts of reagents (polymerase, primer, unit nucleic acid, etc.) relative to a template gene to be amplified are necessary in the early stages of the reaction in order to amplify the gene by about million times. Therefore, nonspecific hybridizations are very likely to occur (Chou et al., Nucleic Acids Res., 20, 1717 (1992)). Furthermore, even in the PCR method which is believed to be the best in terms of specificity, it is difficult to perform highly specific amplification when the amount of template nucleic acid is extremely small, or the presence of large quantities of impurities derived from a sample. Also, primer dimers were found to cause an unexpected problem in the PCR method (Li et al., Proc. Natl. Acad. Sci. USA. 87, 4580-4584 (1990)).
To solve the above problems, the Hot Start Method (Chou et al., Nucleic Acids Res. 20, 1717-1723 (1992)), a method using a polymerase antibody (Kellogg et al., Bio Techniques 16, 1134-1137 (1994); Sharkey et al., BIO/TECHNOLOGY 12, 506-507 (1994)), and others have been proposed. However, none of these methods can sufficiently suppress nonspecific hybridization nor nonspecific extension reaction after the start of the PCR reaction, and cannot be applied to any other amplification method except the PCR method. A nested PCR method using 2 sets of primers (Pierre et al., J. Clin. Microbiol. 29, 712-717 (1991)) definitely improves specificity; however, this method is considered to be procedurally unpractical.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method for suppressing nonspecific hybridization in the primer extension method.
Another object of the present invention is to provide a reagent to suppress nonspecific hybridization of a primer to a nucleic acid template strand in the primer extension method.
According to the present invention, there is provided a primer extension reaction method to form a nucleic acid strand complementary to a nucleic acid template strand by a primer, characterized in that the reaction between the primer and the template strand is carried out in the presence of a nucleic acid or a derivative thereof which is complementary to said primer and has an affinity for said primer which is equivalent to or less than that of said primer for the nucleic acid template strand (primer-complementary nucleic acid).
According to the present application, there is also provided a reagent to suppress nonspecific hybridization of a primer with a nucleic acid template strand comprising a nucleic acid or a derivative thereof which is complementary to said primer and has an affinity for said primer which is equivalent to or less than that of said primer for the nucleic acid template strand.
According to the present invention, nonspecific hybridization in the primer
REFERENCES:
patent: 4675283 (1987-06-01), Roninson
patent: 4683202 (1987-07-01), Mullis et al.
patent: 4965188 (1990-10-01), Mullis et al.
patent: 5043272 (1991-08-01), Hartley
patent: 5348853 (1994-09-01), Wang et al.
patent: 5409818 (1995-04-01), Davey et al.
patent: 5411875 (1995-05-01), Jones
patent: 5556752 (1996-09-01), Lockhart et al.
patent: 5565340 (1996-10-01), Chenchik et al.
patent: 5605793 (1997-02-01), Stemmer
patent: 5674683 (1997-10-01), Kool
patent: 5681702 (1997-10-01), Collins et al.
Evander et al., J. of Virological Methods 31 : 239-250 (1991).
Chou et al., Nucleic Acids Research 20(7) : 1717-1723 (1992).
Jones W. Gary
Wakunaga Seiyaku Kabushiki Kaisha
Whisenant Ethan
LandOfFree
Method for suppressing nonspecific hybridization in primer exten does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for suppressing nonspecific hybridization in primer exten, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for suppressing nonspecific hybridization in primer exten will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-361433