Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-02-13
2004-11-02
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S091200
Reexamination Certificate
active
06811974
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to primer-specific and mispair extension assay for identifying gene variations, such as in different genotypes or subtypes of a given genotype.
2. State of the Art
Current genotyping systems are technically complex, time-consuming and error-prone in the detection of a single nucleotide variation and low level heterozygotes.
Despite current genotyping systems, such as restriction fragment length polymorphism analysis (RFLP), hybridization for example, line probe assay (“LiPA”), selective DNA amplification by PCR-type-specific primers (Okamoto, H., et al.,
J. Gen. Virol.
73: 673, 678, 1992) and direct DNA sequencing having been useful in general, some technical problems still remain and limit their applications.
Development of viral resistance to antiviral drugs used for treatment of HIV-1 infection is an important cause of treatment failure (Coffin J M.,
Sciences,
267: 483-489, 1995). In addition, drug-resistant mutations can give rise to selective cross-resistance to other antiviral drugs which has limited the options available for alternative antiviral regimens (Cohen O J, and Fauci A S.,
N. Engl. J. Med.,
339: 341-243, 1998). Thus, genotypic drug resistance testing plays an important role in selecting an initial antiviral regimen and changing therapy with alternate regimens as the need arises. However, current genotyping assays, including direct DNA sequencing, yield information on only the predominant viral quasispecies due to the inability of these assays to detect low levels of viral variants (Günthard, H. F., et al.,
AIDS Res. Hum. Retroviruses,
14: 869-876, 1998). An ABI automated sequencer was able to detect mutants at levels of 10-50% in an artificially mixed sample only when the mixes were analyzed by editing the sequences manually. Hybridization-based high-density oligonucleotide arrays (GeneChip) (Deeks S G, Abrams D I.,
Lancet,
349: 1489-1490, 1997) were less sensitive than the ABI and were able to detect mutations at levels of 25-75% in the mix (Günthard, H. F., et al,
AIDS Res. Hum. Retroviruses,
14: 869-876, 1998). Similar results were obtained using an automated DNA sequencer (Visible Genetics) (Hu Y W, et al., Reliable detection of mixed HCV genotype infections using a novel genotyping assay. 5
th
International Meeting on Viral Hepatitis C Virus and Related Viruses, Molecular Virology and Pathogenesis, Venezia, Italy, 1998). Line Probe Assay (“LiPA”), which uses reverse hybridization technology, is relatively rapid and could detect mutants at levels as low as 5% (Stuyver L, et al.,
Antimicrob. Agents Chemother.,
41: 284-291, 1997), but again, may not be suitable for detection of mixed genotypes because it may not give results due to nearby polymorphisms that impair hybridization (Stuyver L, et al.,
Antimicrob. Agents Chemother.,
41: 284-291, 1997). Moreover, in 40% of the samples tested, LiPA failed to yield correct results for some of the drug-resistant mutations (Puchhammer-Stockl E, et al,
J. Med. Virol.,
57: 283-289, 1999). Population based sequencing, for example, cloning and sequencing, is the gold standard method for detection of minor drug-resistant mutants. Unfortunately, it is impractical for clinics and large cohort studies.
For most indirect DNA sequencing genotyping systems, a common weakness is that they are not as accurate as direct DNA sequencing analysis, particularly for detection of a single nucleotide mutation or variation, resulting in considerable instances of errors or inconsistent results (Andonov, A., et al.,
J. Clin. Microbiol.
32: 2031-2034, 1994; Tuveri, R., et al.,
Journal of Medical Virology
51: 36-41, 1997; Okamoto, H., et al.,
J. Virol. Methods
57: 31 45 002-16, 1996). Although direct DNA sequencing is the most reliable method for genotyping, this is not practical for large cohort studies.
For example, International Publication No. WO 91/13075 describes a method for detecting variable nucleotides based on primer extension and incorporation of detectable nucleoside triphosphate using T7 polymerase for extending the primer. However, T7 polymerase does not have the proofreading activity and the 3′-5′ exonuclease activity of pfu results in false positive or false negative reactions. Moreover, the method uses ddNTPs for terminating extension.
Another major limitation of all current genotyping systems, including direct DNA sequencing, is that they cannot reliably detect low levels of heterozygotes (Tuveri, R., et al,
Journal of Medical Virology
51: 36-41, 1997; Lau, J. Y., et al.,
J. Infect. Dis.
171: 281-289, 1995; Forns, X., et al.,
J. Clin. Microbiology.
34-10: 2516-2521, 1996) or mixed genotype infections (Tuveri, R., et al.,
Journal of Medical Virology
51: 36-41, 1997; Lau, J. Y., et al.,
J. Infect. Dis.
171: 281-289, 1995; Forns, X., et al.,
J. Clin. Microbiology.
34-10: 2516-2521, 1996).
International Publication No. WO 96/30545 discloses a method for simultaneously analyzing a genetic mutation and a corresponding wild-type sequence within a sample. The method utilizes ddNTPs for terminating primer extension. However, the use of ddNTPs increases the background of the assay, reducing its sensitivity and capacity to detect low levels of heterozygotes, similarly as in WO 91/13075.
HCV was recognized as the major etiologic agent of blood borne non-A, non-B hepatitis soon after the virus was identified in 1989. As an RNA virus, HCV shows great genetic variability, resulting in the existence of types, subtypes and quasispecies. At present, 11 types and at least 50 subtypes have been described. However, types 1a, 1b, 2a, 2b and 3a have been found to be generally the most prevalent (Simmonds, P.,
Hepatology
21: 570-582, 1995). Subtype 1b is the most common genotype found in Japan (Okamoto, H., et al.,
J. Gen. Virol
73: 673, 678, 1992) and European countries while subtypes 1a and 1b are the most common genotypes in the United States (Lau, J. Y., et al.,
J. Infect. Dis.
171: 281-289, 1995) and Canada, (Andonov, A., et al.,
J. Clin. Microbiol.
32: 2031-2034, 1994). Viruses of various genotypes contain different antigenic properties, which have potentially important consequences for the development of a vaccine and for antibody screening tests. Also, the disease severity and response to interferon may be influenced by the virus types and subtypes (Simmonds,
Hepatology
21: 570-582, 1995). Subtype 1b was reported to be associated with a high severity of the disease and low response to interferon (Simmonds,
Hepatology
21: 570-582, 1995). It is apparent that a rapid, simple, accurate and inexpensive genotyping method is urgently needed.
Amplification refractory mutation system (“ARMS”) (Newton, C. R., et al.,
Nucl. Acids Res.
17: 2503-2516), improved the methods used in the prior art for typing the five most common genotypes (Pistello, M., et al.,
J. Clin. Microbiol.
32: 232-234, 1994). ARMS was developed for PCR detection of any point mutation in DNA using Taq DNA polymerase (Newton, C. R., et al.,
Nucl. Acids Res.
17: 2503-2516) and is based on the principle that oligonucleotides with a mismatched 3′-residue would not function as primers in PCR under controlled conditions. In some cases, however, the specificity of ARMS was insufficient to give a correct diagnosis. The problem with nonspecific reactivities still remains with the type-specific primer PCR method for HCV genotyping, even with the improvement using ARMS.
It is apparent that the major cause of the nonspecific reactivities found in these assays is related to the use of Taq DNA polymerase due to its lack of 3′→5′ exonuclease activity. This inaccuracy results in base substitutions, transitions, tranversions, frame shifts or deletion mutations during DNA synthesis. Consequently, mispairs can be frequently formed, and Taq polymerase would be able to continue synthesizing DNA by addition of the next correct nucleotide on the template (Lau, J. Y., et al.,
J. Infect Dis.
171: 281-289, 1995). Even after reaching the end o
Canadian Blood Services
Fredman Jeffrey
Strzelecka Teresa
TraskBritt
LandOfFree
Primer-specific and mispair extension assay for identifying... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Primer-specific and mispair extension assay for identifying..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Primer-specific and mispair extension assay for identifying... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3278337