Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-01
2002-11-26
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C422S050000, C422S068100, C536S023100, C536S024300, C536S024310, C536S024320
Reexamination Certificate
active
06485906
ABSTRACT:
TECHNICAL FIELD
This application is in the field of molecular biology relating to the use of oligonucleotides as probes and primers. It relates further to the use of modified nucleic acid bases to improve the hybridization properties and discriminatory abilities of oligonucleotides that are used as probes and primers.
BACKGROUND
Many techniques currently in use in molecular biology utilize oligonucleotides as probes and/or primers. It is often advantageous, in the practice of these techniques, to be able to distinguish between two or more sequences which are related but which differ by one or more nucleotides. For example, many mutations of clinical significance differ by only a single nucleotide from the wild-type sequence. Polymorphisms in mammalian genomes are also often characterized by sequence differences of one or a few nucleotides. The ability to make such a distinction is known as mismatch discrimination. In practical terms, mismatch discrimination describes the property by which a defined sequence oligonucleotide, at a given stringency, hybridizes strongly (one manifestation of which is that the hybrids have a high melting temperature) to a target sequence with which it is complementary along its entire length (a perfect hybrid or perfect match), but hybridizes detectably more weakly to a target sequence that is non-complementary to the sequence of the oligonucleotide at one or a few nucleotides (a mismatch). The differences in hybridization strength are such that a particular stringency can be selected at which a perfect match is detectable as a hybrid and a mismatch fails to form a hybrid.
In a nucleic acid duplex, each base pair contributes to stability. Hence, the shorter the duplex, the greater the relative contribution of each individual base pair to the stability of the duplex. As a result, the difference in stability between a perfect match and a mismatch will be greater for shorter oligonucleotides. However, short oligonucleotides hybridize weakly, even to a perfectly complementary sequence, and thus must be hybridized under conditions of reduced stringency. Thus, the potential discriminatory power of short oligonucleotides cannot be easily realized except under conditions of low stringency, which counteract their discriminatory ability. It would constitute a substantial advance in the art if it were possible to achieve mismatch discrimination, particularly for single-nucleotide mismatches, under conditions of high stringency; for example, at the elevated temperatures characteristic of most amplification reactions.
Stabilization of duplexes by pyrazolopyrimidine base analogues has been reported. Seela et al. (1988)
Helv. Chim. Acta
. 71:1191-1198; Seela et al. (1988)
Helv. Chim. Acta
. 71:1813-1823; and Seela et al. (1989)
Nucleic Acids Res
. 17:901-910. Pyrazolo[3,4-d]pyrimidine residues in oligonucleotides are also useful as sites for attachment of various pendant groups to oligonucleotides. See co-owned PCT Publication WO 90/14353, Nov. 29, 1990. In addition, oligonucleotides in which one or more purine residues have been substituted by pyrazolo[3,4-d]pyrimidines display enhanced triplex-forming ability, as disclosed, for example, in Belousov et al. (1998)
Nucleic Acids Res
. 26:1324-1328. Pyrazolopyrimidines, when incorporated into an oligonucleotide, may provide improved duplex and triplex formation. U.S. Pat. No. 5,594,121.
DISCLOSURE OF THE INVENTION
It is an object of the present invention to provide new oligonucleotide compositions with improved properties related to hybridization and mismatch discrimination. It is a further object of the present invention to provide improved methods for hybridization, primer extension, hydrolyzable probe assays, PCR, single-nucleotide mismatch discrimination, nucleotide sequence analysis, array analysis and related techniques involving the use of oligonucleotides as probes and/or primers.
Accordingly, in one aspect, the present invention provides modified oligonucleotide compositions comprising one or more pyrazolo[3,4-d]pyrimidine base analogues substituted for at least one purine. In preferred embodiments, the guanine analogue 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (ppG) is substituted for guanine, and/or the adenine analogue 4-amino-1H-pyrazolo[3,4-d]pyrimidine (ppA) is substituted for adenine. In other embodiments, the guanine analogue 1H-pyrazolo[3,4-d]pyrimidin-4(5H) one (ppI) is substituted for guanine. The pyrazolo[3,4-d]pyrimidine-substituted oligonucleotides can comprise, in addition, other moieties such as detectable labels and/or minor groove binders and/or other types of modified bases or base analogues.
Another aspect of the invention is a method for hybridization of nucleic acids, wherein at least one of the nucleic acids is a modified nucleic acid wherein one or more purine residues are substituted with a pyrazolo[3,4-d]pyrimidine base analogue. This method provides higher melting temperatures and enhanced mismatch detection. The improved hybridization methods provided by the present invention can be used in techniques which include, but are not limited to, hybridization, primer extension, single-nucleotide polymorphism detection, hydrolyzable probe assays, cDNA synthesis, nucleotide sequence determination, amplification reactions, and other techniques such as are known to those of skill in the art.
When the guanine bases in an oligonucleotide are replaced by the guanine analogue ppG, the T
m
values of probes containing the analogues are slightly higher than those of oligonucleotide probes containing guanine. Hence, G-containing and ppG-containing oligonucleotides perform similarly in hybridization assays. However, when ppG-substituted oligonucleotides are used as hydrolyzable probes (described infra and see U.S. Pat. No. 5,210,015), two properties are significantly enhanced. First, ppG-substituted probes are more effective at mismatch discrimination, as measured by higher signal-to-noise values comparing the fluorescent signal obtained from a perfectly-matched hybrid with that from a hybrid containing a single-nucleotide mismatch. In addition, ppG-substituted probes provide higher absolute signal from a perfectly-matched target.
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Afonina Irina A.
Kutyavin Igor V.
Meyer, Jr. Rich B.
Epoch Pharmaceuticals, Inc.
Marschel Ardin H.
Townsend and Townsend / and Crew LLP
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