Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1996-07-05
2001-09-25
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C530S350000
Reexamination Certificate
active
06294325
ABSTRACT:
BACKGROUND OF THE INVENTION
The polymerase chain reaction (PCR) is one of the most important technologies for genome analysis. One of the weaknesses of PCR is that primer extension from mismatched primers occurs. Extension from mismatched primers limits allele-specific amplification and detection of mutations and polymorphisms to some extent with homogeneous DNA samples (e.g. for genotyping), but to a greater extent for heterogeneous DNA samples (e.g. for detection of cancer mutations). Another of the weaknesses of PCR is much poorer fidelity than observed during in vivo DNA replication, as reflected in (1) a rather high rate of nucleotide misincorporation, leading to difficulty in using PCR for faithful cloning and (2) the production of multiple bands when di- and trinucleotide repeats are amplified. An order of magnitude improvement in PCR specificity and fidelity could increase accuracy in genotyping and somatic mutation detection and open up new uses for PCR, including the reproducible and faithful cloning of genomic DNA fragments up to several kilobases in length. The present invention provides such an improvement in PCR.
The ligase chain reaction (LCR) and its variations (e.g., oligonucleotide ligation assay (OLA), ligase detection reaction (LDR)) are alternative techniques for genome analysis. A commonly recognized source of spurious background signal in LCR and its variations, as well as in PCR and its variations, is the hybridization of an oligonucleotide such as a probe or a primer, to regions of the nucleic acid not intended to be amplified. Generally, these hybridizations occur because the target sample contains, in addition to the target sequence itself, other sequences with some similarity to the target nucleic acid. Although hybridization of probe or primer to these similar sequences is not as probable as to the target sequence, some hybridization can occur. When such unintended non-specific hybridization occurs, it is possible that sequences other than the targeted sequence will be amplified. If these limitations of PCR and LCR could be reduced or eliminated, the methods would be even more useful than they presently are.
SUMMARY OF THE INVENTION
The invention relates to isolated nucleic acids which encode a thermostable protein that enhances specific binding of a thermostable mismatch binding protein to bulge loops in a heteroduplex nucleic acid. As used herein, bulge loops include mispaired bases and frameshifts of 1-4 nucleotides or more. A protein which enhances specific binding of a thermostable mismatch binding protein to bulge loops in a heteroduplex nucleic acid is defined herein to include proteins which increase the occurrence of binding to bulge loops in a heteroduplex nucleic acid by a thermostable mismatch binding protein and proteins which increase the stability of complexes produced by binding of a thermostable mismatch binding protein to a bulge loop in a heteroduplex nucleic acid. A complex produced by binding of a thermostable mismatch binding protein to a bulge loop in a heteroduplex nucleic acid is referred to herein as a “thermostable bulge loop-binding protein-heteroduplex nucleic acid complex”.
In one embodiment, the invention relates to nucleic acids which encode thermostable MutL proteins. Such nucleic acids include, for example, nucleic acids encoding
Aquifex pyrophilus
MutL,
Thermotoga maritima
MutL or
Thermus thermophilus
MutL, and nucleic acids which hybridize to these nucleic acids and encode a thermostable protein that enhances binding of a thermostable mismatch binding protein to bulge loops in a heteroduplex nucleic acid. In another embodiment, the invention relates to nucleic acids which hybridize to nucleic acids encoding
Aquifex pyrophilus
MutL,
Thermotoga maritima
MutL or
Thermus thermophilus
MutL and are useful as probes or primers to detect and/or recover homologous genes from other hyperthermophilic or thermophilic bacteria, including homologous genes from members of the genus Aquifex other than
Aquifex pyrophilus
, from members of the genus Thermotoga other than
Thermotoga maritima
and from members of the genus Thermus other than
Thermus thermophilus
. The invention further relates to recombinant constructs and vectors comprising nucleic acids that encode
Aquifex pyrophilus
MutL,
Thermotoga maritima
MutL or
Thermus thermophilus
MutL, or nucleic acids which hybridize thereto.
The invention also relates to proteins isolated from hyperthermophilic and thermophilic bacteria that enhance binding of thermostable mismatch binding proteins to bulge loops in a heteroduplex nucleic acid. As used herein, the phrase “isolated from” or “isolated nucleic acid” refers to nucleic acid obtained from (isolated from) naturally occurring sources as well as nucleic acids produced by recombinant methods or chemical synthesis, or by combinations of biological and chemical methods. Isolated nucleic acids produced by recombinant methods (e.g., genetic engineering methods) or synthesized chemically can also be referred to, respectively, as recombinantly produced nucleic acids and chemically synthesized or synthetic nucleic acids.
The invention further relates to isolated MutL proteins from hyperthermophilic or thermophilic bacteria. “Isolated” MutL proteins from hyperthermophilic or thermophilic bacteria include those obtained from naturally-occurring sources, as well as those produced by recombinant methods or chemical synthesis, or by combinations of biological and chemical methods.
The invention also relates to isolated thermostable proteins or polypeptides that enhance binding of thermostable mismatch binding proteins to bulge loops in a heteroduplex nucleic acid. Recombinant thermostable proteins that enhance binding of thermostable mismatch binding proteins to bulge loops in a heteroduplex nucleic acid can be produced in host cells using cells and methods described herein.
Another embodiment of the invention relates to a method of reducing DNA misincorporation (i.e., improving fidelity of DNA replication) in an amplification reaction by including a thermostable mismatch binding protein with a thermostable protein that enhances binding of the thermostable mismatch binding protein to bulge loops in the reaction. The thermostable mismatch binding protein binds to bulge loops in a heteroduplex nucleic acid formed as a result of misincorporation of deoxynucleoside triphosphates during the amplification reaction. This results in formation of a thermostable bulge loop-binding protein-heteroduplex nucleic acid complex. Binding of the thermostable protein prevents nucleic acids which include misincorporated deoxynucleoside triphosphates from acting as templates in subsequent rounds of the amplification reaction. Thus, amplification of nucleic acids which include misincorporated deoxynucleoside triphosphates is prevented, resulting in a reduction in overall DNA misincorporation. The thermostable protein that enhances binding of the thermostable mismatch binding protein to bulge loops in a heteroduplex nucleic acid improves this reaction. As used herein, “thermostable bulge loop-binding protein” refers to a thermostable mismatch binding protein.
The present invention further relates to a method for detecting a target nucleic acid which includes a specific sequence comprising combining a thermostable mismatch binding protein which binds specifically to bulge loops in a heteroduplex nucleic acid, a thermostable protein that enhances binding of the thermostable mismatch binding protein to the bulge loops, and an amplification reaction mixture, to produce a test combination. The individual components of an amplification reaction mixture can each be added, together or separately (e.g., individually), in any order, prior to, subsequent to or simultaneously with the thermostable mismatch binding protein which binds specifically to bulge loops in a heteroduplex nucleic acid, and/or the thermostable protein that enhances binding of the thermostable mismatch binding protein to the bulge loops. The resulting test combination is maintained u
Campbell Eggerton A.
Hamilton Brook Smith & Reynolds P.C.
The Mount Sinai School of Medicine of the City University of New
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