Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-01-30
2004-09-21
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091100, C435S005000, C536S024330
Reexamination Certificate
active
06794133
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to novel polymerase chain reaction (PCR) amplification techniques and their use, for example, for identifying single nucleotide polymorphisms.
Dense linkage maps are invaluable tools for genetic and genomic analysis. They facilitate high resolution genetic mapping, positional cloning of monogenic traits, genetic dissection of polygenic traits, fine-structure linkage disequilibrium studies, and the construction of genome-wide physical maps. Historically, genetic maps were constructed with visible markers, but it is difficult to examine many such markers in a single cross. The recognition that distantly related individuals differ in DNA sequence throughout their genome (Botstein et al., Am. J. Hum. Genet. 32: 314-331, 1980) led to the rapid incorporation of DNA markers into mapping strategies. Useful DNA markers have the following general characteristics: (1) they are inherited in a Mendelian fashion; (2) they are present in most individuals analyzed and recognize a sequence that is polymorphic; (3) they correspond to a single site in the genome; (4) the probe used to recognize the marker hybridizes selectively and efficiently, even under conditions of low stringency; and (5) they can be distributed throughout a community, either as clones or as DNA sequences.
Until recently, the most commonly used DNA markers were restriction fragment length polymorphisms (RFLPs), anonymous single copy-number genomic clones that reveal a polymorphism in the length of a restriction fragment, typically by DNA blot hybridization. RFLP mapping is well-suited for determining the genetic location of any newly-cloned DNA sequence; the DNA fragment can be used as a hybridization probe (assuming it detects an RFLP) against the DNA filters used to construct the RFLP map. However, in many cases, new genes are identified by mutations, and mapping such a mutation onto an RFLP map can be a lengthy and arduous procedure.
SUMMARY OF THE INVENTION
In general, the invention features a method for determining whether a nucleic acid sequence includes a particular allele of a polymorphic sequence, involving:
(a) contacting a nucleic acid sequence, in the same or a separate reaction, with a first pair of PCR primers and a second pair of PCR primers under conditions that allow hybridization of the PCR primers to the nucleic acid sequence, the first pair of PCR primers hybridizing to opposite strands of the nucleic acid sequence and bordering the position of the polymorphic sequence, and the second pair of PCR primers hybridizing to opposite strands of the nucleic acid sequence and bordering the position of the polymorphic sequence, the PCR primers being characterized as follows:
(i) one of the first pair of PCR primers (a) being complementary at its 3′-terminal nucleotide to a first allele of the polymorphic sequence (allele A), (b) being non-complementary at its 3′-terminal nucleotide to a second allele of the polymorphic sequence (allele B), and (c) being non-complementary to the nucleic acid sequence at a single non-complementary nucleotide in its 3′-terminal nucleotides 2-6; and
(ii) one of the second pair of PCR primers (a) being complementary at its 3′-terminal nucleotide to the first allele of the polymorphic sequence (allele A), (b) being non-complementary at its 3′-terminal nucleotide to the second allele of the polymorphic sequence (allele B), and (c) being non-complementary to the nucleic acid sequence at one (and, preferably, two) or more nucleotides in its 3′-terminal nucleotides 2-6;
(b) carrying out the amplification reactions; and
(c) detecting an amplification product as an indication of the presence, in the nucleic acid sequence, of the first allele of the polymorphic sequence (allele A).
If desired, the method may involve the further steps of:
(a) contacting the nucleic acid sequence, in the same or a separate reaction, with a third pair of PCR primers and a fourth pair of PCR primers under conditions that allow hybridization of the PCR primers to the nucleic acid sequence, the third pair of PCR primers hybridizing to opposite strands of the nucleic acid sequence and bordering the position of the polymorphic sequence, and the fourth pair of PCR primers hybridizing to opposite strands of the nucleic acid sequence and bordering the position of the polymorphic sequence, the PCR primers being characterized as follows:
(i) one of the third pair of PCR primers (a) being complementary at its 3′-terminal nucleotide to the second allele of the polymorphic sequence (allele B), (b) being non-complementary at its 3′-terminal nucleotide to the first allele of the polymorphic sequence (allele A), and (c) being non-complementary to the nucleic acid sequence at a single nucleotide in its 3′-terminal nucleotides 2-6; and
(ii) one of the fourth pair of PCR primers (a) being complementary at its 3′-terminal nucleotide to the second allele of the polymorphic sequence (allele B), (b) being non-complementary at its 3′-terminal nucleotide to the first allele of the polymorphic sequence (allele A), and (c) being non-complementary to the nucleic acid sequence at one (and, preferably, two) or more nucleotides in its 3′-terminal nucleotides 2-6;
(b) carrying out the amplification reactions; and
(c) detecting an amplification product as an indication of the presence, in the nucleic acid sequence, of the second allele of the polymorphic sequence (allele B).
In a related aspect, the invention features kits for carrying out the method of the invention. One particular kit for determining whether a nucleic acid sequence includes a particular allele of a polymorphic sequence includes (a) a first pair of PCR primers and a second pair of PCR primers, the first pair of PCR primers hybridizing to opposite strands of the nucleic acid sequence and bordering the position of the polymorphic sequence, and the second pair of PCR primers hybridizing to opposite strands of the nucleic acid sequence and bordering the position of the polymorphic sequence, the PCR primers being characterized as follows: (i) one of the first pair of PCR primers (a) being complementary at its 3′-terminal nucleotide to a first allele of the polymorphic sequence (allele A), (b) being non-complementary at its 3′-terminal nucleotide to a second allele of the polymorphic sequence (allele B), and (c) being non-complementary to the nucleic acid sequence at a single non-complementary nucleotide in its 3′-terminal nucleotides 2-6; and (ii) one of the second pair of PCR primers (a) being complementary at its 3′-terminal nucleotide to the first allele of the polymorphic sequence (allele A), (b) being non-complementary at its 3′-terminal nucleotide to the second allele of the polymorphic sequence (allele B), and (c) being non-complementary to the nucleic acid sequence at one (and, preferably, two) or more nucleotides in its 3′-terminal nucleotides.
If desired, the kit may also include (a) a third pair of PCR primers and a fourth pair of PCR primers, the third pair of PCR primers hybridizing to opposite strands of said nucleic acid sequence and bordering the position of the polymorphic sequence, and the fourth pair of PCR primers hybridizing to opposite strands of the nucleic acid sequence and bordering the position of the polymorphic sequence, the PCR primers being characterized as follows: (i) one of the third pair of PCR primers (a) being complementary at its 3′-terminal nucleotide to the second allele of said polymorphic sequence (allele B), (b) being non-complementary at its 3′-terminal nucleotide to the first allele of the polymorphic sequence (allele A), and (c) being non-complementary to the nucleic acid sequence at a single nucleotide in its 3′-terminal nucleotides 2-6; and (ii) one of the fourth pair of PCR primers (a) being complementary at its 3′-terminal nucleotide to the second allele of the polymorphic sequence (allele B), (b) being non-complementary at its 3′-terminal nucleotid
Ausubel Frederick M.
Drenkard Eliana
Mindrinos Michael
Clark & Elbing LLP
Horlick Kenneth R.
The General Hospital Corporation
Tung Joyce
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