Real time pcr detection of single nucleotide polymorphisms

Abstract

Disclosed are methods and kits for the detection of a polymorphism during real-time PCR. Real-time PCR amplification of a target nucleic acid sequence is performed using PCR primer primers that anneal to sequences flanking a single nucleotide polymorphism (SNP) of interest. The real-time PCR reaction includes a labeled probe comprising a RNA sequence that is designed to anneal to DNA sequences at the location of the SNP. An RNA:DNA heteroduplex can then form between the SNP in the PCR fragment and the probe's RNA sequences that are complementary to the SNP. RNase H cleavage of the RNA sequence in the RNA:DNA heteroduplex results in increase in intensity of the signal generated from the label that is indicative of the presence of an SNP in the target nucleic acid.

Description

technical field

[0001] This application claims U.S. Provisional Patent Application No. 61 / 389,412 filed on October 4, 2010, U.S. Provisional Patent Application No. 61 / 390,701 filed on October 7, 2010, and Priority to US Patent Application No. 13 / 158,593, which is hereby incorporated by reference in its entirety.

[0002] This disclosure describes the use of single nucleotide polymorphism (SNP) specific CataCleave TM Real-time PCR detection of probes to SNPs. Background technique

[0003] The completion of the Human Genome Project (HGP) has paved the way for a better understanding of genetic diversity within a given human population and how this diversity is related to the initiation and predisposition of genetic diseases. For example, single nucleotide differences between individuals known as single nucleotide polymorphisms (SNPs) can be responsible for large differences in phenotypes, which in some cases can predict who will develop a certain disease or Who will respond ...

Examples

example 1

[0189] Example 1: Isothermal detection of synthetic SNPs in the Salmonella invA gene

[0190] Construction of artificial single nucleotide polymorphisms (SNPs) in the invA gene (SEQ ID NO: 33) of Salmonella to test CataCleave TM The ability of a probe to distinguish single nucleotide sequence differences in a target DNA sequence. A single nucleotide change produced a T to G transversion at position 116 of the SalmonellainvA coding sequence (SEQ ID NO: 33). Design of two similar 19-nucleotide CataCleave TM Probes, wherein each probe is doubly labeled to generate FRET pairs such that the probes will base pair across the region of invA comprising the SNP nucleotide. The wild-type specific probe inv-CC probe 2 (SEQ ID NO: 1) is completely complementary to the wild-type sequence of invA, and the SNP-specific probe inv-CC probe 2-2C (SEQ ID NO: 2) is completely complementary to the invA The mutant form is fully complementary. Design probes such that CataCleave TM The second of ...

example 2

[0192] Example 2: Real-time PCR detection of synthetic SNPs in the Salmonella invA gene

[0193] Plasmid DNA containing a 267 nucleotide invA sequence containing wild-type or mutated bases (described above) was synthesized. 40 pg of wild-type plasmid, mutant plasmid or a mixture of both plasmids was used as template for multiplex real-time PCR reactions containing differentially labeled probes complementary to wild-type or mutant sequences. The final concentration of each component in the reaction was as follows: 800 nM forward primer Salmonella-F1 (SEQ ID NO: 5), 800 nM reverse primer sal-invR2 (SEQ ID NO: 6), 200 nM wild-type specific probe Needle inv-CC probe 2 (SEQ ID NO: 1), 200 nM SNP-specific probe inv-CC probe 2-2C (SEQ ID NO: 2), 80 uM each dNTP, 10 mM Tris acetic acid (pH 8.6) , 50 mM Potassium Acetate, 2.5 mM Magnesium Acetate, 1 mM DTT, 2.5u Platinum Taq DNA Polymerase (Life Technologies) and 2.5u Hybridase Thermostable RNase HI (Epicentre). The PCR reaction was ...

example 3

[0194] Example 3: Isothermal detection of the A1 and A2 alleles of the bovine b casein gene

[0195] Two completely natural variants or forms of b casein exist in the milk of cows, called A2 and A1b casein. The difference between A1 and A2b caseins is a single amino acid at position 67. In the A1 variant, the base is T, and in the A2 variant, the base is G. The A1 variant b casein in milk is unique among all mammalian b caseins in having a histidine at this position. Milk of other species containing beta-casein may be considered A2 category due to having proline at the equivalent position in their beta-casein chain. Buffalo, yak, goat, and human breast milk all contain the A2-form of beta-casein.

[0196] In this example, two similar 19-nucleotide CataCleaves were designed TM Probes, wherein each probe is doubly labeled to generate FRET pairs such that they will base pair across the position of the A1 / A2 SNP nucleotide in the bovine b casein gene. A1-CC probe 2-RC (SEQ ID...

technical field

[0001] This application claims U.S. Provisional Patent Application No. 61 / 389,412 filed on October 4, 2010, U.S. Provisional Patent Application No. 61 / 390,701 filed on October 7, 2010, and Priority to US Patent Application No. 13 / 158,593, which is hereby incorporated by reference in its entirety.

[0002] This disclosure describes the use of single nucleotide polymorphism (SNP) specific CataCleave TM Real-time PCR detection of probes to SNPs. Background technique

[0003] The completion of the Human Genome Project (HGP) has paved the way for a better understanding of genetic diversity within a given human population and how this diversity is related to the initiation and predisposition of genetic diseases. For example, single nucleotide differences between individuals known as single nucleotide polymorphisms (SNPs) can be responsible for large differences in phenotypes, which in some cases can predict who will develop a certain disease or Who will respond ...

Examples

example 1

[0189] Example 1: Isothermal detection of synthetic SNPs in the Salmonella invA gene

[0190] Construction of artificial single nucleotide polymorphisms (SNPs) in the invA gene (SEQ ID NO: 33) of Salmonella to test CataCleave TM The ability of a probe to distinguish single nucleotide sequence differences in a target DNA sequence. A single nucleotide change produced a T to G transversion at position 116 of the SalmonellainvA coding sequence (SEQ ID NO: 33). Design of two similar 19-nucleotide CataCleave TM Probes, wherein each probe is doubly labeled to generate FRET pairs such that the probes will base pair across the region of invA comprising the SNP nucleotide. The wild-type specific probe inv-CC probe 2 (SEQ ID NO: 1) is completely complementary to the wild-type sequence of invA, and the SNP-specific probe inv-CC probe 2-2C (SEQ ID NO: 2) is completely complementary to the invA The mutant form is fully complementary. Design probes such that CataCleave TM The second of ...

example 2

[0192] Example 2: Real-time PCR detection of synthetic SNPs in the Salmonella invA gene

[0193] Plasmid DNA containing a 267 nucleotide invA sequence containing wild-type or mutated bases (described above) was synthesized. 40 pg of wild-type plasmid, mutant plasmid or a mixture of both plasmids was used as template for multiplex real-time PCR reactions containing differentially labeled probes complementary to wild-type or mutant sequences. The final concentration of each component in the reaction was as follows: 800 nM forward primer Salmonella-F1 (SEQ ID NO: 5), 800 nM reverse primer sal-invR2 (SEQ ID NO: 6), 200 nM wild-type specific probe Needle inv-CC probe 2 (SEQ ID NO: 1), 200 nM SNP-specific probe inv-CC probe 2-2C (SEQ ID NO: 2), 80 uM each dNTP, 10 mM Tris acetic acid (pH 8.6) , 50 mM Potassium Acetate, 2.5 mM Magnesium Acetate, 1 mM DTT, 2.5u Platinum Taq DNA Polymerase (Life Technologies) and 2.5u Hybridase Thermostable RNase HI (Epicentre). The PCR reaction was ...

example 3

[0194] Example 3: Isothermal detection of the A1 and A2 alleles of the bovine b casein gene

[0195] Two completely natural variants or forms of b casein exist in the milk of cows, called A2 and A1b casein. The difference between A1 and A2b caseins is a single amino acid at position 67. In the A1 variant, the base is T, and in the A2 variant, the base is G. The A1 variant b casein in milk is unique among all mammalian b caseins in having a histidine at this position. Milk of other species containing beta-casein may be considered A2 category due to having proline at the equivalent position in their beta-casein chain. Buffalo, yak, goat, and human breast milk all contain the A2-form of beta-casein.

[0196] In this example, two similar 19-nucleotide CataCleaves were designed TM Probes, wherein each probe is doubly labeled to generate FRET pairs such that they will base pair across the position of the A1 / A2 SNP nucleotide in the bovine b casein gene. A1-CC probe 2-RC (SEQ ID...

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