Compositions, methods and kits for allele discrimination
First Claim
1. A purified polynucleotide selected from the group consisting of SEQ ID NOS. 1-31.
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Abstract
The invention relates to sequence-specific polynucleotide probes, pairs of probes, the design of pairs of probes in relation to the strands of target nucleic acid, and coordinate sequence-specific pairs of polynucleotide primers, and also relates to kits containing such probes, probe pairs, primers, and primer pairs. The invention thus relates to kits and methods employing such polynucleotides. The invention relates to six specific allele discrimination kits. The specific kits are developed for the detection of four single base substitutions in the human CCR2, SDF1, Factor V, MTHFR, Factor XIII genes, and a 32-bp deletion in the human CCR5 gene. The capability of using two allele-specific molecular beacons in the same PCR solution enables the simultaneous determination of three possible allelic representations of a given sequence change in target DNA (Allele 1/Allele 1, Allele 2/Allele 2, Allele 1/Allele 2). It also definitively differentiates a true negative signal that is due to the absence of an allele from a false negative signal that results from PCR failure.
9 Citations
37 Claims
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1. A purified polynucleotide selected from the group consisting of SEQ ID NOS. 1-31.
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2. A pair of polynucleotides for allele discrimination, said polynucleotides selected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 2;
- SEQ ID NO.1 and SEQ ID NO. 4;
SEQ ID NO. 3 and SEQ ID NO. 2;
SEQ ID NO. 3 and SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6;
SEQ ID NO. 7 and SEQ ID NO. 8;
SEQ ID NO. 9 and SEQ ID NO. 10;
SEQ ID NO. 11 and SEQ ID NO. 12; and
SEQ ID NO. 30 and SEQ ID NO 31. - View Dependent Claims (3, 4, 6, 9, 10, 11, 12, 13, 14, 29, 34)
- SEQ ID NO.1 and SEQ ID NO. 4;
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5. A pair of polynucleotide primers for a polymerase chain reaction selected from the group consisting of SEQ ID NO. 13 and SEQ ID NO. 14;
- SEQ ID NO. 15 and SEQ ID NO. 16;
SEQ ID NO. 17 and SEQ ID NO. 18;
SEQ ID NO. 19 and SEQ ID NO. 20;
SEQ ID NO. 19 and SEQ ID NO. 21;
SEQ ID NO. 22 and SEQ ID NO. 23;
SEQ ID NO. 24 and SEQ ID NO. 25;
SEQ ID NO. 26 and SEQ ID NO. 27; and
SEQ ID NO. 28 and SEQ ID NO. 29. - View Dependent Claims (7, 8, 33, 36, 37)
- SEQ ID NO. 15 and SEQ ID NO. 16;
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15. A kit for allele discrimination, comprising a pair of polynucleotides for allele discrimination of the CCR2 gene, wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO:
- 1 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NOS;
2 or 4, a pair of polynucleotides for PCR of a region of the CCR2 gene wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO;
13 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
14, a DNA polymerase, and a buffer suitable for allele discrimination and polymerase chain reaction. - View Dependent Claims (25, 26, 27)
- 1 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NOS;
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16. A kit for allele discrimination, comprising a pair of polynucleotides for allele discrimination of the CCR2 gene, wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NOS:
- 3 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NOS;
2 or 4, a pair of polynucleotides for PCR of a region of the CCR2 gene wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO;
13 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
14, a DNA polymerase, and a buffer suitable for allele discrimination and polymerase chain reaction.
- 3 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NOS;
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17. A kit for allele discrimination, comprising a pair of polynucleotides for allele discrimination of the CCR5 gene, wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO:
- 5 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
6, a pair of polynucleotides for PCR of a region of the CCR5 gene wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO;
15 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
16, a DNA polymerase, and a buffer suitable for allele discrimination and polymerase chain reaction.
- 5 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
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18. A kit for allele discrimination, comprising a pair of polynucleotides for allele discrimination of the CCR5 gene, wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO:
- 5 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
6, a pair of polynucleotides for PCR of a region of the CCR5 gene wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO;
17 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
18, a DNA polymerase, and a buffer suitable for allele discrimination and polymerase chain reaction.
- 5 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
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19. A kit for allele discrimination, comprising a pair of polynucleotides for allele discrimination of the SDF1 gene, wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO:
- 7 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
8, a pair of polynucleotides for PCR of a region of the SDF1 gene wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO;
19 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NOS;
20, a DNA polymerase, and a buffer suitable for allele discrimination and polymerase chain reaction.
- 7 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
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20. A kit for allele discrimination, comprising a pair of polynucleotides for allele discrimination of the SDF1 gene, wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO:
- 7 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
8, a pair of polynucleotides for PCR of a region of the SDF1 gene wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO;
19 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NOS;
21, a DNA polymerase, and a buffer suitable for allele discrimination and polymerase chain reaction.
- 7 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
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21. A kit for allele discrimination, comprising a pair of polynucleotides for allele discrimination of the Factor V gene, wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO:
- 9 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
10, a pair of polynucleotides for PCR of a region of the Factor V gene wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO;
22 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
23, a DNA polymerase, and a buffer suitable for allele discrimination and polymerase chain reaction.
- 9 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
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22. A kit for allele discrimination, comprising a pair of polynucleotides for allele discrimination of the MTHFR gene, wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO:
- 11 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
12, a pair of polynucleotides for PCR of a region of the MTHFR gene wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO;
24 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
25, a DNA polymerase, and a buffer suitable for allele discrimination and polymerase chain reaction. - View Dependent Claims (28, 30, 31, 32)
- 11 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
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23. A kit for allele discrimination, comprising a pair of polynucleotides for allele discrimination of the MTHFR gene, wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO:
- 11 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
12, a pair of polynucleotides for PCR of a region of the MTHFR gene wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO;
26 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
27, a DNA polymerase, and a buffer suitable for allele discrimination and polymerase chain reaction.
- 11 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
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24. A kit for allele discrimination, comprising a pair of polynucleotides for allele discrimination of the Factor XIII gene, wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO:
- 30 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
31, a pair of polynucleotides for PCR of a region of the Factor XIII gene wherein a first polynucleotide of said pair has the sequence presented in SEQ ID NO;
28 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
29, a DNA polymerase, and a buffer suitable for allele discrimination and polymerase chain reaction.
- 30 and a second polynucleotide of said pair of polynucleotides has the sequence presented in SEQ ID NO;
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35. A pair of polynucleotide probes wherein a first probe of said pair is complementary to the positive strand of a DNA duplex and a second probe of said pair is complementary to the negative strand of said DNA duplex.
Specification