Method for determining the likelihood that a test polynucleotide sequence differs from a driver polynucleotide
The present invention relates generally is a method for determining the likelihood that a test polynucleotide sequence differs from a driver polynucleotide sequence. More particularly, the present method uses fluorescence-based technology in the assessment of the results of competitive hybridization between polynucleotide sequences. The present method does not require nucleotide sequencing or gel electrophoresis and is capable of being multiplexed and automated. The methods of the present invention will find broad application in the analysis of polynucleotides, inter alia in genetic analysis, specific locus testing, genotyping, mutation detection, the discovery and detection of single nucleotide polymorphisms (SNPs) and mapping.
1. A method of determining a single nucleotide mutation or a single nucleotide difference between a test polynucleotide, from a first nucleic acid sample from a first subject, and a driver polynucleotide sequence, from a second nucleic acid sample from a second subject, the method comprising:
(a) producing the test polynucleotide which corresponds to the driver polynucleotide by amplification from the first nucleic acid sample with a pair of forward and reverse primers including a first label whereby the test polynucleotide is labeled with a first reporter molecule capable of providing a first identifiable signal;
(b) producing complementary driver and target polynucleotides by amplification from the second nucleic acid sample with a pair of forward and reverse primers that amplify the same region in (a) and include a second label whereby the driver polynucleotide is labeled with a second reporter molecule different from the first reporter molecule and capable of providing a second identifiable signal
(c) preparing a composition comprising the labeled test and driver polynucleotides in solution and a target polynucleotide immobilized on a solid support and unlabeled with the first or second reporter molecules wherein the ratio of labeled test to driver polynucleotides present in the composition is from 2:1 to 10:1; and
(d) flow cytometry screening to detect immobilized first and second identifiable signals after subjecting said composition to competitive hybridization conditions comprising a temperature gradient, wherein the driver polynucleotide will more preferentially hybridize to the target polynucleotide than said test polynucleotide when said test polynucleotide differs from said driver polynucleotide by at least one nucleotide;
(e) determining said test polynucleotide differs from said driver polynucleotide by at least one nucleotide when the (D:T)comp is greater than the (D:T)non-comp and said test polynucleotide sequence is not different from said driver polynucleotide sequence when (D:T)comp is not greater than the (D:T)non-comp,
wherein
D is the driver-label;
T is the test-label;
(D:T)comp is the D:T ratio under the competitive hybridization conditions; and
(D:T)non-comp is the D:T ratio under the non-competitive hybridization conditions
wherein the complete nucleotide sequence of either the test polynucleotide and/or the driver polynucleotide is unknown.
2. The method of claim 1 , wherein the single base mutation is centrally-located in said test polynucleotide.
3. The method of claim 1 , wherein the single base mutation is less than 10 bases from either end of the test polynucleotide.
4. The method of claim 1 , wherein the non-competitive hybridization conditions is from 25° C.-42° C.
5. The method of claim 1 , wherein the competitive hybridization conditions is at least 65° C.
6. The method of claim 1 , wherein the temperature gradient comprises a period of about 0.2-10 minutes per degree drop.
7. The method of claim 1 , wherein the temperature gradient is from about Tm+10° C. to about Tm−20 to −25° C.
8. The method of claim 1 , wherein the amounts of the target, driver and test polynucleotides are provided in a ratio of 1:2:10.
9. The method of claim 1 , wherein the amounts of the driver and test polynucleotides are provided in a ratio of 1:5.
10. The method of claim 1 wherein the single base mutation is in one allele.