Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
The present invention relates to a method for identifying a target nucleotide sequence. This method involves forming a ligation product on a target nucleotide sequence in a ligation detection reaction mixture, amplifying the ligation product to form an amplified ligation product in a polymerase chain reaction (PCR) mixture, detecting the amplified ligation product, and identifying the target nucleotide sequence. Such coupling of the ligase detection reaction and the polymerase chain reaction permits multiplex detection of nucleic acid sequence difference.
1. A method comprising:
providing a sample potentially containing one or more target nucleotide sequences;
forming primary products in a reaction process comprising a polymerase reaction, wherein each primary product comprises a 5′ primer-specific portion, a target nucleotide sequence-specific portion, and a 3′ primer-specific portion, wherein the 5′ primer-specific portion and the 3′ primer-specific portion differ from each other and are not the same as or complementary to the target-specific portion;
providing one or more oligonucleotide primer sets, each oligonucleotide primer set characterized by a first and second oligonucleotide primer, wherein said first oligonucleotide primer is capable of hybridizing to the 3′ primer-specific portion of a primary product and said second oligonucleotide primer is capable of hybridizing to a 3′ primer specific portion of a complement of the primary product; and
forming secondary products in a polymerase reaction mixture comprising the one or more oligonucleotide primer sets, wherein said secondary products are complementary to each of the formed primary products or complements thereof.
2. The method of claim 1 further comprising:
sequencing the secondary products.
3. The method of claim 1 further comprising:
coupling a label to the secondary products and
detecting the label coupled to the secondary products formed in the polymerase chain reaction mixture.
4. The method of claim 3 further comprising:
distinguishing, based on said detecting, two or more target nucleotide sequences in the sample that differ by one or more single-base changes, insertions, deletion, or translocations.
5. The method of claim 3 further comprising:
sequencing, based on said detecting, the secondary products.
6. The method of claim 1 , wherein the primary products comprise a further portion.
7. The method of claim 6 , wherein the further portion of one primary product differs from the further portion of a different primary product.
8. The method of claim 1 , wherein the secondary products are immobilized on a solid support.
9. A method comprising:
providing a sample potentially containing one or more target nucleotide sequences and complements thereof;
adding a 5′ primer-specific portion and a 3′ primer-specific portion, which differ from each other, to the one or more target nucleotide sequences and complements thereof in the sample in a reaction process comprising a polymerase reaction to form one or more primer labeled target nucleotide sequences and complements thereof; and
forming extension products that are complementary to the one or more primer labeled target nucleotide sequences and complements thereof in a polymerase reaction mixture comprising one or more oligonucleotide primer sets, each oligonucleotide primer set characterized by a first and second oligonucleotide primer, wherein said first oligonucleotide primer is capable of hybridizing to a 3′ primer-specific portion of a primer labeled target nucleotide sequence and said second oligonucleotide primer is capable of hybridizing to a 3′ primer specific portion of the complement of the primer labeled target nucleotide sequence.
10. The method of claim 9 further comprising:
sequencing the extension products.
11. The method of claim 9 further comprising:
coupling a label to the extension products and
detecting the label coupled to the extension products formed in the polymerase reaction mixture.
12. The method of claim 11 further comprising:
distinguishing, based on said detecting, two or more target nucleotide sequences or complements thereof in the sample that differ by one or more single-base changes, insertions, deletion, or translocations.
13. The method of claim 11 further comprising:
sequencing, based on said detecting, the extension products.
14. The method of claim 9 , wherein the primer labeled target nucleotide sequences and complements thereof comprise a further portion.
15. The method of claim 14 , wherein the further portion of one primer labeled target nucleotide sequence or complement thereof differs from the further portion of a different primer labeled target nucleotide sequence or complement thereof.
16. The method of claim 9 , wherein the extension products are immobilized on a solid support.