Detection of nucleic acid sequence differences using coupled 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;
providing one or more primary oligonucleotide primer sets, each set characterized by (a) a first oligonucleotide primer, having a target-specific portion and a 5′ upstream secondary primer-specific portion, and (b) a second oligonucleotide primer, having a target-specific portion and a 5′ upstream secondary primer-specific portion;
subjecting a primary polymerase chain reaction mixture, comprising the sample, the one or more primary oligonucleotide primer sets, and a polymerase, to two or more polymerase chain reaction cycles to form primary extension products;
providing one or a plurality of secondary oligonucleotide primer sets, each set characterized by (a) a first secondary primer containing the same sequence as the 5′ upstream portion of a first primary oligonucleotide primer, and (b) a second secondary primer containing the same sequence as the 5′ upstream portion of a second primary oligonucleotide primer from the same primary oligonucleotide primer set as the first primary oligonucleotide primer contained by the first secondary primer; and
subjecting a secondary polymerase chain reaction mixture, comprising the primary extension products, the one or plurality of secondary oligonucleotide primer sets, and the polymerase to two or more polymerase chain reaction cycles to form secondary extension products complementary to the primary extension products.
2. The method of claim 1 further comprising:
sequencing the secondary extension products.
3. The method of claim 1 further comprising:
coupling a label to the secondary extension products and
detecting the label coupled to the secondary extension products.
4. The method of claim 3 , wherein said detecting comprises:
distinguishing two or more of a plurality of 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 , wherein said detecting comprises:
sequencing the secondary extension products.
6. The method of claim 1 , wherein one of the primary oligonucleotide primers of a primary oligonucleotide primer set comprises a further portion.
7. The method of claim 6 , wherein the further portion of one primary primer in a primary oligonucleotide primer set differs from the further portion of another primary primer in a different primary oligonucleotide primer set.
8. The method of claim 1 , wherein the secondary extension products are immobilized on a solid support.
9. A method comprising:
providing a sample potentially containing one or more target nucleotide sequences;
forming primary products complementary to each of the one or more target nucleotide sequences in a reaction mixture comprising one or more primary oligonucleotide primer sets, each primary oligonucleotide primer set characterized by a first primary oligonucleotide primer comprising a 5′ secondary primer-specific portion and a second primary oligonucleotide primer comprising a 5′ secondary primer-specific portion, wherein each primary product comprises a 5′ secondary primer-specific portion, a target-specific portion, and a 3′ secondary primer-specific portion, wherein the sequence of the 5′ secondary primer-specific portion differs from the sequence of the 3′ secondary primer-specific portion; and
forming secondary extension products that are complementary to each of the primary products in a secondary reaction mixture comprising one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set characterized by a first and second secondary oligonucleotide primer, each first and second secondary primer capable of hybridizing to a 3′ secondary primer-specific portion of primary products formed from a primary oligonucleotide primer set.
10. The method of claim 9 further comprising:
sequencing the secondary extension products.
11. The method of claim 9 further comprising:
coupling a label to the secondary extension products and
detecting the label coupled to the secondary extension products formed in the secondary reaction mixture.
12. The method of claim 11 , wherein said detecting comprises:
distinguishing two or more target nucleotide sequences in the sample that differ by one or more single-base changes, insertions, deletion, or translocations.
13. The method of claim 11 , wherein said detecting comprises:
sequencing the secondary extension products.
14. The method of claim 9 , wherein the primary products comprise a further portion.
15. The method of claim 14 , wherein the further portion of one primary product differs from the further portion of a different primary product.
16. The method of claim 9 , wherein the secondary extension products are immobilized on a solid support.