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:
subjecting a reaction mixture to a ligation process to produce one or more ligation products, wherein the reaction mixture comprises:
a ligase;
one or more target nucleotide sequences; and
one or more single-stranded oligonucleotide probe sets, each probe set comprising (a) a first single-stranded oligonucleotide probe comprising a first target-specific portion capable of hybridizing to a first portion of a corresponding target nucleotide sequence and (b) a second single-stranded oligonucleotide probe comprising a second target-specific portion capable of hybridizing to a second portion of the corresponding target nucleotide sequence, wherein each of the one or more ligation products comprises a ligated sequence which includes (1) the first target-specific portion of the first oligonucleotide probe in a corresponding probe set and (2) the second target-specific portion of the second oligonucleotide probe in the corresponding probe set; and
subjecting the one or more ligation products to one or more polymerase chain reaction cycles to produce one or more extension products comprising the ligation products and/or complements thereof.
2. The method according to claim 1 , wherein the one or more extension products are used to identify one or more target nucleotide sequences in the sample.
3. The method according to claim 1 , wherein the target-specific portions of the oligonucleotide probes each have a hybridization temperature of 50-85° C.
4. The method according to claim 1 , wherein the target-specific portions of the oligonucleotide probes are 20-28 nucleotides long.
5. The method according to claim 1 , wherein the oligonucleotide probe sets are selected from the group consisting of ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar backbone oligonucleotides, nucleotide analogues, and mixtures thereof.
6. The method according to claim 1 , wherein the ligase is selected from the group consisting of a Thermus aquaticus ligase, a Thermus thermophilus ligase, an E. coli ligase, T4 ligase, and a Pyrococcus ligase.
7. A method comprising:
forming a ligation product from two single-stranded oligonucleotide probes hybridized to complementary regions of a target nucleotide sequence in a reaction mixture, wherein the ligation product comprises an upstream primer portion and a downstream primer portion, wherein the upstream primer portion and the downstream primer portion are not complementary with the target nucleotide sequence; and
amplifying the ligation product to form an amplified ligation product in a polymerase chain reaction (PCR) mixture, wherein the PCR mixture comprises an upstream primer that binds to the upstream primer portion of the ligation product and a downstream primer that binds to the downstream primer portion of the ligation product.
8. The method according to claim 7 , wherein the amplified ligation product is used to identify the target nucleotide sequence.
9. The method according to claim 7 , wherein the ligation product results from the ligation of two single-stranded oligonucleotide probes, wherein the two single-stranded oligonucleotide probes are two different molecules.
10. The method according to claim 7 , wherein the upstream primer or the downstream primer contains a label.
11. The method according to claim 7 , wherein the target nucleotide sequence is one of a plurality of nucleotide sequences comprising one or more single-base changes, insertions, deletions, or translocations.
12. The method according to claim 1 , wherein the target nucleotide sequence is one of a plurality of nucleotide sequences comprising one or more single-base changes, insertions, deletions, or translocations.