Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
The present invention describes a method for identifying one or more of a plurality of sequences differing by one or more single base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences. The method includes a ligation phase, a capture phase, and a detection phase. The ligation phase utilizes a ligation detection reaction between one oligonucleotide probe, which has a target sequence-specific portion and an addressable array-specific portion, and a second oligonucleotide probe, having a target sequence-specific portion and a detectable label. After the ligation phase, the capture phase is carried out by hybridizing the ligated oligonucleotide probes to a solid support with an array of immobilized capture oligonucleotides at least some of which are complementary to the addressable array-specific portion. Following completion of the capture phase, a detection phase is carried out to detect the labels of ligated oligonucleotide probes hybridized to the solid support.
1. A group of beads, wherein each bead of the group comprises one type of capture oligonucleotide from a collection of capture oligonucleotides, wherein each type of capture oligonucleotide is 20-24 nucleotides in length, and wherein each type of capture oligonucleotide comprises a nucleotide sequence that differs from the nucleotide sequence of other types of capture oligonucleotides of the collection by at least 25% when aligned, and wherein each capture oligonucleotide in the collection hybridizes to a nucleic acid molecule comprising a complementary nucleotide sequence under uniform hybridization conditions, wherein hybridized capture oligonucleotides comprise a melting temperature (T m ) that is >55° C. when calculated using the formula T m =4(G+C)+2(A+T) ° C.
2. The group of beads of claim 1 , wherein each type of capture oligonucleotide hybridizes to the nucleic acid molecule comprising a complementary nucleotide sequence at a temperature of 60° C.
3. The group of beads of claim 1 , wherein each type of capture oligonucleotide hybridizes to the nucleic acid molecule comprising a complementary nucleotide sequence at a temperature of 65° C.
4. The group of beads of claim 1 , wherein each type of capture oligonucleotide hybridizes to the nucleic acid molecule comprising a complementary nucleotide sequence at a temperature of 70° C.
5. The group of beads of claim 1 , wherein each type of capture oligonucleotide is 24 nucleotides in length.
6. The group of beads of claim 1 , wherein the beads are configured on an addressable array.
7. The group of beads of claim 1 , wherein each type of capture oligonucleotide within the collection of oligonucleotides exhibits minimal cross-hybridization to a complement of another type of capture oligonucleotide within the collection at 35° C.
8. The group of beads of claim 1 , wherein each type of capture oligonucleotide has a sequence configured to hybridize to the nucleic acid molecule having a complementary nucleotide sequence when incubated for 15 minutes at 60° C. in a solution comprising 0.5 M Na 2 HPO 4 having a pH of 7.2, 1% BSA, 1 mM EDTA, and 7% SDS.
9. The group of beads of claim 8 , wherein each type of capture oligonucleotide has a sequence configured to remain hybridized to its complementary nucleic acid molecule following washing at 60° C. in a low stringency wash buffer comprising 300 mM sodium chloride, 30 mM sodium citrate, and 0.1% SDS, and washing at 60° C. in a high stringency wash buffer comprising 30 mM sodium chloride, 3 mM sodium citrate, and 0.1% SDS.
10. The group of beads of claim 8 , wherein each type of capture oligonucleotide has a sequence configured to remain hybridized to its complementary nucleic acid molecule following washing at 65° C. in a low stringency wash buffer comprising 300 mM sodium chloride, 30 mM sodium citrate, and 0.1% SDS, and washing at 65° C. in a high stringency wash buffer comprising 30 mM sodium chloride, 3 mM sodium citrate, and 0.1% SDS.
11. The group of beads of claim 8 , wherein each type of capture oligonucleotide has a sequence configured to remain hybridized to its complementary nucleic acid molecule following washing at 70° C. in a low stringency wash buffer comprising 300 mM sodium chloride, 30 mM sodium citrate, and 0.1% SDS, and washing at 70° C. in a high stringency wash buffer comprising 30 mM sodium chloride, 3 mM sodium citrate, and 0.1% SDS.
12. The group of beads of claim 1 , wherein each type of capture oligonucleotide has a sequence configured to hybridize to the nucleic acid molecule having a complementary nucleotide sequence when incubated for 15 minutes at 65° C. in a solution comprising 0.5 M Na 2 HPO 4 having a pH of 7.2, 1% BSA, 1 mM EDTA, and 7% SDS.
13. The group of beads of claim 1 , wherein each type of capture oligonucleotide has a sequence configured to hybridize to the nucleic acid molecule having a complementary nucleotide sequence when incubated for 15 minutes at 70° C. in a solution comprising 0.5 M Na 2 HPO 4 having a pH of 7.2, 1% BSA, 1 mM EDTA, and 7% SDS.
14. A group of beads, wherein each bead of the group comprises one type of capture oligonucleotide from a collection of capture oligonucleotides, wherein each type of capture oligonucleotide is 20-24 nucleotides in length and comprises a guanosine/cytosine (GC) content that is >50%, and wherein each type of capture oligonucleotide comprises a nucleotide sequence that differs from the nucleotide sequence of other types of capture oligonucleotides of the collection by at least 25% when aligned, and wherein each capture oligonucleotide in the collection hybridizes to a nucleic acid molecule comprising a complementary nucleotide sequence under uniform hybridization conditions.
15. The group of beads of claim 14 , wherein each type of capture oligonucleotide hybridizes to the nucleic acid molecule comprising a complementary nucleotide sequence at a temperature of 60° C.
16. The group of beads of claim 14 , wherein each type of capture oligonucleotide hybridizes to the nucleic acid molecule comprising a complementary nucleotide sequence at a temperature of 65° C.
17. The group of beads of claim 14 , wherein each type of capture oligonucleotide hybridizes to the nucleic acid molecule comprising a complementary nucleotide sequence at a temperature of 70° C.
18. The group of beads claim 14 , wherein each type of capture oligonucleotide is 24 nucleotides in length.
19. The group of beads of claim 14 , wherein the beads are configured on an addressable array.
20. The group of beads of claim 14 , wherein each type of capture oligonucleotide within the collection of oligonucleotides exhibits minimal cross-hybridization to a complement of another type of capture oligonucleotide within the collection at 35° C.
21. The group of beads of claim 14 , wherein each type of capture oligonucleotide has a sequence configured to hybridize to a nucleic acid molecule having a complementary nucleotide sequence when incubated for 15 minutes at 60° C. in a solution comprising 0.5 M Na 2 HPO 4 having a pH of 7.2, 1% BSA, 1 mM EDTA, and 7% SDS.
22. The group of beads of claim 21 , wherein each type of capture oligonucleotide has a sequence configured to remain hybridized to its complementary nucleic acid molecule following washing at 60° C. in a low stringency wash buffer comprising 300 mM sodium chloride, 30 mM sodium citrate, and 0.1% SDS, and washing at 60° C. in a high stringency wash buffer comprising 30 mM sodium chloride, 3 mM sodium citrate, and 0.1% SDS.
23. The group of beads of claim 21 , wherein each type of capture oligonucleotide has a sequence configured to remain hybridized to its complementary nucleic acid molecule following washing at 65° C. in a low stringency wash buffer comprising 300 mM sodium chloride, 30 mM sodium citrate, and 0.1% SDS, and washing at 65° C. in a high stringency wash buffer comprising 30 mM sodium chloride, 3 mM sodium citrate, and 0.1% SDS.
24. The group of beads of claim 21 , wherein each type of capture oligonucleotide has a sequence configured to remain hybridized to its complementary nucleic acid molecule following washing at 70° C. in a low stringency wash buffer comprising 300 mM sodium chloride, 30 mM sodium citrate, and 0.1% SDS, and washing at 70° C. in a high stringency wash buffer comprising 30 mM sodium chloride, 3 mM sodium citrate, and 0.1% SDS.
25. The group of beads of claim 14 , wherein each type of capture oligonucleotide has a sequence configured to hybridize to a nucleic acid molecule having a complementary nucleotide sequence when incubated for 15 minutes at 65° C. in a solution comprising 0.5 M Na 2 HPO 4 having a pH of 7.2, 1% BSA, 1 mM EDTA, and 7% SDS.
26. The group of beads of claim 14 , wherein each type of capture oligonucleotide has a sequence configured to hybridize to a nucleic acid molecule having a complementary nucleotide sequence when incubated for 15 minutes at 70° C. in a solution comprising 0.5 M Na 2 HPO 4 having a pH of 7.2, 1% BSA, 1 mM EDTA, and 7% SDS.