Method to determine single nucleotide polymorphisms and mutations in nucleic acid sequence
A genotyping method and a prepared oligomicroarray as device is to determine single nucleotide polymorphism (SNP) and mutations are provided. The method uses two specific APEX-2 primers per each SNP or mutations to be determined. The same primers are used in amplification phase (primer extension and PCR with universal primer) and in the single base extension phase on an array. All SNP-containing sequences can be genotyped and amplified in one reaction tube and visualized on a microarray.
1. A method to determine single nucleotide polymorphism (SNP) in a target nucleic acid sequence, said method comprising the steps of:
a) providing a template nucleic acid, consisting of a first and a second strand and having one unknown nucleotide pair;
b) APEX-2 (Arrayed Primer Extension using Universal primer) oligonucleotide primer;
said first APEX-2 primer consisting of a first specific oligonucleotide sequence in its 3′-end and a universal sequence in its 5′end, said universal sequence further having a modification in its 5′end, and said first specific oligonucleotide sequence being complementary to nucleotides before the unknown nucleotide in the first strand of the template nucleic acid;
said second APEX-2 primer consisting of a second specific oligonucleotide sequence in its 3′-end and a universal sequence in its 5′end, said universal sequence optionally having a modification in its 5′end and, said second specific oligonucleotide sequence being complementary to nucleotides before the unknown nucleotide in the second strand of the template nucleic acid;
c) amplifying the unknown nucleotide of the first and the second strand of the template nucleic acid by running a multiplex PCR primer extension reaction with the first and the second APEX-2 primers as templates, thereby producing a first amplification product, said first amplification product consisting of a first and a second strand, said first strand consisting of sequence of the first APEX-2 primer and a complementary sequence to the second APEX-2 primer and the unknown nucleotide between them, and the second strand consisting of complementary sequence to the APEX-2 first primer and the sequence of the second APEX-2 primer and the unknown nucleotide between them;
d) amplifying the first amplification product in a PCR reaction with universal primer as template, thereby producing a second amplification product;
e) providing a microarray of probes, where probes are identical to the first and the second APEX-2 primers of step b) and where the probes are immobilized on the array by attaching them to a solid surface from the modified 5′end;
f) letting the second amplification products anneal with immobilized probes and providing labeled or modified terminator nucleotides for single base extension reaction; and
g) identifying terminating nucleotide that has been added to the probes, said terminating nucleotides corresponding to the unknown nucleotides of the template nucleic acid.
2. The method according to claim 1 , wherein the template nucleic acid has a first and a second pair of unknown nucleotides in proximity of 2 to 25 bp to each other and wherein the first specific oligonucleotide sequence is complementary to nucleotides before unknown nucleotide of the first nucleotide pair on the first nucleic acid strand and the second specific oligonucleotide sequence is complementary to nucleotides before unknown nucleotide of the second nucleotide pair of the second nucleic acid strand.
3. The method according to claim 1 , wherein the universal sequence of both the first and the second APEX-2 primers have a modification in its 5′ end.
4. The method of claim 1 , wherein only two cycles of amplification are used to produce the first amplification product in step c).
5. The method of claim 1 , wherein the modification in the 5′end of the first specific primer and/or the second specific primer are attached with a linker.
6. The method of claim 5 , wherein the linker is Amine-C6.
7. The method of claim 1 , wherein the template nucleic acid is selected from a group consisting of genomic DNA, cDNA and RNA.
8. The method of claim 1 , wherein the solid surface is made of a material selected from the group consisting of glass, plastic, beads, and paper.
9. The method of claim 1 , wherein the unknown nucleotide pair is di, -tri or quatro allelic mutation or SNP.
10. The method of claim 1 , wherein the unknown nucleotide pair is an insertion and the identifying terminating nucleotide of step g) corresponds to first nucleotide of insertion sequence.
11. The method of claim 1 , wherein the unknown nucleotide pair is a deletion and the identifying terminating nucleotide of step g) corresponds to first nucleotide of opposite APEX-2 primer.
12. The method according to claim 1 , wherein the universal sequence is according to SEQ ID NO:1.
13. The method according to claim 2 , wherein the universal sequence of both the first and the second APEX-2 primers have a modification in its 5′ end.
14. The method of claim 2 , wherein only two cycles of amplification are used to produce the first amplification product in step c).
15. The method of claim 2 , wherein the modification in the 5′end of the first specific primer and/or the second specific primer are attached with a linker.
16. The method of claim 2 , wherein the template nucleic acid is selected from a group consisting of genomic DNA, cDNA and RNA.
17. The method of claim 2 , wherein the solid surface is made of a material selected from the group consisting of glass, plastic, beads, and paper.
18. The method of claim 2 , wherein the unknown nucleotide pair is di, -tri or quatro allelic mutation or SNP.
19. The method of claim 2 , wherein the unknown nucleotide pair is an insertion and the identifying terminating nucleotide of step g) corresponds to first nucleotide of insertion sequence.
20. The method of claim 2 , wherein the unknown nucleotide pair is a deletion and the identifying terminating nucleotide of step g) corresponds to first nucleotide of opposite APEX-2 primer.
21. The method according to claim 2 , wherein the universal sequence is according to SEQ ID NO: 1.