Method for identifying polymorphisms
The present invention relates to methods for the detection of polymorphism in polynucleotides by using hybridization of fragments of segments of a polynucleotide suspected of containing a polymorphism with an oligonucleotide having a sequence complementary to a fragment identifying the polymorphism and subsequent detection of incorporated labels in the oligonucleotide-fragment duplex.
1 . A method for detecting polymorphism in a polynucleotide, comprising:
providing a polynucleotide suspected of containing a polymorphism;
amplifying a segment of the polynucleotide encompassing the suspected polymorphism wherein amplification comprises replacing one or more natural nucleotide(s), one of which is a nucleotide involved in the suspected polymorphism, at substantially each point of occurrence in the segment with a modified nucleotide or, if more than one natural nucleotide is replaced, with different modified nucleotides to form an amplified modified segment;
cleaving the amplified modified segment into fragments by contacting it with a reagent or reagents that cleave(s) the segment at substantially each point of occurrence of the modified nucleotide(s);
hybridizing the fragments to an oligonucleotide; and,
analyzing the hybridized fragments for an incorporated detectable label identifying the suspected polymorphism.
2 . The method of claim 1 , wherein the detectable label is incorporated during amplification.
3 . The method of claim 2 , wherein incorporating the detectable label during amplification comprises using a detectably labeled primer.
4 . The method of claim 3 , wherein the detectably labeled primer comprises a radioactive primer or a primer containing a fluorophore.
5 . The method of claim 1 , wherein incorporating the detectable label during amplification comprises using a detectably labeled, modified nucleotide.
6 . The method of claim 5 , wherein the detectably labeled, modified nucleotide comprises a radioactive modified nucleotide or a modified nucleotide containing a fluorophore.
7 . The method of claim 5 , wherein the detectably labeled, modified nucleotide is a detectably labeled, modified ribonucleotide.
8 . The method of claim 7 , wherein the detectably labeled, modified ribonucleotide comprises a radioactive modified ribonucleotide or a modified ribonucleotide containing a fluorophore.
9 . The method of claim 1 , wherein incorporating the detectable label during amplification comprises replacing a natural nucleotide, that is different than the natural nucleotide(s) being replaced with a modified nucleotide(s), at one or more point(s) of occurrence in the segment with a detectably labeled nucleotide.
10 . The method of claim 9 , wherein the detectably labeled nucleotide comprises a radioactive nucleotide or a nucleotide containing a fluorophore.
11 . The method of claim 9 , wherein the detectably labeled nucleotide comprises a detectably labeled ribonucleotide.
12 . The method of claim 11 , wherein the detectably labeled ribonucleotide comprises a radioactive ribonucleotide or a ribonucleotide containing a fluorophore.
13 . The method of claim 1 , wherein the detectable label is incorporated during cleavage.
14 . The method of claim 13 , wherein incorporating the detectable label during cleavage comprises using detectably labeled tris(carboxyethyl)phosphine (TCEP).
15 . The method of claim 14 , wherein using detectably labeled TCEP comprises using radioactive TCEP or TCEP containing a fluorophore.
16 . The method of claim 13 , wherein incorporating the detectable label during cleavage comprises using a detectably labeled secondary amine.
17 . The method of claim 16 , wherein using a detectably labeled secondary amine comprises using a radioactive secondary amine or a secondary amine containing a fluorophore.
18 . The method of claim 1 , wherein the detectable label is incorporated during hybridization.
19 . The method of claim 18 , wherein incorporating the detectable label during hybridization comprises hybridizing a second, detectably labeled oligonucleotide to the fragments hybridized to the oligonucleotide.
20 . The method of claim 19 , wherein the second, detectably labeled oligonucleotide comprises a radioactive oligonucleotide or an oligonucleotide containing a fluorophore.
21 . The method of claim 1 , wherein the detectable label is incorporated after cleavage or after hybridization, the method comprising:
cleaving using a reagent comprising TCEP or a secondary amine; and,
substituting the TCEP or secondary amine with a radioactive molecule or a fluorophore after cleavage or after hybridization.
22 . The method of claim 1 , wherein the polymorphism is selected from the group consisting of a single nucleotide polymorphism (SNP), a deletion or an insertion.
23 . The method of claim 1 , wherein amplifying the segment comprises a polymerase chain reaction (PCR).
24 . The method of claim 1 , wherein amplifying the segment comprises replacing one natural nucleotide that is involved in the suspected polymorphism at each point of occurrence in the segment with a modified nucleotide to form a modified segment.
25 . The method of claim 24 , wherein the modified nucleotide comprises a labeled, modified nucleotide.
26 . The method of claim 25 , wherein the labeled modified nucleotide comprises a radioactive modified nucleotide or a modified nucleotide containing a fluorophore.
27 . The method of claim 24 , wherein the modified nucleotide comprises a modified ribonucleotide.
28 . The method of claim 24 , wherein the modified nucleotide comprises a labeled, modified ribonucleotide.
29 . The method of claim 28 , wherein the labeled, modified ribonucleotide comprises a radioactive ribonucleotide or a ribonucleotide containing a fluorophore.
30 . The method of claim 1 , wherein hybridizing the fragments to an oligonucleotide comprises using an oligonucleotide that is immobilized on a solid support.
31 . The method of claim 1 , wherein the incorporated detectable label comprises fluorescence resonance energy transfer (FRET).