Compositions and methods for amplifying a nucleic acid sequence comprising a primer oligonucleotide with a 3′-terminal region comprising a 2′-modified nucleotide
View Patent ↗The present invention features compositions and methods for amplifying a target oligonucleotide in a sample comprising one or more primer oligonucleotides comprising a 5′ nicking enzyme recognition site and a 3′-terminal region comprising a 2′-modified nucleotide. These methods are compatible with target oligonucleotides amplified using a nicking amplification reaction.
1. A method of amplifying a polynucleotide, the method comprising:
(a) contacting a target nucleic acid molecule under substantially isothermal conditions with a polymerase, two or more primer oligonucleotides, each of which specifically binds to a complementary sequence on the target nucleic acid molecule, and a nicking enzyme, wherein each of the primer oligonucleotides comprises one or more 2′ modified nucleotides positioned at the 3′ end of the sequence complementary to the target nucleic acid molecule; and
(b) generating amplicons comprising at least a portion of said target nucleic acid molecule.
2. The method of claim 1 , further comprising detecting the amplicons.
3. The method of claim 2 , wherein the detection comprises binding the amplicons with a detectable probe.
4. The method of claim 1 , wherein one or more 2′ modified nucleotides positioned at the 5′ terminus of the sequence complementary to the target nucleic acid molecule are separated from the nick site by 1, 2, 3, 4, 5 or more unmodified nucleotides.
5. The method of claim 1 , wherein two or more 2′ modified nucleotides are contiguous.
6. The method of claim 1 , wherein 5 contiguous 2′-O-methyl modified nucleotides are positioned at the 3′ end of the sequence complementary to the target nucleic acid molecule.
7. The method of claim 1 , wherein 5 contiguous 2′-O-methyl modified nucleotides are positioned at the 5′ end of the sequence complementary to the target nucleic acid molecule.
8. A method of amplifying a polynucleotide, the method comprising:
(a) contacting a target nucleic acid molecule under substantially isothermal conditions with a polymerase, two primer oligonucleotides, each of which specifically binds to a complementary sequence on the target nucleic acid molecule, and a nicking enzyme, wherein each of the primer oligonucleotides comprises 5 contiguous 2′-O-methyl modified nucleotides positioned at the 3′ end of the sequence complementary to the target nucleic acid molecule; and
(b) generating amplicons comprising at least a portion of said target nucleic acid molecule.
9. A method of amplifying a polynucleotide derived from a pathogen, the method comprising:
(a) contacting a polynucleotide derived from a pathogen under substantially isothermal conditions with a polymerase, two or more primer oligonucleotides, each of which specifically binds to a complementary sequence on the target nucleic acid molecule, and a nicking enzyme, wherein each of the primer oligonucleotides comprises one or more 2′ modified nucleotides positioned at the 3′ end of the sequence complementary to the target nucleic acid molecule; and
(b) generating amplicons comprising at least a portion of said target nucleic acid molecule.
10. The method of claim 9 , further comprising detecting the amplicons with a detectable probe.
11. The method of claim 9 , wherein the pathogen is a virus, bacteria, yeast or fungus.
12. The method of claim 9 , wherein the pathogen is present in a biological sample.
13. The method of claim 12 , wherein the biological sample is selected from the group consisting of blood, serum, plasma, amniotic fluid, sputum, urine, semen, vaginal secretion, cerebrospinal fluid, lymph, tear fluid, feces, and gastric fluid.
14. The method of claim 9 , wherein the pathogen is present in an environmental sample.