Methods and compositions for amplification and detection of MicroRNAs
View Patent ↗A method and system for amplifying non-coding RNA, microRNA, and small polynucleotide sequences through the generation of a pool of signature sequences to the target sequences. The target sequences can be amplified through DNA synthesis, RNA synthesis, or the combination of DNA and RNA synthesis. The amplification of signature sequences provides an efficient and reproducible mechanism to determine the presence or absence of the miRNAs, to analyze the quantities of the target miRNAs, and for miRNA profiling. The method may also be used for screening for unknown non-coding RNAs, including novel miRNAs.
1. A method of generating a signature sequence of a target ncRNA, comprising the steps of:
hybridizing the target ncRNA to a first oligonucleotide to form a hybridized molecule;
extending the hybridized molecule to form a double-stranded RNA/DNA hybrid molecule;
cleaving the double-stranded RNA/DNA hybrid molecule with a ribonuclease to form a cleaved molecule with an overhang at the 3′ end that is at least partially complementary to the target ncRNA;
hybridizing a second oligonucleotide to the overhang at the 3′ end of the cleaved molecule to form a complex; and
generating a signature sequence from the complex.
2. The method of claim 1 , wherein the step of hybridizing the target ncRNA to the first oligonucleotide comprises incubating a sample containing the target ncRNA with the first oligonucleotide.
3. The method of claim 2 , wherein the first oligonucleotide is single stranded with a binding molecule conjugated at 5′ end.
4. The method of claim 2 , wherein the first oligonucleotide is partially double stranded and the sense strand has a 3′ overhang complementary to the ncRNA and the antisense strand has a binding molecule conjugated at 3′ end.
5. The method of claim 1 , wherein the step of extending the hybridized ncRNA sequence and first oligonucleotides to generate double stranded RNA/DNA hybrid molecule is performed in the presence of a DNA polymerase.
6. The method of claim 1 , wherein the step of hybridizing a second oligonucleotide to the cleaved RNA/DNA hybrid molecule comprises incubating a sample containing the RNA/DNA hybrid molecule with a second oligonucleotide.
7. The method of claim 6 , wherein step of hybridizing the target ncRNA to a first oligonucleotide comprises incubating the sample containing the target ncRNAs with a pool of first oligonucleotides.
8. The method of claim 1 , wherein the step of generating a signature sequence is performed in the presence of DNA polymerase.
9. The method of claim 1 , further comprising the step of removing any free first oligonucleotides before incubating the cleaved double stranded RNA/DNA hybrid molecule with the second oligonucleotide.
10. The method of claim 9 , wherein the step of removing any free first oligonucleotides comprises enzymatically digesting any single stranded first oligonucleotides with an exonuclease.
11. The method of claim 9 , wherein the step of removing any free first oligonucleotides comprises a pair of binding molecules present in the free first oligonucleotides and an insoluble support.
12. The method of claim 9 , wherein the step of removing any free first oligonucleotides comprises a combination of digesting the double stranded RNA/DNA hybrid molecule and clearing any free first oligonucleotide with a pair of binding molecules present in the free first oligonucleotides and an insoluble support.
13. A method of amplifying at least one target ncRNA sequence, comprising the steps of:
hybridizing the at least one target ncRNA to a first oligonucleotide to form a hybridized molecule;
extending the hybridized molecule to form a double-stranded RNA/DNA hybrid molecule;
cleaving the double-stranded RNA/DNA hybrid molecule to form a cleaved molecule with an overhang at the 3′ end that is at least partially complementary to the target ncRNA;
hybridizing a second oligonucleotide to the overhang at the 3′ end of the cleaved molecule to form a complex;
generating a signature sequence from the complex; and
amplifying the signature sequence.
14. The method of claim 13 , wherein the signature sequence is amplified using at least one amplification process selected from the group consisting of one directional polymerase chain reaction, limited cycle polymerase chain reaction, in vitro transcription, and combinations thereof.
15. The method of claim 13 , further comprising the step of removing any free second oligonucleotides before amplifying the signature sequence.
16. The method of claim 13 , wherein the second oligonucleotide is single stranded with a binding molecule conjugated at 5′ end.
17. The method of claim 13 , wherein the second oligonucleotide is partially double stranded with a sense strand having a 3′ overhang complementary to the ncRNA and an antisense strand having a binding molecule conjugated at 3′ end.
18. The method of claim 16 , further comprising the step of removing any free second oligonucleotides before amplifying the signature sequence.
19. The method of claim 18 , wherein step of removing any free second oligonucleotides comprises enzymatically digesting any single stranded second oligonucleotides with an exonuclease.