Method to Quantify siRNAs, miRNAs and Polymorphic miRNAs
AssigneeLife Technologies Corporation
InventorsRuoying TAN, Caifu Chen, Karl J. Guegler
Life of this patent
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Abstract
The present teachings provide methods, compositions, and kits for quantifying target, polynucleotides. In some embodiments, a reverse stem-loop ligation probe is ligated to the 3′ end of a target polynucleotide, using a ligase that can ligate the 3′ end of RNA to the 5′ end of DNA using a DNA template, such as T4 DNA ligase. Following digestion to form an elongated target polynucleotide with a liberated end, a reverse transcription reaction can be performed, followed by a PCR. In some embodiments, the methods of the present teachings can discriminate between polymorphic polynucleotides that vary by as little as one nucleotide.
Claims12
A method of amplifying a target polynucleotide, the method comprising;
- forming a first reaction complex comprising a reverse stem-loop ligation probe hybridized to the target polynucleotide, wherein the reverse stem-loop ligation probe comprises a loop, a stem, and a 3′ target-specific portion;
- ligating the reverse stem-loop ligation probe to the target polynucleotide to form an elongated target polynucleotide;
- removing the hybridized 3′ target-specific portion from the elongated target polynucleotide to form an elongated target polynucleotide with a liberated end;
- forming a second reaction complex comprising a reverse primer hybridized to the liberated end of the elongated target polynucleotide;
- extending the reverse primer to form a first strand, wherein the first strand is hybridized to the elongated target polynucleotide to form a double stranded complex;
- and, amplifying the double stranded complex.
The method according to claim t wherein the target polynucleotide is an RNA molecule, wherein the reverse stem-loop ligation probe is a DNA molecule and wherein the ligase is T4 DNA ligase.
The method according to claim 11 wherein the extension blocker is an amine group.
12-31. (canceled)
Dependencies are inferred from the claim text.