Detection and amplification of rna using target-mediated ligation of dna by rna ligase

Abstract

Disclosed are techniques for detection of nucleic acids, amplification of nucleic acids, or both, involving ligation by T4 RNA ligase of DNA strands hybridized to an RNA strand. These techniques are particularly useful for the detection of RNA sequences and for amplification of nucleic acids from, or dependent on, RNA sequences. It has been discovered that T4 RNA ligase can efficiently ligate DNA ends of nucleic acid strands hybridized to an RNA strand. In particular, this ligation is more efficient than the same ligation carried out with T4 DNA ligase. Thus, techniques involving ligation of DNA ends of nucleic acid strands hybridized to RNA can be performed more efficiently by using T4 RNA ligase. Many known ligation-based detection and amplification techniques are improved through the use of T4 RNA ligase acting on DNA strands or ends. Such techniques include ligase chain reaction (LCR), ligation combined with reverse transcription polymerase chain reaction (RT PCR), ligation-mediated polymerase chain reaction (LMPCR), polymerase chain reaction/ligation detection reaction (PCR/LDR), ligation-dependent polymerase chain reaction (LD-PCR), oligonucleotide ligation assay (OLA), ligation-during-amplification (LDA), ligation of padlock probes, open circle probes, and other circularizable probes, and iterative gap ligation (IGL).