MODIFIED STEM-LOOP OLIGONUCLEOTIDE MEDIATED REVERSE TRANSCRIPTION AND BASE-SPACING CONSTRAINED QUANTITATIVE PCR

• Main Authors: ANWAR AZLINDA B, TOO HENG PHON
• Format: Patent
• Language: English
• Published: 11.07.2013
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS OR TEST PAPERS THEREFOR; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL ORENZYMOLOGICAL PROCESSES; ENZYMOLOGY; MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEICACIDS OR MICROORGANISMS; METALLURGY; MICROBIOLOGY; MUTATION OR GENETIC ENGINEERING; PROCESSES OF PREPARING SUCH COMPOSITIONS; SPIRITS; VINEGAR; WINE

There is provided a method for detecting a target RNA molecule in a sample. The method comprises reverse transcribing the target RNA contained in the sample using an RT oligonucleotide, the RT oligonucleotide comprising a stem-loop portion containing one or more nucleotides modified or modifiable to block DNA polymerase extension and a target annealing portion that is complementary to a downstream portion of the target RNA, the target annealing portion located 3' to the stem-loop portion, to produce a reverse transcription product that comprises the RT oligonucleotide and a 3' extended region; amplifying the reverse transcription product using (i) a first amplification primer that anneals to a downstream portion of the 3' extended region of the reverse transcription product and (ii) a second amplification primer that anneals to an interface portion of a DNA strand complementary to the reverse transcription product, the interface portion comprising a region that is complementary to a 3' portion of the RT oligonucleotide and a 5' portion of the 3' extended region in the reverse transcription product, to produce an amplification product; and detecting the amplification product; wherein the stem-loop portion adopts a stem-loop structure under conditions used for said reverse transcribing but does not adopt the stem-loop structure under conditions used for said amplifying and wherein when the stem-loop portion contains one or more nucleotides that are modifiable to block DNA polymerase extension, the method further comprises modifying the modifiable nucleotide prior to said amplifying.