Methods, compositions and kits for the improved detection of small rna molecules
OwnerIgor Vassily Kutyavininventor
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Abstract
The present invention provides compositions, methods and kits for use in the detection of small RNA sequences, which allow for rapid and robust amplification and detection. The methods provide improved sensitivity and efficiency in the amplification-based detection of small RNA sequences by incorporating one or more base-modified duplex-stabilizing dNTPs during reverse transcription and/or amplification.
Claims35
A method for reverse transcription of a small RNA target sequence less than about 100 nucleotides in length, which comprises:
- (a) contacting a sample comprising the small RNA target sequence with:
- (i) a primer that is sufficiently complementary the small RNA target sequence to hybridize thereto, and
- (ii) an RNA-dependent DNA polymerase having reverse transcriptase activity;
- and
- (b) synthesizing cDNA complementary to the small RNA target sequence in the presence of a mixture of dNTPs comprising at least one base-modified, duplex-stabilizing dNTP that is a substrate for the RNA-dependent DNA polymerase.
A method for amplifying a small RNA target sequence less than about 100 nucleotides in length, which comprises:
- (a) contacting a sample comprising the small RNA target sequence with:
- (i) a primer that is sufficiently complementary the small RNA target sequence to hybridize thereto, and
- (ii) an RNA-dependent DNA polymerase having reverse transcriptase activity;
- and
- (b) synthesizing cDNA complementary to the small RNA target sequence in the presence of a mixture of dNTPs comprising at least one base-modified, duplex-stabilizing dNTP that is a substrate for the RNA-dependent DNA polymerase.
- (c) performing a nucleic acid amplification reaction using amplification primers that are sufficiently complementary to the small RNA target sequence and its complement to hybridize thereto and produce amplification products in the presence of DNA polymerase.
A reaction mixture comprising:
- (a) a sample comprising a small RNA target sequence;
- (b) a primer that is sufficiently complementary the small RNA target sequence to hybridize therewith,
- (c) an RNA-dependent DNA polymerase having reverse transcriptase activity;
- and
- (d) a mixture of deoxynucleoside 5′-triphosphates containing at least one base-modified duplex-stabilizing dNTP that is a substrate for the RNA-dependent DNA polymerase.
A kit comprising
- (a) an RNA-dependent DNA polymerase having reverse transcriptase activity;
- and
- (b) at least one base-modified, duplex-stabilizing deoxyribonucleotide triphosphates that is a substrate for the RNA-dependent DNA polymerase.
Dependencies are inferred from the claim text.