Method of eliminating background amplification of nucleic acid targets
AssigneesThe Foundation for the Promotion of Industrial Science, Centre National de la Recherche Scientifique (CNRS)
InventorsYannick Rondelez, Guillaume Gines, Kevin Montagne, Teruo Fujii
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Abstract
An object is to provide a method of eliminating background amplification in detection of rare nucleic acids based on isothermal amplification. The method comprising steps of: preparing a mixture including solution and enzymes, and adding a first and a second template into the mixture, wherein the first template is an amplification template, and the second template is a leak absorption template. Methods to use this approach for multiplexed, ultrasensitive and ultra-specific detection of nucleic acids are presented.
Claims20
A method of isothermal amplification of a nucleic acid target sequence to obtain an amplified sequence, the method comprising:
- (a) obtaining a mixture of:
- (i) buffer comprising a polymerase, nicking enzyme and exonuclease,
- (ii) a first oligonucleotide which is an amplification oligonucleotide having a nucleic acid sequence between 10 and 40 nucleotides, wherein the sequence includes a partial repeat structure containing a nicking enzyme recognition site,
- (iii) a second oligonucleotide which is a leak absorption oligonucleotide having a nucleic acid sequence between 10 and 35 nucleotides, a 3′ end that is complementary to the amplified sequence, and a 5′ end that encodes a sequence of a tail which is appended to the amplified sequence, the sequence of the tail having a length between 2 and 6 nucleotides and being different from the amplified sequence, and
- (iv) a third oligonucleotide which is a target-specific conversion oligonucleotide having a 3′ end that is complementary to the nucleic acid target sequence and a 5′ end at least partially complementary to 3′ end of the amplification oligonucleotide;
- (b) adding the mixture of (a) to a sample to be analyzed;
- (c) incubating the sample at constant temperature;
- and
- (d) detecting the nucleic acid target sequence through amplification of the amplified sequence, while eliminating background amplification by the leak absorption oligonucleotide.
Dependencies are inferred from the claim text.