Preferential amplification of mRNA over DNA using chemically modified primers
First Claim
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1. A method of selective amplification of a messenger RNA (mRNA) target in the presence of corresponding genomic DNA contaminant comprising the steps of:
- a. providing a sample that comprises said mRNA target and said corresponding genomic DNA contaminantb. hybridizing a first oligonucleotide to said mRNA target and performing RNA-directed DNA synthesis using at least one enzyme capable of RNA-directed synthesis, wherein said first oligonucleotide;
i. comprises at least one nucleotide with a base covalently modified at the exocyclic amino group,ii. is at least partially complementary to said mRNA target and to said corresponding genomic DNA contaminant, and,iii. spans an exon-exon junction in the target;
c. amplifying the product of step b) using said first oligonucleotide and a second oligonucleotide with at least one enzyme capable of DNA-directed DNA synthesis;
wherein said second oligonucleotide is at least partially complementary to said mRNA target and to said corresponding genomic DNA contaminant;
wherein said mRNA target is selectively amplified over said corresponding genomic DNA contaminant when compared to amplifying with an unmodified first oligonucleotide.
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Abstract
The present invention relates to a method, oligonucleotides, reaction mixtures and kits for the selective amplification of a messenger RNA target comprising an exon-exon junction, using an oligonucleotide that comprises at least one nucleotide modified at the exocyclic amino group.
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7 Claims
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1. A method of selective amplification of a messenger RNA (mRNA) target in the presence of corresponding genomic DNA contaminant comprising the steps of:
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a. providing a sample that comprises said mRNA target and said corresponding genomic DNA contaminant b. hybridizing a first oligonucleotide to said mRNA target and performing RNA-directed DNA synthesis using at least one enzyme capable of RNA-directed synthesis, wherein said first oligonucleotide; i. comprises at least one nucleotide with a base covalently modified at the exocyclic amino group, ii. is at least partially complementary to said mRNA target and to said corresponding genomic DNA contaminant, and, iii. spans an exon-exon junction in the target; c. amplifying the product of step b) using said first oligonucleotide and a second oligonucleotide with at least one enzyme capable of DNA-directed DNA synthesis;
wherein said second oligonucleotide is at least partially complementary to said mRNA target and to said corresponding genomic DNA contaminant;wherein said mRNA target is selectively amplified over said corresponding genomic DNA contaminant when compared to amplifying with an unmodified first oligonucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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Specification