Methods and materials for detection of mutations
AssigneePentaBase ApS
InventorUlf Bech Christensen
Life of this patent
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Abstract
Provided herein are specially modified blocking nucleotides allowing for the sensitive detection of low copies of variant sequences, while significantly reducing signals from non-variant sequences that are similar but not identical to the variant sequence. These nucleotides can be used to detect rare variants in a sample mixture, as described in the present methods.
Claims21
A method for detecting the presence of a variant sequence in a target nucleic acid sequence comprising nucleotide(s) of interest (NOI), wherein said NOI may consist of said variant sequence, of other variant sequences or of a reference NOI sequence, said method comprising the steps of
- a) Providing a sample comprising nucleic acids
- b) Providing a blocking oligonucleotide comprising a sequence of in the range of 10 to 50 nucleotides (referred to as “blocking sequence”) into which in the range of 2 to 10 hydrophobic nucleotides have been inserted
- wherein
- at least one hydrophobic nucleotide is positioned at the 5′ end of said sequence or within 4 nucleotides from the 5′ end;
- and
- at least one hydrophobic nucleotide is positioned at the 3′ end of said sequence or within 4 nucleotides from the 3′ end;
- and
- wherein the hydrophobic nucleotide has the structure
- X-Y-Q
- wherein
- X is a nucleotide or nucleotide analogue or a backbone monomer unit capable of being incorporated into the backbone of a nucleic acid or nucleic acid analogue,
- Q is a intercalator which is not taking part in Watson-Crick hydrogen bonding;
- and
- Y is a linker moiety linking said nucleotide or nucleotide analogue or backbone monomer unit and said intercalator;
- and
- wherein the blocking sequence is identical to a consecutive stretch of the target nucleic acid sequence comprising the reference NOI sequence,
- c) Providing a set of primers consisting of primer 1 and primer 2, wherein the set of primers together are capable of amplification of the target nucleic acid sequence comprising the variant sequence, and wherein primer 1 comprises a sequence of at least 15 nucleotides positioned at the 3′ end of said primer, which is identical to a consecutive stretch of the target nucleic acid sequence comprising the variant sequence expect for up to one mismatch;
- d) performing a polymerase chain reaction in the presence of said sample, said blocking oligonucleotide;
- said set of primers and PCR reagents;
- e) detecting a product of said polymerase chain reaction, wherein the presence of a product of said polymerase chain reaction indicates the presence of a target nucleic acid sequence comprising the variant sequence in said sample.
A kit-of-parts comprising
- a) a blocking oligonucleotide consisting of a sequence of in the range of 10 to 50 nucleotides (referred to as “blocking sequence”) into which in the range of 2 to 10 hydrophobic nucleotides have been inserted,
- wherein
- at least one hydrophobic nucleotide is positioned at the 5′ end of said sequence or within 4 nucleotides from the 5′ end;
- and
- at least one hydrophobic nucleotide is positioned at the 3′ end of said sequence or within 4 nucleotides from the 3′ end;
- and
- Z is a hydrophobic nucleotide of the structure
- X-Y-Q
- wherein
- X is a backbone monomer unit capable of being incorporated into the backbone of a nucleic acid or nucleic acid analogue,
- Q is an intercalator, which is not taking pa son-Crick hydrogen bonding;
- and
- Y is a linker moiety linking said nucleotide or nucleotide analogue or backbone monomer unit and said intercalator;
- and
- wherein the blocking sequence is identical to a consecutive stretch of a target nucleic acid sequence comprising a reference NOI sequence;
- and
- b) a set of primers consisting of primer 1 and primer 2, wherein the set of primers together are capable of amplification of the target nucleic acid sequence comprising a variant sequence, and wherein primer 1 contains a sequence of at least 15 nucleotides positioned at the 3′ end of said primer, which is identical to a consecutive stretch of the target nucleic acid sequence comprising the variant sequence expect for up to one mismatch.
The method or the kit according to any one of the preceding claims, wherein primer 1 comprises a sequence which is identical to a consecutive sequence of the blocking sequence of at least 3, preferably of at least 6 nucleotides except for up to one mismatch.
The method or the kit according to any one of the preceding claims, wherein the most 3′ nucleotide(s) of primer 1 are identical to the variant sequence.
The method or the kit according to any one of the preceding claims, wherein the variant sequence is a single nucleotide variant.
The method or the kit according to any one of the preceding claims, wherein said mismatch is positioned at position 2, 3 or 4 from the 3′ end of primer 1.
The method or the kit according to any one of the preceding claims, wherein primer 2 comprises a sequence of at least 15 nucleotides, which is complementary to a consecutive sequence of the target nucleic acid sequence.
The method, the kit or the blocking oligonucleotide according to any one of the preceding claims, wherein at least one hydrophobic nucleotide comprises an intercalator which is pyrene or pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4(1H)-one or 7,9-dimethyl-pyrido[3′,2′,4,5]thieno[3,2-d]pyrimidin-4(3H)-one.
The method, the kit or the blocking oligonucleotide according to any one of the preceding claims, wherein at least one backbone monomer unit X is selected from the group consisting of phosphoramidites.
Dependencies are inferred from the claim text.