Back to results
US 10,988,810 B2Granted
Methods and materials for detection of mutations
AssigneePentaBase ApS
InventorUlf Bech Christensen
Life of this patent
Estimating the term…
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.
Claims20
A method for detecting the presence of a more than one of a number of variant sequences in a target nucleic acid sequence comprising nucleotide(s) of interest (NOI), wherein said NOI may consist of said variant sequences, 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 one 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,
- 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 sets of primers comprising at least two different primers including a first primer 1 and a primer 2, wherein the first primer 1 together with the primer 2 is capable of amplification of the target nucleic acid sequence comprising a first variant sequence, and wherein first 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 said first variant sequence except for up to one mismatch, and a second primer 1 together with the primer 2 is capable of amplification of a target nucleic acid sequence comprising a second variant sequence, wherein said second 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 said second variant sequence except for up to one mismatch;
- and wherein the sets of primers optionally comprise one or more further primer 1, which together with the primer 2 are capable of amplification of a target nucleic acid sequence comprising one or more further variant sequence(s), wherein said further primer 1(s) comprise 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 said further variant sequence(s) except for up to one mismatch;
- d) performing a polymerase chain reaction in the presence of said sample, said blocking oligonucleotide;
- and said set of primers;
- 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.
18 more elements
A kit-of-parts comprising
- a) one 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,
- Q is an 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 a target nucleic acid sequence comprising a reference nucleotide(s) of interest (NOI) sequence;
- and
- b) sets of primers comprising at least two different primer 1 together with a primer 2, wherein a first primer 1 together with the primer 2 is capable of amplification of a target nucleic acid sequence comprising a first variant sequence, wherein said first 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 said first variant sequence except for up to one mismatch, and a second primer 1 together with the primer 2 is capable of amplification of a target nucleic acid sequence comprising a second variant sequence, wherein said second 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 said second variant sequence except for up to one mismatch;
- and wherein the sets of primers optionally comprise one or more further primer 1, which together with the primer 2 are capable of amplification of a target nucleic acid sequence comprising one or more further variant sequence(s), wherein said further primer 1 comprise 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 said further variant sequence(s) except for up to one mismatch.
15 more elements
A blocking oligonucleotide having the following structure
- 5′-(N)a—Z—(N)i—Z—(N)j—Z—(N)l—Z—(N)l—Z—(N)b-3′
- wherein
- N is any nucleotide or nucleotide analogue;
- and
- Z is a hydrophobic nucleotide having the structure X—Y-Q wherein X is a nucleotide or nucleotide analogue or a backbone monomer unit, 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
- a and b individually are integers in the range from 0 to 4;
- and
- j, k and l individually are integers in the range from 1 to 17;
- and
- a+b+i+j+k+l is at least 10 and at the most 50;
- and
- (N)a—(N)i—(N)j—(N)k—(N)l—(N)b is identical to a stretch of the target nucleic acid sequence comprising a reference nucleotide(s) of interest NOI sequence;
- or
- having the following structure
- 5′-(N)a—Z—(N)m—Z—(N)n—Z—(N)o—Z—(N)p—Z—(N)q—Z—(N)b-3′
- wherein
- N is any nucleotide or nucleotide analogue;
- and
- Z is a hydrophobic nucleotide having the structure X—Y-Q wherein X is a nucleotide or nucleotide analogue or a backbone monomer unit, 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
- a and b individually are integers in the range of 0 to 4;
- and
- m, n, o, p and q individually are integers in the range of 1 to 16;
- and
- a+b+m+n+o+p+q is at least 10 and at the most 50;
- and
- (N)a—(N)m—(N)n—(N)o—(N)p—Z—(N)q—(N)b is identical to a stretch of the target nucleic acid sequence comprising a reference NOI sequence.
25 more elements
Dependencies are inferred from the claim text.