Allele-specific amplification of nucleic acids using blocking oligonucleotides for wild type suppression

• US 9,909,169 B2
• Filed: 12/17/2014
• Issued: 03/06/2018
• Est. Priority Date: 12/17/2014
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Will, Stephen G.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/574,181
• Publication Number:
  US 20160177372A1
• Time in Patent Office: 1,175 Days
• Field of Search: 435 61
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2537/163 blocking probe

Abstract

A method is provided for allele-specific amplification, utilizing a blocking oligonucleotide including at least one nucleotide with a base covalently modified at the exocyclic amino group, the blocking oligonucleotide being perfectly complementary to a wild type (WT) sequence when hybridized forming a first complex having a first melting temperature (Tm), the blocking oligonucleotide being partially non-complementary, at one or more nucleotides, to a target mutant (MT) sequence when hybridized forming a second complex having a second melting temperature (Tm), wherein the first Tm is higher than the second Tm and having at least one nucleotide with a base covalently modified at the exocyclic amino group, wherein the blocking oligonucleotide becomes unhybridized from the target MT sequence during amplification but remains hybridized with the WT sequence inhibiting amplification of the WT sequence utilizing a polymerase lacking 5′-3′ nuclease activity.

Claims

1. A method of allele-specific amplification of a target sequence, which exists in the form of several variant sequences in a sample, comprising:

providing a blocking oligonucleotide comprising a 5′

terminus, a 3′

terminus, and at least one nucleotide with a base covalently modified at the exocyclic amino group, the blocking oligonucleotide being perfectly complementary to a wild type (WT) sequence when hybridized forming a first complex having a first melting temperature (Tm), the blocking oligonucleotide being partially non-complementary, at one or more nucleotides, to a target mutant (MT) sequence when hybridized forming a second complex having a second melting temperature (Tm), wherein the first Tm is higher than the second Tm, the blocking oligonucleotide being blocked at the 3′

terminus prohibiting extension; and

performing an amplifying step at a temperature higher than the second Tm but lower than the first Tm utilizing a polymerase lacking 5′

-3′

nuclease activity, the amplifying step comprising contacting the sample with a set of primers to produce an amplification product if the WT sequence and/or the target MT sequence is present in the sample, wherein the blocking oligonucleotide becomes unhybridized from the target MT sequence during the amplification step but remains hybridized with the WT sequence inhibiting amplification of the WT sequence.

2. The method of claim 1, wherein the nucleotide having a covalently modified base at the exocyclic amino group is located at positions −

5, −

4, −

3, −

2, −

1, or 0 relative to the site of non-complementary nucleotide of the blocking oligonucleotide.

3. The method of claim 1, wherein the base covalently modified at the exocydic amino group is selected from a group consisting of N⁶-benzyl-adenine, N⁶-para-tert-butyl-benzyl adenine, N²-alkyl-guanine and N⁴-benzyl-cytosine.

4. The method of claim 1, further comprising a step of detecting the amplification product.

5. The method of claim 1, wherein the polymerase is selected from a group consisting of Stoffel fragment of Taq DNA polymerase, Δ

Z05 DNA polymerase, and Δ

Z05-Gold DNA polymerase.

6. The method of claim 1, wherein the structure of the nucleotide with a base covalently modified at the exocydic amino group is selected from the group consisting of:

7. The method of claim 1, wherein the base covalently modified at the exocydic amino group comprises a modifier of the following formula:

8. The method of claim 2, wherein the modifier has the following formula:

9. The method of claim 8, wherein the modifier is selected from the group consisting of: