Allele-specific amplification

• US 8,586,299 B2
• Filed: 10/20/2009
• Issued: 11/19/2013
• Est. Priority Date: 10/20/2008
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Will, Stephen G., Tsan, Alison, Newton, Nicolas
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US12/582,068
• Publication Number:
  US 20100099110A1
• Time in Patent Office: 1,491 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension

Abstract

The present invention includes a method of allele-specific amplification, utilizing an allele-specific oligonucleotide, at least partially complementary to more than one variant of the target sequence, but having a 3′-terminal nucleotide complementary to only one variant of the target sequence and having at least one nucleotide with a base covalently modified at the exocyclic amino group, wherein the allele-specific oligonucleotide is extended by a nucleotide-incorporating biocatalyst predominantly when hybridized to the variant of the target sequence for which it has said complementary 3′-terminal nucleotide.

Claims

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

(a) providing a sample, possibly containing at least one variant of a target sequence;

(b) providing a first oligonucleotide, at least partially complementary to one or more variants of the target sequence;

(c) providing a second oligonucleotide, at least partially complementary to one or more variants of the target sequence, having a 3′

-terminal nucleotide complementary to only one variant of the target sequence;

wherein said second oligonucleotide incorporates at least one nucleotide with a base covalently modified at the exocyclic amino group;

(d) providing conditions suitable for the hybridization of said first and second oligonucleotides to at least one variant of the target sequence; and

(e) providing conditions suitable for the oligonucleotide extension by a nucleotide-incorporating biocatalyst;

wherein said biocatalyst is capable of extending said second oligonucleotide when it is hybridized to the variant of the target sequence for which it has said complementary 3′

-terminal nucleotide, and substantially less when said second oligonucleotide is hybridized to the variant of the target sequence for which it has a non-complementary 3′

-terminal nucleotide.

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 or −

1 relative to the 3′

-terminus of the second oligonucleotide.

3. The method of claim 1, wherein said nucleotide-incorporating biocatalyst in step (e) is capable of extending said second oligonucleotide, exclusively when said oligonucleotide is hybridized to the variant of the target sequence to which it has said complementary 3′

-terminal nucleotide.

4. The method of claim 1, wherein said base, covalently modified at the exocyclic 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.

5. The method of claim 1, further comprising a step (f) of detecting the product of said amplification.

6. The method of claim 1 wherein said nucleotide incorporating biocatalyst is substantially lacking the 5′

-3′

nuclease activity.

7. The method of claim 1, wherein said nucleotide-incorporating biocatalyst is selected from a group consisting of Taq DNA polymerase, Z05 DNA polymerase, Δ

Z05 DNA polymerase and Δ

Z05-Gold DNA polymerase.

8. The method of claim 1, wherein said target sequence, which exists in the form of several variant sequences is SEQ ID NO:

2 and variants thereof.

9. The method of claim 1, wherein the second oligonucleotide has a scorpion or scorpion-like format.

10. The method of claim 7, wherein said variant of the sequence in step (a) is SEQ ID NO:

1.

11. The method of claim 7, wherein said first oligonucleotide is SEQ ID NO:

6.

12. The method of claim 7, wherein said second oligonucleotide is selected from a group consisting of SEQ ID NOs:

4, 5, 7, 8, 10, 11, 12, 13, 17 and 18.

13. A method of detecting a variant of a target sequence in a sample, which exists in the form of several variant sequences comprising

(a) hybridizing a first and second oligonucleotides to at least one variant of the target sequence;

wherein the first oligonucleotide is at least partially complementary to one or more variant of the target sequence and the second oligonucleotide is at least partially complementary to one or more variant of the target sequence and has a 3′

-terminal nucleotide complementary to only one variant of the target sequence, said second oligonucleotide incorporating at least one nucleotide with a base covalently modified at the exocyclic amino group;

(b) extending the second oligonucleotide with a nucleotide-incorporating biocatalyst;

wherein said biocatalyst is capable of detectably extending only the oligonucleotide, hybridized to the variant of the target sequence for which it has said complementary 3′

-terminal nucleotide; and

(c) detecting the products of said second oligonucleotide extension, wherein the extension signifies the presence of the variant of a target sequence to which the oligonucleotide has a complementary 3′

-terminal nucleotide.