Detecting nucleic acid variations within populations of genomes

• US 9,169,514 B2
• Filed: 12/01/2011
• Issued: 10/27/2015
• Est. Priority Date: 12/03/2010
• Status: Active Grant

• Current Assignee: Brandeis University
• Original Assignee: Brandeis University
• Inventors: Jia, Yanwei, Sanchez, J. Aquiles, Rice, John E., Wangh, Lawrence J.
• Primary Examiner(s): Mummert, Stephanie K
• Assistant Examiner(s): PRIEST, AARON A
• Application Number:
  US13/991,056
• Publication Number:
  US 20140024033A1
• Time in Patent Office: 1,426 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

This disclosure relates to amplification and detection of a rare variant or variants of a DNA sequence in an abundant variant of the sequence, such as detection of a low-level somatic mutations and minority alleles in an excess of normal nucleic acid target sequences.

Claims

1. A primer-dependent DNA amplification method for selectively amplifying a rare second variant of a DNA target sequence in a sample containing an abundance of a first variant of the target sequence, where the second variant differs from the first variant by at least one nucleotide, comprising a) preparing a reaction mixture comprising the sample and amplification reagents that include a first linear primer and a second linear primer that bracket the target sequence, and additionally a primer-blocking hairpin oligonucleotide whose binding site on the target sequence is downstream from the target binding site of the first primer and includes said at least one nucleotide difference, wherein the target-binding sequence of the primer-blocking hairpin oligonucleotide is perfectly complementary to the first variant of the target sequence, wherein the binding site on the target sequence of the primer-blocking hairpin oligonucleotide overlaps by at least one nucleotide the binding site on the target sequence of the first primer, wherein the primer-blocking hairpin oligonucleotide has a concentration-adjusted melting temperature at the start of amplification versus the first variant of the target in the target template sequence that is at least 5°

C. higher than its corresponding melting temperature versus the second variant of the target sequence, and wherein the primer-blocking hairpin oligonucleotide hybridizes to the first variant of the target sequence at a temperature at which the primers do not hybridize to the target sequence but at a rate that is at least five times slower than the rate at which the first primer binds to the target sequence; and

b) subjecting the reaction mixture to a thermal-cycling protocol that includes repeated cycles of the steps of strand melting, primer annealing and primer extension, wherein at least the initial cycles include additionally, before the primer-annealing step, a blocker-binding step that has a temperature at which the primer-blocking hairpin oligonucleotide binds only to the first variant of the target sequence and the primers do not bind to the target sequence and that has a duration sufficient for the primer-blocking hairpin oligonucleotide to saturate in order to block extension on the first variant of the target sequence, and wherein the primer-annealing step has a duration insufficient for the primer-blocking hairpin oligonucleotide to bind to the second variant of the target sequence.

2. The method according to claim 1, wherein at the start of amplification the primer-blocking hairpin oligonucleotide has a concentration-adjusted melting temperature versus the first variant of the target in the target template sequence that is at least 7°

C. higher than its corresponding melting temperature versus the second variant of the target sequence.

3. The method of claim 1 that is a non-symmetric amplification method wherein the first primer is a limiting primer and the second primer is an excess primer, wherein the ratio of excess primer to limiting primer is at least 5:

1, and wherein the concentration-adjusted melting temperature of the limiting primer at the start of amplification is at least equal to the concentration-adjusted melting temperature of the excess primer at the start of amplification.

4. The method of claim 3 wherein the concentration-adjusted melting temperature of the limiting primer at the start of amplification is 3-10°

C. higher than the concentration-adjusted melting temperature of the excess primer at the start of amplification.

5. The method of claim 1 wherein the reaction mixture includes at least one homogeneous detection reagent, and the method includes homogeneous detection of at least one amplified product.

6. The method of claim 5 wherein the primer-blocking hairpin oligonucleotide is labeled so as to participate is said homogeneous detection.

7. The method of claim 6 wherein the primer-blocking hairpin oligonucleotide includes a quencher at its 3′

end, and the reaction mixture includes a short-stem molecular beacon probe that includes a fluorophore at its 5′

end, that includes a quencher at its 3′

end, and that hybridizes immediately downstream of the primer-blocking hairpin oligonucleotide.

8. The method of claim 1 wherein the thermal-cycling protocol includes multiple terminal cycles that do not include the blocker-binding step.

9. The method of claim 8 wherein the initial cycles that include the blocker-binding step are interrupted by one to ten cycles that do not include the blocker-binding step.

10. The method of claim 9 wherein the blocker-binding step is included in not more than two cycles following said interruption.

11. The method of claim 1 wherein the thermal cycling protocol includes at least ten initial cycles that include said blocker-binding step and a primer-annealing temperature sufficiently low for the first primer to bind but not sufficiently low for the second primer to bind, whereby the second variant is linearly amplified by binding and extension of the first primer prior to initiation of exponential amplification utilizing both primers.

12. The method of claim 1 wherein at least one of the primers is modified to have two concentration-adjusted melting temperatures during amplification, a low melting temperature versus the initial target sequence and a high melting temperature versus a product strand produced in the reaction, and wherein the thermal cycling protocol includes at least one cycle that has a primer annealing temperature sufficiently low for the at least one primer to bind at its low melting temperature and multiple cycles that have a primer annealing temperature sufficiently high for the at least one primer it bind only at its high melting temperature.

13. The method of claim 12 wherein the at least one modified primer is the second primer, and the second primer is mismatched to the target sequence.

14. The method of claim 12 wherein at least one primer is a tailed primer.

15. The method of claim 12, wherein the first and second primers are tailed primers having a linear 3′

segment and a 5′

tail that is not complementary to the target sequence, and wherein the thermal cycling protocol comprises not more than ten cycles that include the blocker-binding step and multiple subsequent cycles having a primer-annealing temperature sufficiently high that amplicon strands containing complements of the primer tails are exponentially amplified but other strands are not.

16. The method of claim 15 wherein at least one of the primers is a flip-tail primer.

17. The method of claim 16 wherein not more than two initial cycles include the blocker-binding step.