Primers, probes and methods for nucleic acid amplification

• US 20060177841A1
• Filed: 10/17/2005
• Published: 08/10/2006
• Est. Priority Date: 10/18/2004
• Status: Active Grant

• Current Assignee: Brandeis University
• Original Assignee: Brandeis University
• Inventors: Rice, John, Wangh, Lawrence J., Salk, Jesse, Hartshorn, Cristina, Sanchez, J. Aquiles, Pierce, Kenneth, Reis, Arthur
• Granted Patent: US 7,632,642 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2531/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

Homogenous detection during or following PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled primers and probes, improves reproducibility and quantification. Low-temperature homogeneous detection during or following non-symmetric PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled mismatch-tolerant probes permits analysis of complex targets. Sequencing sample preparation methods following LATE-PCR amplifications reduce complexity and permit “single-tube” processing.

Claims

1. A method for analyzing at least one single-stranded amplification product of a non-symmetric nucleic acid amplification process that generates both double-stranded and single-stranded amplicons by extension of oligonucleotide primers by a DNA polymerase and that includes at least one primer-annealing temperature, comprising

detecting double-stranded amplicons by homogeneous fluorescence detection,

detecting said at least one single-stranded amplification product by sequence-specific homogeneous fluorescence detection at a temperature below the at least one primer-annealing temperature, and

calculating the ratio fluorescence of said single-stranded product to the fluorescence of said double-stranded amplicons.

2. The method according to claim 1 wherein detection reagents are added at the beginning of the nucleic acid amplification process.

3. The method according to claim 2 wherein detection of the double-stranded amplicons is by means of a fluorescent DNA dye.

4. The method according to claim 3 wherein detection of said at least one single-stranded product includes a fluorophore-labeled hybridization probe that binds to said single-stranded product during the low-temperature detection step but not at said at least one primer-annealing temperature.

5. The method according to claim 4 wherein the hybridization probe for said at least one single-stranded amplification product is an allele-discriminating quenched double-stranded probe.

6. The method according to claim 5 comprising at least two probes for different single-stranded amplification products that are labeled with the same fluorophore but have different melting temperatures with respect to their targets, wherein said low-temperature detection step includes detecting emission from said fluorophore at a temperature at which only one probe binds to its target and at a temperature wherein at least two probes bind to their respective targets.

7. The method according to claim 3 wherein the primer whose extension generates single-stranded amplicons is labeled with a fluorophore that is stimulated indirectly by fluorescence emission from said dye, and wherein detection of said at least one single-stranded product includes stimulation of the dye and detecting fluorescence emitted by said primer.

8. The method according to of claim 3 comprising at least one mismatch-tolerant hybridization probe that binds to at least two possible single-stranded amplification products to form hybrids having different melting temperatures below the temperature utilized to anneal primers in said amplification and that is labeled with a fluorophore that is stimulated indirectly by fluorescence emission from said dye, wherein detection of single-stranded product includes low-temperature detection at multiple temperatures determined by said different melting temperatures.

9. The method according to claim 8 wherein said mismatch-tolerant hybridization probe is a linear hybridization probe that forms secondary structure comprising a double-stranded region 1-4 nucleotides in length during said low-temperature detection, wherein fluorescence resulting from said secondary structure is internally quenched.

10. The method of claim 8 wherein said mismatch-tolerant hybridization probe is a molecular beacon probe.

11. The method according to claim 8 comprising a plurality of differently fluorescently labeled low-temperature hybridization probes, each of which has a melting temperature with respect to any target that is below the temperature of primer annealing and that in combination hybridize to multiple possible target sequences, at least two probes being mismatch-tolerant probes that hybridize to a plurality of possible targets at different temperatures and that have fluorophores that are excited by fluorescence emission from said dye, wherein detection of single-stranded amnplicons includes stimulating said reaction mixture at at least three temperatures below said temperature of primer annealing with light that excites the dye but not the fluorophores of said mismatch-tolerant probes and detecting emissions from the probes.

12. The method of claim 1 wherein the amplification process is a linear-after-the-exponential PCR (LATE-PCR) amplification process.

13. The method of any of claims 1-12 wherein said detection is an end-point detection following the completion of the amplification process.

14. A kit of reagents for performing the method of claim 1 comprising at least all primers and probes to be utilized in the method.

15. The kit according to claim 14 further including all amplification reagents.

16. A double-stranded nucleic acid hybridization probe for a preselected nucleic acid target sequence comprising

a) a first oligonucleotide comprising a first sequence perfectly complementary to said target sequence and having a calculated melting temperature (T_m) with respect to said target sequence of 30-55°

C.;

b) a second oligonucleotide comprising a second sequence that is complementary to said first sequence but is shorter than said first sequence by up to ten nucleotides;

c) a fluorophore label attached to said first sequence that is excitable by fluorescence emission from a fluorescent DNA dye but is not excitable at the maximum absorption wavelength of said dye; and

d) a non-fluorescent quencher attached to said second sequence.

17. An oligonucleotide set comprising a pair of LATE-PCR primers for amplifying a preselected target sequence and a probe according to claim 16.

18. A low-temperature, mismatch tolerant, single-stranded oligonucleotide hybridization probe having a first calculated melting temperature with respect to its perfect complement of not more than 60°

C., preferably not more than 50°

C., said probe including a stem-loop hairpin having a stem whose calculated melting temperature is at least 10°

C. lower than said first melting temperature but not lower than 30°

C., said probe including a fluorophore capable of absorption of fluorescence energy emitted by a fluorescent DNA dye and means to quench fluorescence that would result from exposing said probe to said dye and exciting the dye in the absence of a target for the probe.

19. A low-temperature linear oligonucleotide hybridization probe

a) that is mismatch tolerant at temperatures in the range of 30-60°

C.;

b) that forms secondary structure in the temperature range of 30°

C. to 50°

C.; and

c) is dual-labeled with

(i) a fluorophore that is excitable by fluorescence emission from at least one fluorescent DNA dye but is not excitable at the wavelength appropriate for exciting said dye; and

(ii) a non-fluorescence quencher located so as to substantially quench fluorescence associated with said secondary structure but not florescence associated with a probe-target hybrid.

20. The probe according to claim 19 wherein said secondary structure is a stem formed by complementary terminal nucleotides, said stem having a T_m greater than the T_m of any other secondary structure the probe would form in the absence of the stem.

21. A sequential amplification-Pyrosequencing method comprising,

a) amplifying a DNA substrate by a LATE-PCR amplification reaction to prepare single stranded DNA product;

b) removing pyrophosphate and, if present, leftover dNTPs;

c) if necessary, adding an oligonucleotide to block the excess primer

d) adding Pyrosequencing reagents before adding sequencing primer if pyrophosphate and dNTPs have not already been removed as in b) above;

e) adding sequencing primer; and

f) performing Pyrosequencing.

22. The method of claim 21 wherein step b) comprises adding Pyrosequencing reagents and step d) comprises replenishing such reagents.

23. The method of claim 21 wherein pyrophosphate is removed by adding an enzyme with pyrophosphatase activity.

24. The method of claim 21 wherein dNTPs are removed by adding an enzyme with dNTPase activity.