Primers for melting analysis
Methods and kits are provided for nucleic acid analysis. In an illustrative method a target nucleic acid is amplified using a first primer and a second primer, wherein the first primer comprises a probe element specific for a locus of the target nucleic acid and a template-specific primer region, and the probe element is 5′ of the template-specific primer region, subsequently allowing the probe element to hybridize to the locus to form a hairpin, generating a melting curve for the probe element by measuring fluorescence from a dsDNA binding dye as the mixture is heated, wherein the dye is not covalently bound to the first primer, and analyzing the shape of the melting curve. Kits may include one or more of the first and second primers, the dsDNA binding dye, a polymerase, and dNTPs.
1. A method for nucleic acid analysis comprising the steps of
mixing a target nucleic acid with a first primer, a second primer, and a dsDNA binding dye to form a mixture, the primers configured for amplifying the target nucleic acid, wherein the first primer comprises a probe element specific for a locus of the target nucleic acid and a template-specific primer region, wherein the probe element is 5′ of the template-specific primer region, and wherein the dsDNA binding dye is not covalently bound to the first primer,
amplifying the target nucleic acid in the mixture by means of the primers to generate an amplicon, the amplicon comprising the probe element and the locus,
allowing the probe element of the amplicon to hybridize intramolecularly to the locus of the amplicon to form a hairpin,
heating the mixture to melt the hairpin, and
generating a melting curve for the probe element by measuring fluorescence from the dsDNA binding dye as the mixture is heated to melt the hairpin, wherein the melting curve for the probe element is distinguishable from a melting curve for double-stranded amplicon.
2. The method of claim 1 wherein the first primer is provided in the mixture at a concentration greater than the second primer for asymmetric amplification.
3. The method of claim 1 wherein the amplifying step is amplifying by PCR.
4. The method of claim 1 wherein the first primer further comprises a mismatched region 5′ of the probe element.
5. The method of claim 1 wherein the first primer is an oligonucleotide that does not have any covalently attached dyes or quenchers.
6. The method of claim 5 wherein the first primer does not have an extension blocker.
7. The method of claim 1 wherein the dsDNA binding dye is a saturation dye.
8. The method of claim 7 wherein the dye is present at concentrations sufficient to distinguish heterozygotes in the melting curve for the probe element.
9. The method of claim 1 wherein the first and second primers are provided in the mixture at essentially the same concentration, and wherein the second primer comprises a probe element specific for a second locus of the target nucleic acid and a 30 template-specific primer region, wherein the probe element of the second primer is 5′ of the template-specific primer region.
10. The method of claim 9 further comprising the step of diluting the amplicon prior to generating the melting curve for the probe element.
11. The method of claim 10 further comprising the step of heating the diluted amplicon to at least a denaturation temperature of the amplicon and cooling the heated diluted amplicon to a temperature below a denaturation temperature of the hairpin prior to generating the melting curve for the probe element.
12. The method of claim 11 wherein the cooling is rapid cooling.
13. The method of claim 9 wherein the first primer comprises an extension blocker between the probe element and the template-specific primer region.
14. The method of claim 1 wherein the hairpin has a loop of between 20 and 50 bases.
15. The method of claim 1 wherein the probe element is less than 20 bases.
16. The method of claim 15 wherein the probe element is less than 10 bases.
17. The method of claim 16 wherein the probe element is 6 bases.
18. The method of claim 1 wherein the mixture further comprises a third primer, the third primer comprises a probe element specific for a third locus of the target nucleic acid and a template-specific primer region, wherein the third primer's template-specific primer region is the same as the first primer's template-specific primer region, but the third primer's probe element is specific for a locus that is distinct from the locus of the first primer's probe element.
19. The method of claim 1 wherein the locus has a known single nucleotide polymorphism, and the single nucleotide polymorphism is located no closer than 8 bases from an end of the probe element.
20. The method of claim 1 wherein the mixture further comprises an unlabeled probe configured to hybridize to a different locus of the target nucleic acid.
21. The method of claim 1 wherein
the second primer comprises a probe element specific for a second locus of the target nucleic acid, a template-specific primer region and a covalently bound dye capable of resonance energy transfer with the dsDNA binding dye, wherein the probe element of the second primer is 5′ of the template-specific primer region, and
the fluorescence of the dsDNA binding dye is measured at a first wavelength and the generating step further includes measuring fluorescence from the covalently bound dye at a second wavelength.
22. The method of claim 1 wherein
the target nucleic acid further comprises a second locus, and
the template-specific primer region of the first primer is configured to amplify the target nucleic acid only if a particular allele of the second locus is present.
23. The method of claim 1 further comprising the step of analyzing the shape of the melting curve for the probe element.
24. The method of claim 1 wherein the mixture is adjusted to favor probe element binding intramolecularly prior to generating the melting curve for the probe element.
25. The method of claim 1 wherein amplification is terminated prior to reaching plateau phase, to limit amplicon concentration.
26. A method for nucleic acid analysis comprising the steps of
mixing a target nucleic acid with a first primer, a second primer, and a dsDNA binding dye to form a mixture, the primers configured for amplifying the target nucleic acid, wherein the first primer comprises a probe element specific for a locus of the target nucleic acid and a template-specific primer region, wherein the probe element is 5′ of the template-specific primer region, and wherein the dsDNA binding dye is not covalently bound to the first primer,
amplifying the target nucleic acid in the mixture to generate an amplicon, the amplicon comprising the probe element and the locus,
adjusting the mixture to favor hairpin formation by the probe element binding intramolecularly to the target nucleic acid, and
generating a melting curve for the probe element by measuring fluorescence from the dsDNA binding dye as the mixture is heated to melt the hairpin, wherein the melting curve for the probe element is distinguishable from a melting curve for the amplicon.
27. The method of claim 1 , wherein the amplicon is double-stranded, the melting curve for the probe element being distinguishable from the melting curve for the double-stranded amplicon.
28. The method of claim 26 , wherein the amplicon is double-stranded, the melting curve for the probe element being distinguishable from the melting curve for the double-stranded amplicon.