Allele amplification bias
View Patent ↗Methods are provided for nucleic acid analysis. In an illustrative method, allele amplification bias is used to amplify preferentially a target nucleic acid that is present in a low allele fraction.
1. A method for amplification and allele detection of a biological sample, comprising the steps of:
providing the biological sample comprising a first allele and a second allele of a target nucleic acid, the first allele being present in a higher concentration than the second allele;
adding to the biological sample a thermostable polymerase, an oligonucleotide probe, and a pair of primers configured for amplification of the target nucleic acid in the biological sample, wherein the probe is configured to hybridize to the target nucleic acid without being extended and wherein the probe has a first Tm when hybridized to the first allele and a second Tm when hybridized to the second allele, wherein the first Tm is higher than the second Tm;
amplifying the target nucleic acid in the biological sample in the presence of the probe by thermal cycling through an extension temperature between an annealing temperature and a denaturation temperature at a ramp rate of at least 4° C./sec, wherein the annealing temperature is below the first Tm such that the probe hybridizes to the first allele at the annealing temperature and inhibits amplification of the first allele by the polymerase and the second allele is preferentially amplified during thermal cycling; and
detecting the preferentially amplified second allele through melting curve analysis of the probe and the amplified second allele.
2. The method of claim 1 , wherein the annealing temperature is about half way between the second Tm and the first Tm.
3. The method of claim 1 , wherein the annealing temperature is at least 1.0° C. above the second Tm.
4. The method of claim 1 , further comprising detecting the first allele.
5. The method of claim 4 , further comprising calculating allele fractions of the first allele and the second allele.
6. The method of claim 5 , wherein the calculating step comprises estimating the second allele fraction as F m =wLf(TL)+wHf(TH), where wL and wH are weights and f(TL) and f(T H ) are individual estimates at each temperature peak calculated from standards of a homozygote of the first allele, a homozygote of the second allele, and a 50:50 mix of the first allele and second allele.
7. The method of claim 1 , wherein the probe is a probe element attached to a 5′ end of one of the pair of primers, and wherein a mismatch prevents extension when the probe element is hybridized to the target nucleic acid.
8. A method for amplification and allele detection of a biological sample, comprising:
providing the biological sample comprising a first allele and a second allele of a target nucleic acid, the first allele being present in a higher concentration than the second allele;
adding a thermostable polymerase, a first primer, and a second primer to the biological sample, the primers being 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 configured to be extended by the polymerase,
wherein the probe element is 5′ of the template-specific primer region, wherein the probe element is an oligonucleotide configured to hybridize to the target nucleic acid and the probe element has a first Tm when hybridized to the first allele and a second Tm when hybridized to the second allele, wherein the first Tm is higher than the second Tm;
amplifying the target nucleic acid in the biological sample in the presence of the probe element by thermal cycling between an annealing temperature and a denaturation temperature at a ramp rate of at least 4° C./sec, wherein the annealing temperature is below the first Tm such that the probe element hybridizes to the first allele at the annealing temperature and inhibits amplification of the first allele and the second allele is preferentially amplified; and
detecting the first allele and the preferentially amplified second allele through melting curve analysis of probe melting from each the first allele and the preferentially amplified second allele.
9. The method of claim 8 , wherein the amplifying step further comprises cycling through an extension temperature, the extension temperature being higher than the annealing temperature and below the first Tm.
10. The method of claim 9 , wherein the amplifying step includes a hold at the annealing temperature that is less than 5 seconds.
11. The method of claim 10 , wherein the hold is 1 second.
12. The method of claim 9 , wherein there is a 0 second hold at the extension temperature.
13. The method of any of claims 8 to 12 , wherein the adding step includes adding Mg++ to a concentration less than 2.0 mM.
14. The method of claim 13 , wherein the Mg++ concentration is about 1.5 mM.
15. The method of claim 8 , further comprising calculating allele fractions of the first allele and the second allele.
16. The method of claim 15 , wherein the calculating step comprises estimating the second allele fraction as Fm=wLf(TL)+wHf(TH), where wL and wH are weights and f(TL) and f(TH) are individual estimates at each temperature peak calculated from standards of a homozygote of the first allele, a homozygote of the second allele, and a 50:50 mix of the first allele and second allele.