Allele amplification bias
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 kit for amplification and allele detection of a biological sample wherein the biological sample comprises 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, the kit comprising:
a thermostable polymerase lacking a 5′ to 3′ exonuclease activity, a ds DNA binding dye
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 and the probe has a first Tm when hybridized to the first allele and second Tm when hybridized to the second allele, wherein the first Tm is higher than the second Tm,
an oligonucleotide probe, wherein the probe is a probe element attached to a 5′ end of one primer of the pair of primers, and
instructions for amplifying the target nucleic acid in the biological sample by thermal cycling between a denaturation temperature and an annealing temperature, wherein the annealing temperature is below the first Tm, and thermal cycling through an extension temperature between at least the annealing temperature and the denaturation temperature at a ramp rate of at least 4° C./sec.
2. The kit of claim 1 , further comprising a buffer to provide a Mg++ concentration less than 2.0 mM when added to the biological sample.
3. The kit of claim 1 , wherein the second allele is present in an allele fraction of 10% or less.
4. The kit of claim 1 , wherein the second allele is present in an allele fraction of 5% or less.
5. The kit of claim 1 , wherein the instructions further include instructions for detecting the preferentially amplified second allele through melting curve analysis of the probe and the amplified second allele.
6. The kit of claim 1 , wherein the kit includes only one probe.
7. The kit of claim 1 , wherein the instructions further include instructions for calculating allele fractions of the first allele and the second allele, 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.
8. A kit for amplification and allele detection of a biological sample wherein the biological sample comprises 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, comprising:
a thermostable polymerase lacking a 5′ to 3′ exonuclease activity,
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 and the probe has a first Tm when hybridized to the first allele and second Tm when hybridized to the second allele, wherein the first Tm is higher than the second Tm,
an oligonucleotide probe configured to hybridize to the target nucleic acid without being extended by the thermostable polymerase, wherein the probe is a probe element attached to a 5′ end of one primer of the pair of primers, the probe having 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,
instructions for amplifying the target nucleic acid in the biological sample by thermal cycling between a denaturation temperature and an annealing temperature, wherein the annealing temperature is below the first Tm, and thermal cycling through an extension temperature between at least the annealing temperature and the denaturation temperature at a ramp rate of at least 4° C./sec, and for detecting the preferentially amplified second allele through melting curve analysis of the probe and the amplified second allele.
9. The kit of claim 8 , wherein the kit includes only one probe.
10. The kit of claim 8 , wherein the instructions further include instructions for calculating allele fractions of the first allele and the second allele, 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.