Allele amplification bias
Inventors
Zhou, Luming • Palais, Robert Andrew
Assignees
Biofire Defense LLC • University of Utah Research Foundation Inc
Publication Number
US-10351903-B2
Publication Date
2019-07-16
Expiration Date
2029-11-06
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Abstract
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.
Core Innovation
The invention describes methods for nucleic acid analysis that utilize allele amplification bias to preferentially amplify a target nucleic acid present at a low allele fraction. The approach involves configuring a probe and thermal cycling conditions such that a minor allele, often representing mutations or sequence alterations of clinical significance, can be enriched and readily detected even when present at low levels in a biological sample.
Allele amplification bias is achieved by using probes—such as unlabeled probes or Snapback primers—matched to the higher fraction allele, with melting temperatures (Tms) that differ between the two alleles. By selecting an annealing or extension temperature below the Tm of the matched probe (i.e., the wild type or higher fraction allele), PCR preferentially retards amplification of that predominant allele, thereby enabling enrichment and detection of alleles present at much lower fractions through subsequent melting curve analysis or other detection methods.
The problem addressed is the difficulty in detecting and quantifying low copy number alleles, such as somatic mutations, mitochondrial variants, or fetal DNA in maternal blood, due to the high background of wild type sequences and limitations of standard PCR or sequencing technologies. Existing methods are often complex, expensive, and inadequate in sensitivity for rare allele detection, motivating the need for a simple, rapid, and sensitive method as described in this invention.
Claims Coverage
The patent claims cover two independent inventive features related to kits for amplification and allele detection that exploit allele amplification bias using specific probe and primer configurations.
Kit for allele detection using probe with differential melting temperatures and rapid thermal cycling
A kit comprising: - A thermostable polymerase lacking 5′ to 3′ exonuclease activity. - A dsDNA binding dye. - A pair of primers for amplification of a target nucleic acid, with an oligonucleotide probe configured to hybridize to the target and having a first Tm when hybridized to a majority allele and a lower Tm for a minority allele. - The probe is attached to the 5′ end of one primer. - Instructions for amplifying the nucleic acid by thermal cycling, with an annealing temperature below the Tm of the matched probe, including cycling through an extension temperature between annealing and denaturation at a ramp rate of at least 4°C/sec.
Kit for allele detection with probe not extended by polymerase and instructions for melting curve analysis
A kit comprising: - A thermostable polymerase lacking 5′ to 3′ exonuclease activity. - A pair of primers for amplifying a target nucleic acid, with a probe that hybridizes to the target without being extended by the polymerase. - The probe is attached to a primer’s 5′ end and possesses distinct Tms with the matched and mismatched alleles. - Instructions for amplifying the nucleic acid at an annealing temperature below the matched allele Tm, with an extension temperature between the annealing and denaturation temperatures at a ramp rate of at least 4°C/sec, and instructions for detecting the preferentially amplified low-fraction allele through melting curve analysis.
The claims provide kits and methods for sensitive detection of low concentration alleles using probes with differential Tm, specialized PCR conditions, and analysis protocols, specifically to achieve allele amplification bias and preferential detection of rare alleles.
Stated Advantages
The method lowers costs and simplifies DNA analysis, making it accessible for routine clinical use.
It provides rapid, simple, and inexpensive detection of mutations, even when present at very low fractions.
It enables identification of low copy number alleles, such as rare oncogene mutations or minority DNA in a mixed background, which is important for disease detection and treatment selection.
The approach allows for closed-tube, solution-phase genotyping which reduces contamination risk and minimizes steps.
Methods are compatible with high resolution melting analysis, offering high sensitivity and specificity without the need for additional reagents or separation steps.
Documented Applications
Detection of somatic mutations, such as p53, EGFR, and BRAF mutations, for cancer profiling and diagnostic purposes.
Genotyping for known sequence variants, including single nucleotide polymorphisms (SNPs), in clinical and research settings.
Detection of mutant bacterial infections (e.g., malaria) where rapid identification of minor alleles is important for therapy selection.
Analysis of mitochondrial DNA and fetal DNA present within maternal blood for non-invasive prenatal or disease testing.
Detection and quantification of low levels of epigenetic mutations, such as BRCA1 promoter methylation.
Identification of EGFR exon 19 small deletions in non-small cell lung cancer for targeted therapy decisions.
Allele fraction estimation for applications requiring quantification of rare and common allelic species in a sample.
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