DNA mutation detection employing enrichment of mutant polynucleotide sequences and minimally invasive sampling

1. A method for enriching a target polynucleotide sequence containing a genetic variation, said method comprising:

(a) providing a biological sample;

(b) isolating DNA from said biological sample; said DNA including said target polynucleotide sequence containing a genetic variation;

(c) providing two primer probes targeted to said target polynucleotide sequence said primer probes allowing formation of a PCR process product;

(d) providing a target specific xenonucleic acid clamp oligomer probe specific for a wildtype polynucleotide sequence; having SEQ ID NO: 188; wherein said target specific xenonucleic acid clamp provides a Tm differential of about 15°-20° C. so that during the qPCR process only mutant templates are amplified;

(e) admixing the primer probes and the xenonucleic clamping probe with the target nucleic acid sample;

(f) performing a PCR amplification process in a reaction solution under hybridization conditions thereby generating multiple amplicons; and

(g) detecting said amplicons.

2. The method of claim 1 , wherein said detection employs oligonucleotide probes specific for hybridization of variant polynucleotide amplicon sequences.

3. The method of claim 1 , wherein the target polynucleotide sequence containing a genetic variation is in a gene selected from the group consisting of: KRAS, BRAF, EGFR, TP53, JAK2, NPM1, and PCA3.

4. A method for enriching multiple target polynucleotide sequences containing a genetic variation said method comprising:

(a) providing a biological sample;

(b) isolating DNA from said biological sample; said DNA including said multiple target polynucleotide sequences containing a genetic variation;

(c) providing a library of amplifying primer probes targeted to said multiple target polynucleotide sequences containing a genetic variation; said primer probes allowing formation of PCR process products;

(d) providing a library of target specific xenonucleic acid clamp oligomer probes specific for multiple wildtype polynucleotide sequences selected from the group consisting from SEQ ID NO: 187 to SEQ ID NO: 189; wherein said target specific xenonucleic acid clamps provides a Tm differential of about 15°-20° C. so that during the qPCR process only mutant templates are amplified;

(e) admixing the primer probes and the xenonucleic clamping probes with the multiple target nucleic add samples;

(f) performing a PCR amplification process in a reaction solution under hybridization conditions thereby generating multiple amplicons; and

(g) detecting said amplicons.

5. The method of claim 3 , wherein said detection employs oligonucleotide probes specific for hybridization of variant polynucleotide amplicon sequences.

6. A method for conducting a minimally invasive biopsy in a mammalian subject suspected of a having a neoplastic disease, said method comprising:

(a) providing a biological sample derived from said mammalian subject;

(b) isolating DNA from said biological sample; said DNA including multiple target polynucleotide sequences containing a genetic variation;

(c) providing a library of amplifying primer probes targeted to said multiple target poly-nucleotide sequences containing a genetic variation; said primer probes allowing formation of PCR process products;

(d) providing a library of target specific xenonucleic acid clamp oligomer probes specific for multiple wildtype polynucleotide sequences selected from the group consisting from SEQ ID SEQ ID NO: 187 to SEQ ID NO: 189; wherein said target specific xenonucleic acid clamps provides a Tm differential of about 15°-20° C. so that during the qPCR process only mutant templates are amplified;

(e) admixing the primer probes and the xenonucleic clamping probes with the multiple target nucleic acid samples;

(f) performing a PCR amplification process in a reaction solution under hybridization conditions thereby generating multiple amplicons; and

(f) detecting said amplicons.

7. The method of claim 5 , wherein said biological sample are cells derived from said mammalian subject.

8. The method of claim 5 , wherein said target polynucleotides containing a genetic variation are derived from free circulating cell free polynucleotides derived from said mammalian subject.

9. The method of claim 4 , which includes using multiple Xenonucleic acid clamp probes and amplifying primers targeted to multiple polynucleotide sequences.