Methods for detecting dna mutations using mitra tip extraction

Abstract

The present disclosure provides rapid and non-invasive methods for determining whether a patient exhibiting cancer symptoms, or at risk for hereditary cancers such as breast cancer, ovarian cancer, colon cancer, or skin cancer, will benefit from treatment with one or more therapeutic agents. These methods are based on detecting hereditary cancer-related mutations in small-volume dried biological fluid samples that are collected using a volumetric absorptive microsampling device (e.g., MITRA Tip). Kits for use in practicing the methods are also provided.

1 . A method for detecting the presence or absence of at least one mutation in a plurality of hereditary cancer-related genes in a dried biological fluid sample comprising:
 (a) eluting a dried biological fluid sample from an absorbent tip of a microsampling device by contacting the absorbent tip of the microsampling device with a lysis buffer and Proteinase K;   (b) extracting genomic DNA from the dried biological fluid sample eluted from the absorbent tip of a microsampling device;   (c) generating a library of amplicons comprising a plurality of hereditary cancer-related genes comprising APC, ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16), CHEK2, EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, POLD1, POLE, PTEN, RAD51C, RAD51D, RET, SDHB, SDHC, SDHD, SMAD4, STK11, TP53, and VHL, wherein an index sequence is ligated to at least one end of each amplicon in the library of amplicons; and   (d) detecting the presence or absence of at least one mutation in at least one of the amplicons using high throughput massive parallel sequencing.   
     
     
         2 . The method of  claim 1 , wherein the dried biological fluid sample is obtained from a patient having, or is suspected of having a hereditary cancer. 
     
     
         3 . The method of  claim 1 , wherein the dried biological fluid sample is dried plasma, dried serum, or dried whole blood. 
     
     
         4 . The method of  claim 1 , wherein the dried biological fluid sample on the absorbent tip of the microsampling device is collected from a patient via fingerstick. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein the lysis buffer comprises guanidine hydrochloride, Tris.Cl, EDTA, Tween 20, and Triton X-100. 
     
     
         7 . The method of  claim 1 , wherein elution of the dried biological fluid sample is performed by contacting the absorbent tip of the microsampling device with the lysis buffer for up to 15 minutes at 90° C. 
     
     
         8 . The method of  claim 1 , wherein elution of the dried biological fluid sample is performed by contacting the absorbent tip of the microsampling device with Proteinase K for up to 1 hour at 56° C. 
     
     
         9 . The method of  claim 1 , wherein elution of the dried biological fluid sample is performed by contacting the absorbent tip of the microsampling device with Proteinase K for up to 16-18 hours at 56° C. 
     
     
         10 . The method of  claim 1 , wherein the microsampling device is a MITRA® tip. 
     
     
         11 . The method of  claim 1 , wherein the sample volume of the microsampling device is no more than 10-20 μL. 
     
     
         12 . The method of  claim 2 , wherein the hereditary cancer is breast cancer, ovarian cancer, colon cancer, or skin cancer. 
     
     
         13 . The method of  claim 1 , wherein no more than 400 ng of genomic DNA is eluted from the absorbent tip of the microsampling device. 
     
     
         14 . The method of  claim 1 , wherein about 100 ng to about 400 ng of genomic DNA is eluted from the absorbent tip of the microsampling device. 
     
     
         15 . The method of  claim 1 , wherein the high throughput massive parallel sequencing is performed using pyrosequencing, reversible dye-terminator sequencing, SOLiD sequencing, Ion semiconductor sequencing, Helioscope single molecule sequencing, sequencing by synthesis, sequencing by ligation, or SMRT™ sequencing. 
     
     
         16 . The method of  claim 1 , wherein the adapter sequence is a P5 adapter, P7 adapter, P1 adapter, A adapter, or Ion Xpress™ barcode adapter. 
     
     
         17 . The method of  claim 1 , wherein the plurality of amplicons further comprise a unique index sequence. 
     
     
         18 . The method of  claim 1 , wherein the plurality of amplicons are enriched using a bait set comprising nucleic acid sequences that are complementary to at least one of the plurality of amplicons. 
     
     
         19 . The method of  claim 18 , wherein the nucleic acid sequences of the bait set are RNA baits, DNA baits, or a combination thereof. 
     
     
         20 - 31 . (canceled) 
     
     
         32 . The method of  claim 2  further comprising selecting the patient for treatment with an anti-cancer therapeutic if a mutation in at least one amplicon in the library of amplicons is detected. 
     
     
         33 . The method of  claim 32 , wherein he anti-cancer therapeutic agent is one or more agents selected from the group consisting of cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb, anthracyclines, bevacizumab, aldesleukin, cobimetinib, dabrafenib, dacarbazine, talimogene laherparepvec, imiquimod, recombinant Interferon Alfa-2b, ipilimumab, pembrolizumab, trametinib, nivolumab, peginterferon Alfa-2b, sonidegib, vismodegib, vemurafenib, cetuximab, irinotecan hydrochloride, leucovorin calcium, trifluridine and tipiracil hydrochloride, oxaliplatin, panitumumab, ramucirumab, regorafenib, and ziv-aflibercept. 
     
     
         34 . The method of  claim 2 , wherein patient has or is at risk of developing a hereditary cancer selected from the group consisting of breast cancer, ovarian cancer, skin cancer, or colon cancer.