High Quality, High Integrity Nucleic Acid Extraction from FFPE Tissues

Project Summary Formalin fixed paraffin embeddedFFPEtissue samples have evolved into a valuable resource for genomics research and become a de facto sample type for many molecular cancer testsWhile FFPE processing can adequately stabilize nucleic acids for transport and storageexisting extraction methods struggle to obtain high quality DNA RNA due to cross linkingfragmentationand organic contaminationIn Phase Iwe performed proof of feasibility studies using Nanobind to extract high quality DNA from various types of fresh and fixed tissue samplesNanobind is a novel thermoplastic nanomaterial that can be inexpensively manufactured and is capable of extracting higher quality DNA than any competing methodWhere current extraction methods have struggledwe have demonstrated that with Nanobind it is possible to obtain extremely high qualityhigh molecular weight DNAkbfrom fresh FFPE samplesSeparatelywe discovered that UV spectrometryQubit PicoGreen assaysand electrophoresis provide an incomplete picture of DNA quality and are often poor predictors of performance in sequencing and genome mappingDamage lesionssuch as nicksabasic sitesprotein DNA crosslinksand DNA DNA crosslinksthat are pervasive in FFPE samples cannot be detected by these methodsSuch damage is first generated during fixation and then compounded during subsequent storage and harsh extraction processesDue to the lack of suitable assays to quantify various damage lesionslittle is known about how preanalytical and sample preparation factors impact DNA quality other than their effects on yieldgross impuritiesand integrityIn Phase IIwe will build upon our Phase I studies to develop new assays to quantify specific DNA damage lesions and then use these assays to refine our understanding of FFPE sample processingFirstwe will develop simple fluorescent assays to quantifycommon forms of DNA damagenicksabasic sitesand deaminationSecondwe will utilize these assays to further improve the quality of Nanobind extracted DNA from FFPE tissue samples and to study the upstream effects of FFPE processing on damage lesionsFinallywe will validate Nanobind FFPE DNA extraction performance by isolating DNA from freshfixedand accelerated aging samplescharacterizing the DNA using standard methods and the newly developed DNA damage assaysand comparing against NGS and long read sequencing data