Whole-genome amplified DNA from stored dried blood spots is reliable in high resolution melting curve and sequencing analysis.

Author(s): Winkel BG, Hollegaard MV, Olesen MS, Svendsen JH, Haunsø S, Hougaard DM, Tfelt-Hansen J

Publication: BMC Med Genet, 2011, Vol. 12, Page 22

PubMed ID: 21306642 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine if WGA-DBS stored frozen for 19-28 years have comparable melting curves and sequencing results to fresh blood specimens from patients with atrial fibrillation. DBS obtained at patient birth were stored at -20 degrees C for 19-28 years. DNA was extracted from DBS by Extract-N-amp Blood PCR Kit and amplified with REPLI-g. Fresh blood was obtained from the same patients, and DNA was extracted using the QIAamp mini blood kit. Melting curves were evaluated by high resolution melting curve analysis.

   Summary of Findings:

   Melting curve analysis of 40 amplicons in 10 patients revealed 85 (21%) and 81 (20%) alterations from wild-type using WGA-DBS DNA and fresh genomic DNA, respectively. Although, 64% and 62% of the alterations using WGA-DBS and fresh blood, respectively, were determined to be false positives after sequencing. By sequencing, the same 31 variants were identified using DNA from WGA-DBS and fresh DNA and included 29 heterogeneous and 2 homogeneous changes.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative
   • None (Fresh)
   • Frozen

   Diagnoses:

   • Cardiovascular Disease
   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   DNA	DNA sequencing
   DNA	SNP assay
   DNA	Whole genome amplification

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Air-dried None (fresh) Frozen
   Storage	Storage duration	0 days 19-28 years
   Analyte Extraction and Purification	Analyte isolation method	QIAamp mini kit Extract-N-amp Blood PCR Kit
   SNP assay Specific	Targeted nucleic acid	SCN5A KCNA5
   SNP assay Specific	Nucleic acid amplification	None REPLI-g
   Preaquisition	Diagnosis/ patient condition	Atrial fibrillation
   SNP assay Specific	Technology platform	High resolution melting curve analysis DNA sequencing

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