Characterization of Effect of Repeated Freeze and Thaw Cycles on Stability of Genomic DNA Using Pulsed Field Gel Electrophoresis

Author(s): Shao W, Khin S, Kopp WC

Publication: Biopreservation and Biobanking, 2012, Vol. 10, Page 4-11

Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of blood DNA extraction protocol, sample concentration during storage, freeze-thaw cycling of DNA samples and freezing temperature on genomic DNA size distribution. Acid-citrate dextrose blood was frozen at -70 degrees C until extraction of DNA using each of the three methods. DNA was stored for 4-6 months at 4 degrees C prior to freeze-thaw cycling experiments and returned to 4 degrees C after freeze-thaw cycling. DNA was cycled by freezing slowly (2 h at 4 degrees C, 1 h at -20 degrees C, and then overnight at -70 degrees C) and thawing slowly (-20 degrees C for 1 h, and 4 degrees C for 2 h prior, and then room temperature for 3 h)(protocol A) or cycled between -70 (protocol B), -20 (protocol C) or 4 degrees C (protocol D) overnight and thawing at room temperature for 3 h.

   Summary of Findings:

   DNA extracted using phenol-chloroform was the most intact and had the most uniform size distribution (12 to more than 300 kb), followed by PureGene which yielded DNA of 12-100 kb (average 60 kb). DNA extracted by QIAamp average only 30 kb and ranged from 8-80 kb. After 18 freeze-thaw cycles using protocol B (-70 degrees C/room temperature), the average DNA size was 30 kb and the size distributions were similar, regardless of initial extraction method. While average DNA size was not impacted by cycling between room temperature and 4 degrees C (protocol D), average DNA size decreased with each of the freeze-thaw cycling temperature predominantly due to loss of the largest fragments. Significant declines in DNA size were noted with 3 freeze-thaw cycles and further declines were observed with additional freeze-thaw cycles when PureGene or phenol-chloroform extracted DNA was stored at concentrations of 10 or 50 ug/mL and after 6 or more cycles compared to 0 cycles and 12 cycles compared to 6 or fewer cycles when DNA samples were stored at a concentration of 100 ug/mL. QIAamp extracted DNA was generally more stable than samples extracted using the other two methods, and significant declines in average DNA size were only noted between 0 and 6 and 6 and 12 freeze-thaw cycles, regardless of initial concentration. The changes in mean DNA size were slightly smaller when specimens were frozen at -20 degrees C (protocol C) than -70 degrees C (protocol A or B), but the differences were not significant.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   DNA	Real-time qPCR
   DNA	Electrophoresis
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	QIAamp blood Maxi kit Gentra PureGene Blood Kit Phenol-chloroform
   Storage	Freeze/thaw cycling	0 cycles 3 cycles 6 cycles 9 cycles 12 cycles 15 cycles 18 cycles
   Storage	Storage temperature	4 degrees C -20 degrees C -70 degrees C
   Biospecimen Aliquots and Components	Aliquot size/volume	10 ug/mL DNA 50 ug/mL DNA 100 ug/mL DNA
   Biospecimen Preservation	Type of fixation/preservation	Frozen Refrigeration

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