Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

DNA isolation by a rapid method from human blood samples: effects of MgCl2, EDTA, storage time, and temperature on DNA yield and quality.

Author(s): Lahiri DK, Schnabel B

Publication: Biochem Genet, 1993, Vol. 31, Page 321-8

PubMed ID: 8274138 PubMed 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 storage temperature, freeze-thaw cycling of blood, use of dried blood spots instead of frozen blood, and DNA extraction solution components on the yield and integrity of DNA from leukocytes. Results presented are from DNA extracted from 1 mL aliquots of blood from a single individual, though the authors state extraction from >100 individuals was performed. Freeze-thaw experiments involved freezing of blood on dry ice for 10 min and then thawing at 37°C for 10 min.

   Summary of Findings:

   In whole blood stored for 24 h before DNA extraction, there was no effect of storage temperature (45, 37, 25, 4, -20 or -70°C) on DNA yield or integrity. 2 freeze-thaw cycles of the blood did not impact DNA yield or integrity, but when blood was subjected to 4 or 6 cycles, DNA was degraded. DNA obtained from dried blood spots was comparable to that obtained from frozen or fresh blood. The authors report complete lysis of the red cells with Triton X-100 or NP-40 and increasing DNA yields with increasing concentrations of NP-40 or Triton X-100 ≤1.2, but that use of Brij-35, digitonin, Sarcosyl and Tween-20/-80 did not result in complete lysis. While DNA yield increased with increasing concentrations of MgCl2 in the extraction buffer between 0 and 10 mM, >10mM MgCl2 led to increasing DNA degradation which could be attenuated by the addition of EDTA. The authors report no effect of NaCl addition or Tris concentration between 10 and 100 mM on DNA yield or quality. Further, the authors report no effect of 5-20 mM KCl, but addition of 25 mM KCl resulted in a white precipitate. The authors report lysis of the leukocytes using 0.5-1.0% SDS, but lysis with <0.5% SDS led to insufficient yields, and >1.0% SDS led to a milky white precipitate. The authors recommend that extraction of DNA from leukocytes should include a 10 mM Tris buffer containing 4 mM magnesium (Mg)Cl2, and 10 mM potassium (K)Cl2, with 2 mM EDTA (TKM buffer), use of 1.25% NP-40 to lyse the red blood cells, and lysis of leukocytes in TKM buffer with 1% SDS.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • Other Preservative
   • None (Fresh)

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   DNA	Electrophoresis

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage temperature	45°C 37°C 25°C 4°C -20°C -70°C
   Analyte Extraction and Purification	Analyte isolation method	0-1 M NaCl 0 mM MgCl2 1.5 mM MgCl2 3 mM MgCl2 4 mM MgCl2 5 mM MgCl2 6 mM MgCl2 8 mM MgCl2 10 mM MgCl2 12 mM MgCl2 15 mM MgCl2 20 mM MgCl2 25 mM MgCl2 2 mM EDTA 5 mM EDTA 10 mM EDTA 10-100 mM Tris 5-23 mM KCl 0-10.0% SDS
   Analyte Extraction and Purification	Cell/tissue permeabilization	Triton X-100 NP-40 Brij-35 Digitonin Sarcosyl Tween-20/-80
   Storage	Freeze/thaw cycling	0 cycles 1 cycle 2 cycles 4 cycles 6 cycles
   Biospecimen Preservation	Type of fixation/preservation	Air-dried Frozen None (fresh)

   View more

2. Study Purpose

   The purpose of this study was to investigate the effect of DNA rehydration solution and temperature on DNA yield and integrity. DNA was obtained from an unspecified number of stored blood specimens using an in-house protocol. A single aliquot of DNA was stored in each solution, at each temperature, for 40 or 60 days.

   Summary of Findings:

   DNA integrity and yield were unaffected by storage for 40 days at 25°C, 4°C, -20°C, -70°C, but DNA degraded when stored at 45°C or 37°C and rehydrated in 10 mM Tris-C1 and 2 mM EDTA. However, DNA degradation was partially attenuated when stored at 45°C or 37°C in 10 mM Tris-C1 and 5 mM EDTA and was completely prevented when stored at 45°C or 37°C in 10 mM Tris-C1 and 10 mM EDTA. The authors report similar stability of the DNA at each temperature when stored for 60 days as opposed to 40 days.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • None (Fresh)

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Electrophoresis
   DNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage temperature	37°C 45°C 25°C 4°C -20°C -70°C
   Analyte Extraction and Purification	Rehydration of dried sample/specimen	10 mM Tris-C1 and 2 mM EDTA 10 mM Tris-C1 and 5 mM EDTA 10 mM Tris-C1 and 10 mM EDTA
   Storage	Storage duration	40 days 60 days

   View more