Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods.

Author(s): Sedlackova T, Repiska G, Celec P, Szemes T, Minarik G

Publication: Biol Proced Online, 2013, Vol. 15, Page 5

PubMed ID: 23406353 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effect of DNA fragmentation on the quantification of DNA by spectrophotometric, fluorometric, and PCR based methods. DNA was extracted from K2EDTA blood from 10 volunteers using the QIAamp DSP DNA Blood Mini Kit. DNA was fragmented by ultrasound to achieve specimens with target peaks at 1500 bp, 500 bp, and 150 bp and fragmentation was verified by electrophoresis. DNA was quantified by duplicate measurements using a NanoDrop spectrophotometer, PicoGreen Quant-iT and dsDNA fluoremetry, and real-time PCR amplification of a 115 bp fragment of the Alu element.

   Summary of Findings:

   DNA yield as determined by spectrophotometer was not affected when DNA was fragmented to 150 bp but declined significantly when fragmented to 1500 bp or 500 bp (P<0.0001, both) although the decline was no longer significant when the specimen was diluted 10-fold prior to quantification. The yield of DNA declined with increasing DNA fragmentation as determined by PicoGreen, regardless of DNA dilution (10, 100, or 1000-fold). Importantly, all fragmented specimens had significantly lower amounts of DNA as determined by PicoGreen than unfragmented specimens (P<0.0001, all) except the 10-fold dilution of the DNA fragmented to 1500 bp. In contrast, fragmentation of the DNA to 150 bp resulted in decreased DNA quantification by real-time PCR, regardless of dilution (10, 100 and 1000-fold), but no effects were observed when DNA was fragmented to 1500 bp or 500 bp.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Not specified

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qPCR Specific	Targeted nucleic acid	Alu element
   Real-time qPCR Specific	Template/input amount	Diluted 1:10 Diluted 1:100 Diluted 1:1000
   Spectrophotometry Specific	Template/input amount	Undiluted Diluted 1:10
   Spectrophotometry Specific	Technology platform	PicoGreen Real-time PCR
   Fluorometry Specific	Template/input amount	Diluted 1:10 Diluted 1:100 Diluted 1:1000
   Fluorometry Specific	Template modification	Unfragmented Fragmented to 1500 bp Fragmented to 500 bp Fragmented to 150 bp
   Spectrophotometry Specific	Template modification	Unfragmented Fragmented to 1500 bp Fragmented to 500 bp Fragmented to 150 bp
   Real-time qPCR Specific	Template modification	Unfragmented Fragmented to 1500 bp Fragmented to 500 bp Fragmented to 150 bp

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