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

Sedlackova Tatiana, Repiska Gabriela, Celec Peter, Szemes Tomas, Minarik, Minarik Gabriel

Description

Specific applications and modern technologies, like non-invasive prenatal testing, non-invasive cancer diagnostic and next generation sequencing, are currently in the focus of researchers worldwide. These have common characteristics in use of highly fragmented DNA molecules for analysis. Hence, for the performance of molecular methods, DNA concentration is a crucial parameter; we compared the influence of different levels of DNA fragmentation on the accuracy of DNA concentration measurements. Results In our comparison, the performance of the currently most commonly used methods for DNA concentration measurement (spectrophotometric, fluorometric and qPCR based) were tested on artificially fragmented DNA samples. In our comparison, unfragmented and three specifically fragmented DNA samples were used. According to our results, the level of fragmentation did not influence the accuracy of spectrophotometric measurements of DNA concentration, while other methods, fluorometric as well as qPCR-based, were significantly influenced and a decrease in measured concentration was observed with more intensive DNA fragmentation. Conclusions Our study has confirmed that the level of fragmentation of DNA has significant impact on accuracy of DNA concentration measurement with two of three mostly used methods (PicoGreen and qPCR). Only spectrophotometric measurement was not influenced by the level of fragmentation, but sensitivity of this method was lowest among the three tested. Therefore if it is possible the DNA quantification should be performed with use of equally fragmented control DNA.

Sujets

• DNA fragmentation
• Spectrophotometry
• PCR quantitative

Informations

• Publié par: biomed
• Publié le: 01 janvier 2013
• Nombre de lectures: 13
• Langue: English

Extrait

Sedlackova et al. Biological Procedures Online 2013, 15:5
Biological Procedureshttp://www.biologicalproceduresonline.com/content/15/1/5
Online
RESEARCH Open Access
Fragmentation of DNA affects the accuracy of the
DNA quantitation by the commonly used
methods
1* 1 1,3 2,3 1,3Tatiana Sedlackova , Gabriela Repiska , Peter Celec , Tomas Szemes and Gabriel Minarik
Abstract
Background: Specific applications and modern technologies, like non-invasive prenatal testing, non-invasive cancer
diagnostic and next generation sequencing, are currently in the focus of researchers worldwide. These have
common characteristics in use of highly fragmented DNA molecules for analysis. Hence, for the performance of
molecular methods, DNA concentration is a crucial parameter; we compared the influence of different levels of
DNA fragmentation on the accuracy of DNA concentration measurements.
Results: In our comparison, the performance of the currently most commonly used methods for DNA
concentration measurement (spectrophotometric, fluorometric and qPCR based) were tested on artificially
fragmented DNA samples. In our comparison, unfragmented and three specifically fragmented DNA samples were
used.
According to our results, the level of fragmentation did not influence the accuracy of spectrophotometric
measurements of DNA concentration, while other methods, fluorometric as well as qPCR-based, were significantly
influenced and a decrease in measured concentration was observed with more intensive DNA fragmentation.
Conclusions: Our study has confirmed that the level of fragmentation of DNA has significant impact on accuracy
of DNA concentration measurement with two of three mostly used methods (PicoGreen and qPCR). Only
spectrophotometric measurement was not influenced by the level of fragmentation, but sensitivity of this method
was lowest among the three tested. Therefore if it is possible the DNA quantification should be performed with use
of equally fragmented control DNA.
Keywords: DNA fragmentation, DNA quantitation, Spectrophotometry, PicoGreen, qPCR
Background enzymatic cleavage process during apoptosis [2]. On the
Circulating nucleic acids are currently studied as a po- other hand, the fragment lengths of circulating nucleic
tential diagnostic marker for oncological diseases as well acids vary in size in cases of malignant disease, because
as in relation to non-invasive prenatal diagnosis. Sub- they are released from apoptotic cells as well as necrotic
stantial fragmentation and low concentrations are limit- cells [3,4]. The second mentioned limiting characteristic
ing characteristic features of circulating nucleic acids of cNA is its low concentration. The accuracy of DNA
(cNA). According to a recent study, the cNA are present quantification is crucial for the success of following
in the circulation at sizes lower than 1200 bp and most downstream applications such as (q)PCR, sequencing
of cNA molecules are clustered into two peaks, first at and cloning. Commonly used methods of DNA
concenapproximately 162 bp and second at 340 bp, represent- tration measurements are the evaluation of the intensity
ing a dominant and a minor peak [1]. These molecules of a band on an agarose gel, fluorescence measurements
are released from apoptotic cells after the programmed using various DNA-binding dyes and measurements of
UV absorbance at 260 nm [5], with the latter being the
* Correspondence: [e-mail] most commonly used [6,7]. The disadvantages of the
lat1
Institute of Molecular BioMedicine, Faculty of Medicine, Comenius
ter method are that the absorbance measurement at
University, Sasinkova 4, Bratislava 811 08, Slovakia
260 nm includes signals of a double-stranded and single-Full list of author information is available at the end of the article
© 2013 Sedlackova et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.Sedlackova et al. Biological Procedures Online 2013, 15:5 Page 2 of 8
http://www.biologicalproceduresonline.com/content/15/1/5
stranded DNA oligonucleotides and free nucleotides, the spectrophotometry. The A ratio ranged from 1.83260/280
fact that it does not distinguish between DNA and RNA, ± 0.06 to 1.90 ± 0.04 for undiluted samples and from
and that it has a low sensitivity, reaching 1 ng/μl [6,8,9]. 1.93 ± 0.19 to 1.94 ± 0.23 for 10-fold diluted samples
In contrast, fluorescent dyes selectively measure only (Figure 1).
double-stranded DNA and are much more sensitive Regarding the DNA quantification by PicoGreen
fluor[8,9]. The most commonly used fluorescent dyes are escent dye, it was possible to determine concentrations
Hoechst 33258 and PicoGreen. Hoechst 33258 allows of the 10-fold, 100-fold and 1000-fold diluted samples.
the detection and quantitation of DNA at concentrations The concentration of the undiluted samples could not be
as low as 10 pg/μl [9,10]. The measurement of concen- established because the fluorescence corresponding to
tration using PicoGreen, which is currently very popular, the highest point of the standard curve was lower than
allows the detection of dsDNA in a final concentration
as low as 25 pg/μl [8,9]. The disadvantage is that the
concentration assessment by fluorescent dyes
underestimates the concentration of double-stranded DNA with a
size less than 23 kbp [6]. Another method used for DNA
quantification is qPCR [11,12]. This is a good choice for
qualitative as well as quantitative analysis of DNA
because of its high sensitivity and specificity for typical
molecular applications. The use of multi-copy genes,
such as rDNA genes and Alu repeats, as qPCR targets
can improve the qPCR sensitivity above the limited
sensitivity of ordinary PCR [13,14], as well as fluorometric
methods up to 1 picogram of human DNA [15]. The
aim of our study was to determine whether the degree
of DNA fragmentation affects the measurement of DNA
concentration with the three most commonly used
methods - spectrophotometry, fluorometry and qPCR.
Because of specific sizes of cNA fragments isolated from
plasma samples, we decided to compare measurements
of unfragmented samples (~25 kb fragments) with
artificially fragmented DNA samples at three targeted
fragment sizes - 1500 bp, 500 bp and 150 bp. These three
sizes should cover the whole sample as well as
predominant cNA fractions.
Results
DNA quantification by the spectrophotometric
measurement of absorbance at 260 nm was performed in
undiluted and 10-fold diluted samples. The 100-fold and
1000-fold diluted samples concentrations could not be
measured due to concentrations below the detection
limit of this method. Measurements of undiluted
samples showed that the DNA quantities in samples
with the length of fragments of approximately 1500 bp
and 500 bp were slightly decreased compared to the
concentration of unfragmented samples and those with
Figure 1 UV spectrophotometric quantification of DNA byfragments of approximately of 150 bp. This decrease
NanoDrop. (A) Quantification of undiluted samples showed that the
in DNA concentration was statistically significant
DNA quantity in the samples with the length of fragments approx.
(p< 0.001). There was no difference between the concen- 1500 bp and 500 bp significantly decreased compared to
tration of unfragmented DNA samples and specimens concentration of unfragmented samples. There was no difference
between the concentration of unfragmented DNA samples andwith fragment peaks at 150 bp. DNA concentration was
specimens with the fragment length of 150 bp. (B) The DNAnot affected by the level of fragmentation in the 10-fold
concentration of 10-fold diluted samples was not affected by the
diluted sample. Purity of DNA in all samples was
deterlevel of fragmentation. ns, statistically non-significant; ***, p< 0.0001.
mined based on the A ratio measured by260/280Sedlackova et al. Biological Procedures Online 2013, 15:5 Page 3 of 8
http://www.biologicalproceduresonline.com/content/15/1/5
the fluorescence of undiluted samples. Measurements [5,6,16,19]. Our study was focused on evaluation of the
showed that the concentration of DNA in samples with quantification of DNA using three different methods
different levels of fragmentation was influenced by the with respect to the level of DNA fragmentation.
length of the fragments in all cases of sample dilutions. First, the spectrophotometric measurement at 260 nm,
In case of 10-fold diluted samples, the DNA quantity in which is the most frequently used method, was assessed.
specimens with fragments of approximately 1500 bp was The Shokere et al. [5] group suggested that the
concenreduced only gently compared to intact samples (from tration of DNA increased slightly with increasing
frag39.19 ± 7.79 to 37.07 ± 7.85), but concentrations of sam- mentation of DNA. However, our results show that
ples with targeted fragments of 500 bp and 150 bp were DNA quantification based on A is not significantly260
significantly reduced (from 39.19 ± 7.79 to 34.24 ± 7.62 affected by the level of DNA fragmentation (Figure 1).
or 27.73 ± 5.68 respectively). When it comes to 100-