Post on 25-Apr-2023
Accepted Manuscript
Comparison of DNA extraction methods for multiplex PCR
Triin Viltrop, Kaarel Krjutškov, Priit Palta, Andres Metspalu
PII: S0003-2697(09)00801-X
DOI: 10.1016/j.ab.2009.11.026
Reference: YABIO 9737
To appear in: Analytical Biochemistry
Received Date: 23 August 2009
Revised Date: 16 November 2009
Accepted Date: 17 November 2009
Please cite this article as: T. Viltrop, K. Krjutškov, P. Palta, A. Metspalu, Comparison of DNA extraction methods
for multiplex PCR, Analytical Biochemistry (2009), doi: 10.1016/j.ab.2009.11.026
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Comparison of DNA extraction methods for multiplex PCR
Triin Viltrop1*
, Kaarel Krjutškov1,2*
, Priit Palta2,3
, Andres Metspalu1,2,4
1 Department of Biotechnology, IMCB, University of Tartu, 23 Riia St, 51010 Tartu, Estonia
2 Estonian Biocentre, 23 Riia St, 51010 Tartu
3 Department of Bioinformatics, IMCB, University of Tartu, 23 Riia St, 51010 Tartu
4 Estonian Genome Project of University of Tartu, 61b Tiigi St, 50410, Tartu
* These authors contributed equally to this work
Corresponding author: Triin Viltrop
Tel: + (372) 7 375 034
Fax: +(372) 7 420 286
tviltrop@ut.ee
Department of Biotechnology, IMCB, University of Tartu, 23 Riia St, 51010 Tartu, Estonia
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Abstract
We compared six DNA extraction methods for obtaining DNA from whole blood and saliva for use in
multiplex PCR assays. The aim was to evaluate saliva sampling as an alternative to blood sampling to
obtain DNA for molecular diagnostics, genetic genealogy and research purposes. The DNA quantity,
DNA purity (A260/280), PCR inhibition ratio and mitochondrial DNA/genomic DNA ratio were
measured in order to compare the extraction methods. The different extraction methods resulted in
variable DNA quantity and purity but there were no significant differences in the efficiency of
multiplex PCR and oligomicroarray signals after single base extension on APEX-2
Keywords: Arrayed primer extension, multiplex PCR, saliva DNA
Abbreviations: APEX-2 arrayed primer extension 2, gDNA genomic DNA, mtDNA mitochondrial
DNA, SBE single-base extension
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Numerous protocols for extracting DNA from blood and saliva have been developed and
commercialised. Nevertheless there is still some obscurity whether in high level multiplex PCR
reactions, DNA extracted from saliva could have an equivalent quality to that extracted from blood
and thereby could serve as a reliable alternative DNA source. There are many advantages to using
DNA from saliva. Saliva collection is a painless procedure with no risk of disease transmission and no
requirements for specialized medical personnel. Also, saliva collection allows wider population
sampling as it is possible to collect DNA samples by mail [1; 2]. Here we compare two commercial
saliva DNA extraction kits, three commercial blood DNA extraction kits and an adapted phenol-
chloroform DNA extraction method as “golden standard”. The extraction methods were assessed
based on the yield of total DNA, DNA purity (A260/280), and compatibility with post-extraction
analysis (multiplex PCR). In addition, we estimated the ratio of mitochondrial DNA (mtDNA) to
genomic (gDNA) in the extracted samples. The suitability of the extracted DNA for use in further
analysis was assessed using a 124-plex assay system for genetic testing [3] where the arrayed primer
extension (APEX-2) principle was employed [4] as described previously. After 124-plex PCR, single-
base extension (SBE) on microarrays was performed and the influence of the DNA extraction method
was evaluated by measuring the call rate and the incidence of false signals.
Six different DNA extraction methods were studied. Two kits were used to extract DNA from
saliva: Oragene DNA OG-250 (Oragene, DNA Genotek Inc., Ottawa, Canada) and PSP Saligene
DNA Kit (Saligene, Invitek GmbH, Berlin, Germany). Three kits were used to extract DNA from
blood: QIAamp DNA Mini Kit (QIAamp, Qiagen, Hilden, Germany), PAXgene™ Blood DNA
System (PAXgene, PreAnalytiX GmbH, Hombrechtikon, Switzerland) and Fermentas Genomic DNA
Purification Kit (Fermentas, Fermentas, Vilnius, Lithuania). As a reference, a phenol-chloroform was
to extract DNA from fresh blood. The efficiency of Oragene and QIAamp extraction kits has been
examined previously [2; 5] but the efficiency of Saligene and Fermentas kits has not yet been studied.
Blood samples (4.5 mL) were collected in lavender-top EDTA BD VacutainerTM
tubes (BD,
NY, USA) with informed, written consent from 25 healthy volunteers for all but the PAXgene.
Samples for the PAXgene were collected in 8.5 mL tubes provided by the manufacturer. QIAamp and
PAXgene samples were extracted according to the manufacturers’ instructions. Fermentas blood
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samples (200 L) were extracted using a modified protocol. Briefly, chloroform was added to the
blood sample and the sample was vortexed to homogeneity. All centrifugations were performed at
16 000 g. After the precipitation solution was added (according to manufacturer’s protocol) the
centrifugation lasted for 10 min. Finally, DNA pellet was resuspended in 100 µL (10 mM Tris-HCl,
pH 7.5, 0.1 mM EDTA).
Saliva samples were collected and DNA was extracted from healthy volunteers (25 Saligene,
21 Oragene), following the manufacturer’s instructions. The Oragene kit showed remarkable
variability in DNA concentration (0.9–36 µg) (Figure 1A) and were similar to previous results (0.9–
64 μg) per sample [2]. The QIAamp kit yielded the most reproducible results (3.6–8.6 µg). The
concentration of DNA from the QIAamp kit was relatively low as the elution volume was twice that
of the other kits, but in summary our results for QIAamp are consistent with previously published
results [6]. The wide range of DNA yield, for extractions from saliva, could be caused by variations in
the number of cells in saliva samples from different people or from possible contamination of the
samples with DNA from other organisms in the samples. The Fermentas kit provided the same
median total yield of DNA (5.4 µg from 200 µL of blood) as the QIAamp kit. For the phenol-
chloroform method and Saligene kit (500 µL), the median total yields were 5.4 and 4.8 μg,
respectively.
DNA purity was assessed by measuring the A260/280 of the samples to determine the amount of
organic contamination. Samples with A260/280 of 1.8 to 2.0 are considered relatively free from
contaminants [6]. Some organic contamination was detected for all of the methods studied. All four
DNA isolation methods from blood resulted in A260/280 of 1.54–1.96. A260/280 values of 1.28–1.92 were
measured for the Oragene kit samples, which is consistent with previous results [2]. The Saligene kit
samples showed the most variability of A260/280 ratios, from 1.58–2.33, indicating organic
contamination. Note that for saliva extraction methods, the DNA purity is likely dependent on how
the donor performed the sampling.
To assess if the extraction methods were compatible with PCR reactions, seven samples were
randomly selected for each extraction method and subjected to the APEX-2 forensic assay [3].
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Briefly, extracted DNA (5 µL) was heated at 95°C to evaporate water. PCR was performed in 5 µL
using the pUC18 plasmid (Fermentas, Vilnius, Lithuania) and primers 5’-
GTAAAACGACGGCCAGT-3’, 5’-CAGGAAACAGCTATGAC-3’. After 19 cycles, the PCR
products were loaded on 2.5% agarose gel, separated and visualized using UV for quantification of
the band intensities. The greatest PCR efficiency (minimal PCR inhibition, relative) was obtained
with samples from the phenol-chloroform (100%) and Oragene extraction methods (96%) (Figure
1B). The PAXgene and Fermentas samples gave PCR efficiency of 75–97%. The lowest PCR
efficiency (53–81%) was measured for the QIAamp samples. The statistical significance of observed
differences was assessed using the Tukey's Studentized Range (HSD) test within the GLM procedure
in SAS 9.1 software (SAS Institute Inc. 2004, Cary, NC, USA). Briefly, QIAamp had significantly
(p<0.05) lower relative PCR efficiency than other four extraction methods (Oragene, Fermentas,
phenol-chloroform and PAXgene), leaving Saligene (due to the large variance in its relative PCR
efficiency) in the middle (Figure 1B).
It has been proposed that high levels of mtDNA decrease amplification of autosomal DNA,
especially Y chromosome DNA. To investigate this hypothesis, the ratio of mtDNA to genomic (Y
chromosome SRY region) to was measured from the same extraction samples. Quantitative real-time
PCR was performed on eight randomly selected samples from the six DNA extraction methods and
analyzed using Taqman® mitochondrial (forward, 5’-TGCACGCGATAGCATTGC-3’; reverse, 5’-
TCAAAGACAGATACTGCGACATAGG-3’; probe, 5’-Fam-AGACGCTGGAGCCGG-BHQ-1-3’)
and Y chromosomal (forward, 5’-TTAAGCGTATTCAACAGCGATGA-3’, reverse, 5’-
CCGGAGAGCGGGAATATTCT-3’, probe 5’-Fam-ACAGTCCAGCTGTGCAA-BHQ-1-3’) probes.
Again, the statistical significance of the results was assessed using the Tukey's HSD test. There were
significant (p<0.05) differences among the extraction methods – Saligene and PAXgene kits gave
much higher DNA ratios than the other kits (Figure 1C). The data show that the Saligene and
PAXgene kits yield DNA samples that have higher cycle thresholds (Ct) than the other kits
(mitochondrial Ct 20.23–20.68 versus 18.74–20.73). Higher mitochondrial Ct values indicate that less
mtDNA is obtained. Autosomal Ct values of 27.67–28.14 were obtained for all methods except the
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Oragene kit (28.88–30.21, ratio 0.69), indicating that the Oragene DNA samples contained less
gDNA.
In order to compare the four commercial DNA extraction methods (Oragene, Saligene,
QIAamp and Fermentas), we performed comparative multiplex PCR and genotyping of 25 DNA
samples (17 males and 8 females) using a 124-plex APEX-2 microarray genealogy assay [3]. The call
rate values (defined as the percentage of actual genotype calls related to the possible number of calls)
of all the methods were similar: Oragene 99.92% (2 missing calls), Fermentas 99.90% (3), Saligene
99.83% (5) and QIAamp 99.72% (8). Parallel experiments provided an opportunity to evaluate the
occurrence of false signals after SBE (threshold 25%). In total, out of 11,092 calls, we counted 16
false signals. The false signals were unequally distributed among the extraction methods. The highest
number of false signals (11/16) occurred with the Oragene extraction method. Seven of these false
signals were caused by genotyping failure in one DNA sample (9 ng/µL; A260/280 1.6, smoker). The
remaining four false signals were detected in two other DNA samples. Four false signals occurred
with the Saligene kit in one DNA sample (26 ng/µL; A260/280 2.2). A single false signal occurred with
the QIAamp kit. The Fermentas kit did not yield any false signals.
To evaluate the success and reliability of the extraction methods, DNA was extracted from
saliva and blood, tested using the APEX-2 multiplex PCR assay and finally, genotyping results were
compared. Saliva sampling is less invasive than blood sampling and does not require trained medical
personnel. Also, to store DNA samples in TE buffer at -20ºC, long-term stability and quality of DNA
is ensured [7]. Commercial kits provide saliva preservation for a long period, making saliva sample
sending possible by mail and giving several advantages to this method. The approximate prices (€) of
compared methods for one DNA extraction are the following: Oragene (15), PAXgene (13), Saligene
(3), QIAamp (3), and Fermentas (1). Although DNA from saliva is tested for many applications like
long-range PCR, whole-genome amplification and even for second generation sequencing, no
publications, where more than 12-plex PCR has been performed.
From saliva, the DNA yield of the Oragene kit was twice (11.9 µg) that of the Saligene kit (4.8 µg)
depending on chemistry and bacterial DNA percentage. It is evident that Oragene DNA purified DNA
contain more bacterial DNA than other here compared kits. However, the PCR assay call rates were
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relatively similar (Oragene 99.92%; Saligene 99.83%) and the number of false signals was about
three times less for the Saligene kit sample. As problematic calls occur systematically with low
quality DNA samples the comparison of the two methods is difficult.
Differences in the mtDNA/gDNA ratios (Figure 1C) between different extraction methods support a
previously published report, where different DNA extraction procedures meaningfully influence
quantitative real-time PCR-based mtDNA copy number determination [8]. In context of PCR, copy-
number of target DNA, especially Y chromosome DNA, may influences multiplex PCR outcome if
different targets are used simultaneously. In this report, differences in the mtDNA/gDNA ratios
obtained with the Saligene and Oragene kits did not affect the Y chromosome signal intensities in the
APEX-2 assay. The second parameter, measured here, is relative PCR inhibition in our APEX-2
multiplex PCR conditions. We believe that it can influence primer pair drop-out at higher multiplex
level or lower template DNA concentrations and has an impact if DNA extraction method is replaced
in an already working assay.
In conclusion, we assessed six methods for the extraction of DNA from blood and saliva
based on measurements of the amount of extracted DNA, DNA purity (A260/280), PCR inhibition ratio,
and mtDNA/gDNA ratio. In addition, multiplex PCR reactions were performed to assess the quality
of the extracted DNA for highly complex amplification reactions. While there were variances between
the DNA obtained using the methods, there were no significant differences in the efficiency of
multiplex PCR reactions and oligomicroarray signals after SBE. Thus, DNA extraction from saliva is
a user-friendly and reliable alternative to DNA extraction from blood to obtain DNA for use in further
applications, such as multiplex PCR assays.
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Figure 1. Comparison of six DNA extraction methods (Oragene, Saligene, QIAmp, PAXgene,
Fermantas and phenol-chloroform). (A) The table indicates the number of experiments (n), median
DNA concentration, source of the DNA, total yield and purity. (B) Relative PCR efficiency as a
function of DNA extraction method. Groups represent the results of statistical analysis. (C)
Measurement of mtDNA/gDNA ratio. The Y-axis shows the mtDNA/gDNA cycle threshold ratio.
Groups A and B represent the results of statistical analysis.
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Acknowledgments
We thank Ms Triinu Temberg and Tom Janssens. Microarrays, Oragene kits and oligonucleotides
were provided by Asper Biotech (Tartu, Estonia). This work was supported by Targeted Financing
from the Estonian Government (SF0180142As08) and by the EU through the European Regional
Development Fund (SF0180026s09).
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