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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