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DNA Research Advance Access originally published online on March 27, 2006
DNA Research 2006 13(2):77-88; doi:10.1093/dnares/dsi029
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© The Author 2006. Kazusa DNA Research Institute
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

An Isothermal Method for Whole Genome Amplification of Fresh and Degraded DNA for Comparative Genomic Hybridization, Genotyping and Mutation Detection

Cheryl I. P. Lee, Siew Hong Leong, Adrian E. H. Png1, Keng Wah Choo1, Christopher Syn2, Dennis T. H. Lim3, Hai Yang Law4 and Oi Lian Kon*

Division of Medical Sciences, National Cancer Centre 11 Hospital Drive, Singapore 169610
1 Bioinformatics Group, Nanyang Polytechnic 180 Ang Mo Kio Avenue 8, Singapore 569830
2 Centre for Forensic Science, Health Sciences Authority 11 Outram Road, Singapore 169078
3 Department of General Surgery, Singapore General Hospital Outram Road, Singapore 169608
4 Department of Paediatric Medicine, KK Women's & Children's Hospital 100 Bukit Timah Road, Singapore 229899

Received 5 December 2005; revised 24 January 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgements
 References
 
Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and DNase-degraded DNA using degenerate oligonucleotide-primed PCR or primer extension amplification using a short primer sequence bioinformatically optimized for coverage of the human genome was compared with amplification using current primers by chromosome-based and BAC-array comparative genomic hybridization (CGH), genotyping at short tandem repeats (STRs) and single base mutation detection. Compared with current primers, genome amplification using the bioinformatically optimized primer was significantly less biased on CGH in self–self hybridizations, and replicated tumour genome copy number aberrations, even from FFPE tissue. STR genotyping could be performed on degraded gDNA amplified using our technique but failed with multiple displacement amplification. Of the 18 different single base mutations 16 (89.5%) were correctly identified by sequencing gDNA amplified from clinical samples using our technique. This simple and efficient isothermal method should be helpful for genetic research and clinical and forensic applications.

Key words: whole genome amplification; isothermal; comparative genomic hybridization; genotyping; mutations


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgements
 References
 
Genomic analyses, such as comparative genomic hybridization (CGH), genotyping of polymorphic loci and detection of disease gene mutations, are important in genomic medicine and forensic science. For example, CGH of cancer genomes has identified recurrent copy number aberrations of likely pathogenic significance,1Go certain genotypes are associated with altered disease susceptibility2Go and genotyping of highly polymorphic short tandem repeats (STR) is the most widely used method of human identification.3Go

Genomic techniques require nanogram to microgram quantities of genomic DNA (gDNA). Hence, DNA samples of insufficient quantity must be pre-amplified before the actual genomic analysis can be performed. Multiple displacement amplification (MDA),4Go,5Go a widely used whole genome amplification (WGA) method, is efficient (>1000-fold amplification) and yields minimally biased representation from sub-nanogram quantities of fresh undegraded gDNA. However, MDA performs poorly on degraded gDNA.5Go

Other WGA methods are mainly, though not entirely, PCR based. Degenerate oligonucleotide-based PCR (DOP-PCR),6Go primer extension preamplification7Go and ligation-mediated PCR8Go are known to introduce copy number bias of dispersed genomic regions. Balanced PCR amplification,9Go restriction and circularization-aided rolling circle amplification,10Go and the OmniPlexTM11Go methods require substantial enzymatic manipulations that may be impractical with very small starting samples.

We have adapted an isothermal method originally developed for DNA labelling12Go but which also amplifies input DNA.13Go Our modified method uses a primer that is selected after parsing the human genome for oligomer sequences that have maximum genome coverage with minimal overlaps (A. E. H. Png, K. W. Choo, C. I. P. Lee, S. H. Leong and O. L. Kon, manuscript in preparation). The method we describe here performs better than other current techniques on gDNA from formalin-fixed paraffin-embedded (FFPE) sources and degraded gDNA.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgements
 References
 
2.1. gDNA sources and extraction
Sources of gDNA were peripheral human blood, fresh and FFPE Hs 746T tumour tissue. Tumours were generated by subcutaneously implanting Hs746T human gastric cancer cells (American Type Culture Collection, Manassas, VA, USA) in scid mice. Excised Hs 746T tumours were fresh frozen at –80°C or fixed in neutral buffered 10% formalin for 16 h at room temperature and dehydrated in increasing ethanol concentrations (70–100%), followed by three changes each of absolute ethanol, xylene and paraffin. gDNA was extracted from blood and the fresh tumour using the Blood & Cell Culture DNA kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol except for the following modification for tumour tissue. Ground tissue (300 mg) was incubated for 16–18 h at 50°C in 19 ml Buffer G2 and at least one additional aliquot (0.5 ml) of Qiagen Protease solution was added during this period to obtain a clear lysate.

DNA was extracted from tissue cores (1 mm diameter, total weight of cores 0.5 mg) prepared from paraffin blocks of Hs 746T tumour using the PUREGENE® DNA Purification Tissue Kit (Gentra Systems Inc., Minneapolis, MN, USA) according to the manufacturer's protocol with the following modification. De-paraffinized tissue was incubated with cell lysis solution at 65°C for 60 min with shaking (300 r.p.m.). gDNA was quantified by absorbance at 260 nm and used if the ratio of absorbance at 260 nm/280 nm was 1.8–2.0.

2.2. Primers
The following primers were used: (i) NYP (Nanyang Polytechnic) primer, 5'-NNNTGAGAT-3'; (ii) random octamers and (iii) DOP-PCR primer, 5'-CCGACTCGAGNNNNNNATGTGG-3'.6Go The sequence TGAGAT of the NYP primer was bioinformatically selected for its predicted ability to generate 2–3 kb amplicons uniformly distributed throughout the human genome.

2.3. DNA amplification
Two methods were used. DOP-PCR using the 22-base primer was performed with 40 ng DNA, 2 µM primer, 4 x 200 µM dNTPs, 1.5 mM MgCl2 and 5 U AmpliTaq Gold® DNA polymerase (Applied Biosystems, Foster City, CA, USA) in a reaction volume of 0.1 ml. Thermal cycling conditions were 94°C x 9 min, 9 cycles of 94°C x 1 min/30°C x 1.5 min/72°C x 3 min, 30 cycles of 94°C x 1 min/62°C x 1 min/72°C x 1.5 min and a final extension of 8 min at 72°C. DOP-PCR using NYP primer was performed with 40 ng DNA, 2 µM primer, 4 x 200 µM dNTPs, 1.5 mM MgCl2, 2 units DyNAzymeTM EXT DNA polymerase (Finnzymes Oy, Espoo, Finland) in a reaction volume of 0.1 ml. Thermal cycling conditions were 94°C x 5 min, 40 cycles of 94°C x 1 min/22°C x 1 min/0.1°C per second transition to 72°C/72°C x 1 min and a final extension of 8 min at 72°C. Two more units of DyNAzymeTM EXT DNA polymerase were added to the reaction immediately after completion of the 20th cycle. Amplified DNA was purified using the QIAquick PCR Purification Kit (Qiagen).

Isothermal primer extension amplification12Go was performed by modification of the BioPrime DNA Labeling system (Invitrogen Corporation, USA). A total of 300 ng gDNA were denatured in the presence of 15 µg random octamers or NYP primer by heating to 99°C for 10 min and were snap-cooled on ice. Klenow polymerase (40 U) and 0.12 mM each of dATP, dCTP, dGTP and dTTP were added to the chilled mixture in a total reaction volume of 50 µl. The reaction was incubated at 37°C for 14–16 h. DNA was purified using a Microcon YM-30 centrifugal filter unit (Millipore Corp., USA).

2.4. DNA labelling
Unamplified gDNA and gDNA pre-amplified by primer extension for chromosome CGH were labelled with fluorescein-12-dUTP (test DNA) or Texas Red-5-dUTP (reference DNA) using the Invitrogen's Nick Translation System according to the manufacturer's protocol to obtain 500–2000 bp labelled fragments.

DNA samples for BAC array CGH were labelled with Cyanine 3-dUTP (Cy 3-dUTP for test DNA) or Cyanine 5-dUTP (Cy 5-dUTP for reference DNA) using the BioPrime DNA Labeling System (Invitrogen) and the supplier's protocol. All fluorophore-labelled deoxynucleotides were from PerkinElmer Life Sciences (USA). DNA was purified after labelling with Microcon YM-30 centrifugal filter units.

2.5. CGH
Chromosome CGH was performed as described previously.14Go One microgram each of Hs 746T and reference gDNA (from fresh lymphocytes or paraffin cores of healthy male spleen) were labelled with fluorescein-12-dUTP and Texas Red-5-dUTP, respectively. Labelled DNA and 20 µg Cot-1 DNA (Invitrogen) were co-precipitated with ethanol and dissolved in 10 µl hybridization buffer (50% formamide, 10% dextran sulfate and 2x SSC). The probe mixture was denatured (74°C for 6 min) and hybridized to normal metaphase chromosomes (prepared from phytohemagglutinin-stimulated peripheral lymphocytes using standard protocols) that had been denatured (74°C for 2 min) in 70% formamide/2x SSC and dehydrated in an ethanol series. After a 48 h hybridization, slides were washed once in 50% formamide/2x SSC (10 min), twice in 2x SSC and once in 0.1x SSC (all 5 min each, at 45°C), followed by 1x PBD buffer [12 mM NaHCO3 containing 0.5% (v/v) NP-40] and deionized water at room temperature (5 min each). Slides were mounted in antifade solution with 0.1 µg/ml 4',6-diamidino-2-phenylindole (DAPI).

BAC array CGH was performed on 2464-clone arrays (each clone spotted in triplicate) from the University of California San Francisco Comprehensive Cancer Center Microarray Core. Hybridization was performed as detailed in the ‘Array CGH Hybridization Protocol’ (http://cc.ucsf.edu/microarray/index.asp). Briefly, gDNA was digested for 5 h with DpnII (New England Biolabs, USA), purified using the QIAquick PCR purification kit and labelled as described above. Labelled reference and test DNAs (3–4 µg each) were co-precipitated with 35 µg human Cot-1 DNA. The precipitate was collected by high-speed centrifugation, air-dried and re-dissolved in a final volume of 0.1 ml 1x SSC containing 70% formamide (extra pure grade, Merck KgaA, Germany), 3.4% SDS (Bio-Rad Laboratories, USA) and 5 mg dextran sulfate (sodium salt, MW 500 000, Sigma Chemical Co., USA). DNA was denatured at 73°C for 12 min and snap-cooled before applying to the array slide on which a plastic frame seal (2.3 cm2, Fisher Scientific, USA) had been placed firmly around the region of spotted BAC clones. The array slide was pre-wetted with 50 µl 2x SSC containing 20 mg dextran sulfate and 50% formamide (pre-hybridization solution pre-warmed to 37°C). After the entire array surface was wet, 20 µl of the pre-hybridization solution was withdrawn and the denatured combined reference and test DNA solution (0.1 ml) was quickly applied on the array. The assembled hybridization array slide was placed in a humidified and well-sealed slide chamber box. Uniform hybridization was achieved by rocking the slide on a tilting platform (2 oscillations/min) at 37°C for 72 h. The position of the array slide with respect to the tilting axis was moved by consecutive 90° turns at 24 h intervals.

Post-hybridization washing in dim light was performed in the following sequence: (i) 50% formamide in 2x SSC, pH 7 for 15 min at 50°C; (ii) 0.1 M Na2HPO4 containing 0.1% Nonidet P40, pH 8 for 15 min at room temperature; (iii) 2x SSC for 2 min; (iv) 70%, 85% and absolute ethanol, each for 2 min. The slide was dried by centrifugation (130x g for 2 min) before scanning.

2.6. Imaging and analysis
DAPI, fluorescein and Texas Red images were captured from 8–10 metaphases on chromosome CGH slides using a fluorescence microscope and a charge-coupled device camera. Digital images were analysed by the CV Chromofluor system (Applied Imaging, UK) using 99% standard reference intervals. The mean fluorescence ratio exceeded 1.2 in regions of gain and was below 0.8 in regions of loss. High-level amplifications were chromosomal regions with mean ratios >1.5.

Array CGH slides were scanned on an Axon GenePix 4000B scanner (Axon Instruments, USA) after adjusting the count ratio of channel 1/channel 2 to 1. TIFF images were analysed using the SPOT program, which segmented the spots, performed local background subtraction and generated log2 ratio data of Cy 3/Cy 5 intensities for each spot.15Go SPOT output data were further processed using the SPROC program (http://jainlab.ucsf.edu/Downloads.html), which generated the mean log2 Cy 3/Cy 5 ratio of triplicate spots for each BAC clone that survived quality constraints. SPOT and SPROC data files were exported to a custom program, NEAT, which generated an average intensity grid by grid plot for Cy 3 and Cy 5 to visually inspect hybridization quality and accuracy of the initial count ratio. Cy3/Cy5 ratios were normalized by total intensity normalization and normalized ratio plots were generated for every chromosome. The thresholds for amplification and deletions were +0.33 and –0.33, which are the log2 transformed values corresponding to ratios of 1.25 and 0.75, respectively, of chromosome CGH.

2.7. STR analysis
Control and whole genome amplified DNA were profiled using the commercial AmpFISTR® Identifiler® PCR Amplification Kit (Applied Biosystems) and the supplied protocol. This kit allows amplification/detection of 15 STR loci comprising 13 US CODIS (COmbinded DNA Index System) loci, D2S1338, D19S433 and the gender-determining locus amelogenin. All genotyping reactions were performed on a GeneAmp 9700 PCR system (Applied Biosystems). Where necessary, DNA template quantity was modified for PCR optimization. PCR products were separated by capillary electrophoresis in an ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems). One microlitre of PCR product was injected at 1 kV for 22 s with the minimum allele detection threshold set at 50 relative fluorescence units.

2.8. Mutation detection
A total of 50 ng of lymphocyte gDNA was amplified by primer extension using NYP primer as described above (‘DNA amplification’) followed by purification using the QIAquick PCR Purification Kit. PCR with gene-specific primers (forward primer 5'-GTACGGCTGTCATCACTTAGACCTCA-3'; reverse primer 5'-TCCCATAGACTCACCCTGAA-3'; 2 µM each) was performed on 200 ng of amplified gDNA. Other reaction conditions were 0.8 mM dNTPs, 1.5 mM MgCl2 and 0.02 U DyNAzyme EXT DNA polymerase. Thermal cycling conditions were 94°C for 2 min followed by 30 cycles of 94°C x 30 s/55°C x 30 s/72°C x 60 s and a terminal extension for 10 min at 72°C.

A second PCR using a nested reverse primer (5'-TGCAGCTTGTCACAGTGCAGCTCACT-3') was performed on 1 µl of the completed first reaction product to yield a single 602 bp DNA fragment, which was resolved by electrophoresis on 1.5% agarose and extracted using the QIAquick Gel Extraction Kit (Qiagen). This was sequenced using the CEQ Dye Terminator Cycle Sequencing Quick Start Kit (Beckman Coulter, USA) according to the supplied protocol. After 30 cycles, 5 µl of stop solution/glycogen mixture (1.2 M sodium acetate, 40 mM disodium EDTA and 4 mg glycogen/ml) was added and the sample was ethanol precipitated, vacuum dried for 3 min and resuspended in 20 µl of the provided sample loading solution before loading on the CEQ capillary sequencer (Beckman Coulter).

2.8. Statistical tests
The significance of differences in variance and differences in proportion of outlier clones were determined using Levene's and Fisher's exact tests, respectively (SPSS statistical analysis software, Chicago, USA).


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgements
 References
 
Figure 1 is a comparison of copy number profiles obtained by chromosome CGH after amplification using two different primers. The sample in each experiment was lymphocyte gDNA from the same normal healthy male amplified by DOP-PCR using the bioinformatically optimized NYP primer, 5'-NNNTGAGAT-3', or the widely used primer of Telenius et al.6Go 5'-CCGACTCGAGNNNNNNATGTGG-3'. WGA products were labelled with fluorescein-12-dUTP while the cognate unamplified gDNA was labelled with Texas Red-5-dUTP by nick translation. Self–self hybridization of unamplified and DOP-PCR amplified gDNA showed that WGA products generated using NYP primer resulted in false deletions in only 2 chromosomes, without any spurious copy number gains (Figure 1, upper panel). In contrast, DOP-PCR using the primer of Telenius et al.6Go resulted in major false deletions in no fewer than 11 chromosome arms (Figure 1, lower panel).


Figure 1
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  		Figure 1. Comparison of genomic representation after WGA using NYP (upper panel) or Telenius (lower panel) primer in DOP-PCR. Each panel shows chromosomal CGH self–self hybridization. The same gDNA was used in each experiment. It was extracted from normal lymphocytes of a healthy male. In each CGH experiment, unamplified gDNA was hybridized against itself after DOP-PCR amplification (i.e. unamplified self versus amplified self). Chromosomal CGH was performed as described previously.14Go A colour version of Figure 1 can be found online as supplementary data at http://dnaresearch.oxfordjournals.org

 
WGA in all subsequent experiments was performed by primer extension amplification because DOP-PCR is now recognized to cause significant amplification bias. The extent of gDNA amplification by primer extension was inversely related to the amount of input DNA. About 5500-fold amplification was achieved when input DNA was 1 ng. With 10 and 50 ng input gDNA, the amplification factors were 580 and 144, respectively. The size of WGA products ranged from 200 to >23 kb (Figure 2).


Figure 2
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  		Figure 2. Agarose gel electrophoresis of primer extension amplification products showing varying amounts of fresh gDNA input, DNA size standards and amplification factors.

 
We next compared copy number profiles of unamplified gDNA extracted from fresh frozen Hs 746T tumour tissue (Figure 3A) with unamplified gDNA extracted from the same tumour after formalin fixation and paraffin embedding (FFPE) (Figure 3B) to determine the extent to which tissue preservation alone affects CGH analysis. Reference gDNA was unamplified fresh human lymphocyte gDNA (Figure 3A) or unamplified human spleen gDNA extracted from paraffin blocks (Figure 3B). Figure 3A and B show similar copy number profiles but with several discordant aberrations i.e. spurious copy number losses of 5q, 10p, 12q, 18p and 19q; spurious copy number gain of 20p; and failure to detect 9p loss in CGH of gDNA extracted from FFPE tissue. As neither gDNA sample in Figure 3A and B was amplified, these discrepant copy number changes were most likely due to effects of chemical fixation and embedding on the quality of extracted gDNA e.g. DNA–protein cross-linking and DNA degradation. We proceeded to determine the fidelity of copy number analysis after amplifying gDNA from FFPE Hs 746T tumour tissue. A total of 50 ng of gDNA from FFPE Hs 746T tumour tissue and FFPE normal human spleen (reference DNA) was amplified by primer extension using NYP primer followed by fluorescence labelling using nick translation. Figure 3C shows that the copy number profile of amplified gDNA from FFPE tissue largely replicates the true aberrations of unamplified gDNA prepared from fresh Hs 746T tumour tissue. Copy number gains in 1p, 3pq, 6q, 7q, 8q, 9q, 10q, 11q, 14q and 17q were replicated in gDNA from fixed tumour tissues, as were copy number losses in 1q, 2pq, 4q, 8p, 13q, 15q, 16p, 20p, 21q and 22q. Certain copy number abnormalities, however, only appeared in CGH of amplified gDNA prepared from FFPE tumour tissue (i.e. deletions of 5q, 10p, 12q and 19q; gain of 20q) while gain of 7p and loss of 9p detected in fresh tumour tissue were not detected in gDNA from FFPE tissue. However, most of these apparently artefactual findings were present already in unamplified gDNA extracted from FFPE tissues, i.e. deletions of 5q, 10p, 12q, 18p and gain of 20q (Figure 3B), and cannot be attributed to quantitatively skewed representation caused by WGA.


Figure 3
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  		Figure 3. Primer extension amplification using NYP primer replicates copy number aberrations of unamplified tumour gDNA. cCGH of a xenotumour (Hs 746T) using (A) unamplified gDNA extracted from fresh frozen tissue; (B) unamplified gDNA from FFPE tissue of the same tumour and (C) gDNA from FFPE tumour tissue amplified with NYP primer. Reference gDNA was from normal male lymphocytes (A) or from FFPE normal human spleen (B and C). A colour version of figure 3 can be found online as supplementary data at http://dnaresearch.oxfordjournals.org

 
Array CGH has considerably higher resolution than chromosomal CGH and is increasingly used in preference. We therefore compared the quality of amplified gDNA generated by primer extension amplification performed with NYP primer or random octamers by hybridization with a 2464 BAC clone array. Figure 4 compares normalized log2 Cy3/Cy5 ratios of self–self hybridizations in which normal human lymphocyte gDNA was amplified using NYP primer (upper panel) or random octamers (lower panel). Copy number analysis across the human genome showed significantly less scatter around the zero value (signifying balanced copy number) when WGA was performed with NYP primer compared with random octamers (P < 0.0001 for the difference between variances using Levene's test). Although both datasets included outlier signals, defined as log2 greater than +0.33 or less than –0.33 that falsely signified copy number gain or loss, respectively, there were only 18 such outliers in NYP-primed WGA compared with 65 outliers in random octamer-primed WGA (Table 1, P < 0.0001, Fisher's exact test). We observed that outlier clones did not cluster in genomic regions but were instead uniformly dispersed in all chromosomes.


Figure 4
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  		Figure 4. Primer extension WGA using NYP primer (upper panel) and random octamers (lower panel) compared for artefactually biased representation. Data are normalized log2 Cy3/Cy5 ratios (vertical axis) of self–self hybridizations of normal lymphocyte gDNA plotted against chromosomal positions on the horizontal axis (chromosomes 1 to Y, left to right). Vertical lines denote boundaries between BAC clones that are mapped to consecutive chromosomes. Horizontal grey lines correspond to log2 values of +0.33 and –0.33.

 

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  		Table 1. Comparison of outlier clones in BAC array CGH self–self hybridizations of gDNA amplified using NYP primer or random octamers.

 
We next evaluated the performance of primer extension amplification on degraded gDNA for STR genotyping. Normal human lymphocyte gDNA was digested with DNase I for 2–30 min to generate five different size ranges: (i) 1500–3000 bp, (ii) 800–1500 bp, (iii) 500–800 bp, (iv) 200–500 bp and (v) <200 bp (Figure 5). DNA in each range was gel purified after electrophoresis on 1% agarose and used as the input template for primer extension amplification using NYP primer or random octamers (Figure 6). Figure 7 shows the genotype profile obtained at 15 STR loci and amelogenin X–Y with whole genome amplified DNA generated by NYP primer (panel B) or random octamers (panel C). Compared with reference undegraded gDNA (Figure 7A), 9, 9 and 8 loci were successfully detected in gDNA that had been degraded to 1500–3000 bp, 800–1500 bp (Figure 7B) and 500–800 bp, respectively, and then whole genome amplified with the NYP primer. Seven loci (D8S1179, D3S1358, D19S322, vWA, D13S317, D5S818 and amelogenin) were detected in all three degraded DNA samples. Genotyping of WGA products was not associated with imbalance at biparental alleles if sufficient amplified gDNA was used, and did not increase stutter peaks. In contrast, genotyping the same degraded gDNA after MDA was uniformly unsuccessful in all size ranges and severe imbalances at heterozygous loci were observed (Figure 7D).


Figure 5
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  		Figure 5. DNase-digested gDNA was fractionated by agarose gel electrophoresis into five different size bands: (i) 1500–3000 bp; (ii) 800–1500 bp; (iii) 500–800 bp; (iv) 200–500 bp; (v) <200 bp. Each size range of degraded gDNA was gel purified and used as input DNA for primer extension amplification (shown in Figure 6).

 

Figure 6
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  		Figure 6. Primer extension amplification of degraded gDNA from five different size bands (Figure 5) using random octamers or NYP primer. Each amplified reaction was used for genotyping at 16 STR loci (shown in Figure 7).

 

Figure 7
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  		Figure 7. Genotyping profiles at 16 simple tandem repeat loci (AmpFISTR® Identifiler® panel, Applied Biosystems, USA) of gDNA degraded to the size range of 800–1500 bp (Figure 5) (A) without WGA; (B) after primer extension amplification using NYP primer; (C) after primer extension amplification using random octamers; and (D) after MDA (GenomiPhi Kit, Amersham Biosciences). A colour version of figure 7 can be found online as supplementary data at http://dnaresearch.oxfordjournals.org

 
The ability of primer extension WGA to accurately detect single nucleotide mutations was investigated in a series of 18 clinical samples of lymphocyte gDNA from genetically characterized cases of ß-thalassemia. Each exon-specific PCR product obtained after primer extension amplification with NYP primer was sequenced and the correct mutation was detected in 16 cases (89% accuracy; Table 2). Representative DNA sequence chromatograms of whole genome amplified samples are shown in Figure 8.


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  		Table 2. Detection of single base mutations in WGA samples.

 

Figure 8
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  		Figure 8. Representative examples of whole genome amplified DNA clinical samples showing detection of a single nucleotide substitution (A and B), insertion (C) and deletion (D). All samples were heterozygous. A colour version of figure 8 can be found online as supplementary data at http://dnaresearch.oxfordjournals.org

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgements
 References
 
We have reported a simple isothermal WGA method based on primer extension that performs well for several different genomic analyses, i.e. chromosomal and BAC array CGH, STR genotyping and single base mutation detection. The method is tolerant of degraded DNA and works well on DNA extracted from fixed tissues. It does not require specially modified primers (unlike MDA), ligation steps (unlike balanced PCR, ligation-mediated PCR and rolling circle amplification techniques) or library construction (unlike Omniplex technology).

Parsing the complete human genome sequence made it possible to identify a short primer sequence that is uniformly distributed throughout the entire human genome, thus favouring uniform amplification while at the same time having minimal overlap to avoid over-amplifying certain regions. We further specified that, if used as a primer for PCR-based WGA, the selected sequence should amplify products of 2–3 kb. The hexamer, TGAGAT, met all these criteria but was efficient in priming gDNA only when the 5' end was extended by three degenerate bases. Hence, the WGA primer we used for DOP-PCR and for primer extension amplification was 5'-NNNTGAGAT-3'.

WGA products obtained by DOP-PCR using NYP primer gave minimal deviations from a balanced copy number profile in self–self hybridization on chromosome CGH compared with DOP-PCR with the widely used primer of Telenius et al.6Go that yielded multiple regions of spurious deletions (Figure 1). Identical self–self hybridizations on BAC array CGH also showed that, compared with random octamers, NYP primer generated a more representative and less biased amplification of gDNA (Figure 4).

Although isothermal random primer extension is commonly employed for DNA labelling, it has not been fully investigated as a method for WGA. Our data showed that this reaction could amplify gDNA by several thousand times (Figure 2), confirming an earlier study.13Go

gDNA extracted from FFPE tissues is notoriously difficult to be amplified and analysed using molecular techniques probably because of DNA cross-linking and degradation. We were motivated to test our approach on FFPE tissue because archived human tissues in paraffin blocks are a rich research resource. Our results show that the major characteristic copy number abnormalities can be reproduced in gDNA extracted from FFPE tissue although the process of tissue preservation itself, independent of WGA, introduces artefactual copy number alterations (Figure 3A and B). Despite this, reasonable fidelity of copy number profile was achieved in gDNA amplified with NYP primer from FFPE tumour tissue compared with the same freshly frozen tumour tissue (Figure 3A and C).

Other potentially useful applications of WGA are in forensic science and genetic diagnosis, where samples are frequently of poor quality or available in minute quantities. We were able to genotype DNA that had been degraded to 500–800 bp at a minimum of six standard simple tandem repeat loci using 8 ng of amplified gDNA representing 56 pg of original degraded input DNA, giving a conservatively estimated power of discrimination of 0.999998.16Go,17Go Primers designed to generate smaller amplicons of STR loci have been shown to successfully genotype DNA degraded to <126 bp when standard methods fail.18Go Such an approach, combined with primer extension WGA, should be useful in situations requiring the analysis of highly degraded DNA.

In summary, we have shown that primer extension amplification using a bioinformatically optimized universal primer is a simple and efficient isothermal method for WGA. The same primer can also be used for PCR-based genome amplification and achieves less biased amplification than a widely used DOP-PCR primer.6Go Isothermal primer extension amplification does not require extensive sample processing and manipulation or chemically modified primers. The method we have described performed well in chromosome and BAC array CGH using gDNA from both fresh and archived tissues. It generated usable STR genotype profiles from degraded gDNA and correctly detected a high proportion of single nucleotide mutations in clinical samples. These performance characteristics should make this method useful for practical application in genome science, genomic medicine and forensic science.


   Acknowledgements
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgements
References
 
The authors thank Dr Alvin Eng for guidance with array CGH experiments, Dr Ann Lee for use of the rocker platform, Dr Patrick Tan for use of the Axon GenePix scanner, Dr Balram Chowbay's laboratory for assistance in sequencing and Mark EH Tan for statistical analysis. This work was supported by the National Medical Research Council, Singapore.


   Footnotes
 
*To whom correspondence should be addressed. Tel. +65-6436-8319/8307, Fax. +65-6372-0161, Email: dmskol{at}nccs.com.sg

Communicated by Michio Oishi


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgements
 References
 

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