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American Journal of Epidemiology Advance Access originally published online on March 7, 2008
American Journal of Epidemiology 2008 167(10):1260-1267; doi:10.1093/aje/kwn012
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American Journal of Epidemiology © The Author 2008. Published by the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

PRACTICE OF EPIDEMIOLOGY

Buccal Swabs and Treated Cards: Methodological Considerations for Molecular Epidemiologic Studies Examining Pediatric Populations

Sara M. Beckett1, Stephen J. Laughton2, Luciano Dalla Pozza3, Geoffrey B. McCowage3, Glenn Marshall4, Richard J. Cohn4, Elizabeth Milne5 and Lesley J. Ashton1

1 Children's Cancer Institute Australia for Medical Research, Randwick, New South Wales, Australia
2 Department of Hematology and Oncology, Starship Children's Hospital, Auckland, New Zealand
3 Department of Oncology, The Children's Hospital at Westmead, Westmead, New South Wales, Australia
4 Center for Children's Cancer and Blood Disorders, Sydney Children's Hospital, Randwick, New South Wales, Australia
5 Telethon Institute for Child Health Research, Center for Child Health Research, University of Western Australia, Perth, Western Australia, Australia

Correspondence to Dr. Lesley Ashton, Molecular Epidemiology Group, Children's Cancer Institute Australia for Medical Research, P.O. Box 81, Randwick, NSW 2031, Australia (e-mail: lashton{at}ccia.unsw.edu.au).

Received for publication June 26, 2007. Accepted for publication January 14, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Self-collection of buccal cells provides a noninvasive method for obtaining biologic samples for genetic analyses in pediatric studies. Nevertheless, low yields, microbial contamination, and degradation of buccal samples present challenges for epidemiologic studies incorporating genetic investigations. The aims of this study were to compare the quality and yield of DNA extracted from buccal specimens with BuccalAmp swabs (Epicenter BioTechnologies, Madison, Wisconsin) or FTA cards (Whatman, Inc., Clifton, New Jersey) and to investigate the use of whole-genome amplification (WGA) for increasing DNA yields for single nucleotide polymorphism analyses. Buccal specimens were collected from 55 children with acute lymphoblastic leukemia and 52 control children without acute lymphoblastic leukemia in New South Wales, Australia, in 2003–2004. Real-time polymerase chain reaction was used to evaluate polymorphisms in the genes encoding the cytochrome p450 enzyme CYP3A4 (CYP3A4 A392G, also known as CYP3A4*1B) and the steroid xenobiotic receptor (SXR C25385T). Results showed that DNA could be isolated from buccal specimens collected by use of both methods and that yields could be substantially improved with WGA without introducing genotyping error. However, DNA quality was poorer in samples collected by BuccalAmp swabs, and the presence of polymerase chain reaction inhibitors in these samples reduced the sensitivity of quantitative real-time PCR analysis. These findings show that different methods for collecting buccal samples impact on the downstream success of genetic investigations and influence DNA quality after WGA.

DNA; epidemiologic methods; epidemiology, molecular; genome; mouth mucosa; pediatrics

Abbreviations: MDA, multiple displacement amplification; PCR, polymerase chain reaction; SNP, single nucleotide polymorphism; WGA, whole-genome amplification


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The collection of sufficient quantities of genomic DNA is central to the success of genetic epidemiologic studies. Historically, many of these studies have relied on DNA isolated from whole blood cells collected via venipuncture at designated collection sites or by investigators visiting the homes of study participants (1–3). Although whole blood specimens provide high quality DNA yields, venipuncture is an invasive procedure that is not always practical for infants and young babies; requires specialist collection, processing, and storage facilities; and can necessitate the added expense and inconvenience of travel for families, often resulting in low participation rates (4).

Obtaining human genomic DNA from buccal epithelial cells overcomes many of the problems associated with venipuncture, such as the requirement for a trained phlebotomist to draw blood specimens and the need for study participants to travel to collection sites, leading to improved participation rates (5). Self-collection of buccal specimens is also a more acceptable method for obtaining DNA samples from young children and has been shown to improve recruitment in remote areas or widely dispersed populations (6, 7).

Although collection of buccal cells provides a less invasive technique to obtain DNA samples from children, this approach does present limitations. For example, the yield and quantity of buccal DNA obtained from buccal specimens are lower than those obtained from whole blood cells and can vary depending on how the cells were acquired, stored, and transported (8–10). In addition, studies have also shown that buccal samples obtained from children can have lower DNA yields than those from adults (9, 11).

Various types of buccal swabs, mouthwash, or treated cards have been used to obtain DNA. These include scraping the inside cheek of the mouth with a tongue depressor (12), cotton or synthetic swab (8, 13, 14), or a cytobrush (9, 10, 15). Buccal cells can also be obtained by gargling and using a "swish and spit" method with either water or commercial mouthwash (6, 15–17) or by spitting saliva directly into a collection vial (18).

Previous studies have shown that different methods used to isolate buccal cells can impact on both DNA yield and quality. In adults, collection of buccal cells via the "swish and spit" protocol has been shown to consistently give the highest yields of buccal DNA with the lowest rate of fragmentation (8, 16, 19). However, this method is not always feasible in young children because of the alcohol content of some commercial mouthwashes and the challenge of getting young children to "spit" before swallowing. Similarly, collecting buccal cells from expectorated saliva can be difficult, as infants and young babies may have trouble being directed to supply the saliva sample. Therefore, in young children, swabs or cytobrushes provide the most reliable and accepted method for collection of buccal DNA (20–23).

Delays in transportation of buccal swabs returned by surface mail have also been reported to result in DNA degradation and bacterial contamination (10, 15, 18), which can lead to failed amplification of buccal DNA (7, 12) or decreased allelic discrimination in polymerase chain reaction (PCR) analyses (8). Flinders Technology Associates chemically treated filter papers (FTA cards) provide a novel alternative to sending buccal swabs, brushes, or buccal collection vials through the mail. FTA cards (Whatman, Inc., Clifton, New Jersey) are chemically treated filter papers designed for the collection and room temperature storage of biologic samples for molecular analyses; these cards protect DNA and are impregnated with denaturants that guard against oxidation, nuclease and ultraviolet damage, and both bacterial and fungal degradation. Recent studies have shown that FTA cards provide a convenient and safe method for the collection and storage of DNA from whole blood, tissue, buccal, viral, and bacterial specimens (24–28). Moreover, buccal specimens can be self-collected with FTA cards and returned by regular postal service without the provision of specialized packaging (14).

Despite overcoming the many limitations associated with collecting blood by venipuncture, buccal specimens provide substantially smaller quantities of DNA than do whole blood specimens. The recent development of whole-genome amplification (WGA) provides an attractive solution to this problem. Multiple displacement amplification (MDA) is a WGA technique that uses the Phi29 DNA polymerase enzyme and random exonuclease-resistant primers to uniformly amplify DNA products greater than 10 kilobases in length (11, 29, 30). This technique has been successfully used to extend genomic DNA from human blood (31), buccal samples (31, 32), and laser-dissected neurons (33). Concordance of single nucleotide polymorphism (SNP) genotyping between samples before and after MDA has been reported to be as high as 99.95 percent (34). However, recent studies have suggested that accurate genotyping may be dependent on the amount of DNA used in MDA reactions (35) and that MDA may introduce allele amplification bias in buccal-derived DNA specimens (36, 37).

Although previous investigators have shown that it is possible to use WGA to amplify buccal samples collected with swabs or brushes (11, 32, 38) and FTA cards (26), there have been limited reports on the application of WGA to amplify DNA from buccal samples collected from children. The aims of this study were to compare DNA yields obtained from buccal specimens collected from children by use of FTA cards or BuccalAmp swabs (Epicenter BioTechnologies, Madison, Wisconsin) both before and after WGA and to measure the impact of these methods in real-time PCR analyses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Study population
Cases included 55 children aged less than 16 years who were diagnosed with acute lymphoblastic leukemia between 2001 and 2003 at the Sydney Children's Hospital (n = 31) and the Children's Hospital at Westmead (n = 24) in Sydney, Australia. A control panel of 52 children without acute lymphoblastic leukemia was recruited by random digit dialing from the state of New South Wales and matched for age and sex. The study was approved by the appropriate institutional ethics committees, and informed consent was collected from the parents of all children who participated. Buccal specimens were collected from cases and controls over a 12-month period, commencing in February 2003.

Buccal DNA collection
FTA classic indicator cards (Whatman, Inc.) were used to collect buccal samples from all children in the study. Additional samples were collected with BuccalAmp DNA extraction kits (Epicenter BioTechnologies) from the case children at Sydney Children's Hospital and the control children to assess DNA yields obtained by use of an alternative method. Collection kits were mailed to residential addresses with instructions to collect buccal specimens by both FTA cards and BuccalAmp swabs based on the manufacturers' recommendations. All buccal specimens were collected on the same day, as investigations in our laboratory had previously shown that two swabs from the same cheek on the same day did not decrease the total DNA yields collected or reduce the DNA yields obtained using either method (data not shown).

Collection of buccal specimens was carried out by parents either at home or during a hospital clinic visit. Samples collected at home were posted in certified prepaid postage bags, tracked electronically through the Australian postage system, and shipped to the laboratory for processing within 24 hours, while samples collected or returned to staff during a hospital visit were processed immediately. Neither method was found to induce bleeding in the mouths of the children sampled. Although it was not possible to accurately quantify the total amount of DNA present on a FTA card, all FTA cards were scored for sample "coverage" and defined as having <25 percent, 25–49 percent, 50–74 percent, or 75–100 percent coverage.

BuccalAmp samples were processed according to the manufacturer's instructions and stored at –20°C. FTA cards were stored at room temperature in sterile airtight containers with silicon desiccant. A 1.2-mm disc was punched from FTA cards by use of a Harris Micro-Punch (Whatman, Inc.) and processed according to the manufacturer's instructions immediately before PCR analyses or WGA.

Multiplex PCR analysis
Multiplex PCR analysis was used to assess the quality of DNA in a subset of buccal samples both before and after WGA. DNA products of 100, 200, 300, 400, and 600 base pairs were amplified from four human control genes as described elsewhere (37). The DNA template was either 1 µl of BuccalAmp solution, one FTA hole-punch, or 1 µl of whole-genome amplified solution. All PCR reactions were analyzed with a GeneAmp 9700 PCR system (Applied Biosystems, Foster City, California). PCR products were visualized using polyacrylamide gels stained with SYBR Safe DNA gel stain (Invitrogen, Victoria, Australia).

Real-time PCR analyses
Human genomic DNA concentrations were quantified by real-time PCR for the beta-actin housekeeping gene with the following primer and probe sequences: forward 5'-TCACCCACACTGTGCCCATCTACGA-3'; reverse 5'-CAGCGGAACCGCTCATTGCCAATGG-3'; and 5'-VIC-ATGCCCTCCCCCATGCCATCCTGCGT-TAMRA-3'. Standard dilution curves of human placental DNA (100 ng, 10 ng, 1 ng, 0.1 ng, and 0.01 ng) were generated and used to quantify the DNA concentration in each sample. The lower detection limit of the quantitative real-time PCR assay was 0.01 ng/µl.

SNPs in genes encoding the cytochrome p450 enzyme CYP3A4 (CYP3A4 A392G, also known as CYP3A4*1B) and the steroid xenobiotic receptor (SXR C25385T) were determined by use of real-time PCR, in an ABI 7000 Real-Time system (Applied Biosystems). Sequences for the CYP3A4 A392G primers and probes were obtained from the SNP500 database (39). SNP specific primer sequences used for SXR C25385T included the following: forward 5'-AACTGTGGTCATTTTTTGGCAA-3' and reverse 5'-ACCACGATTGAGCAAACAGGT-3', while the probes were 5'-FAM-CCCAGGTTCTCTTTTC-MGBNFQ-3' and 5'-VIC-CCCAGGTTTTCTTTTC-MGBNFQ-3'. Primer concentrations varied, from 5 ng/µl (beta-actin) and 15 ng/µl (SXR C25385T) to 90 ng/µl (CYP3A4 A392G). All samples were analyzed in duplicate.

Whole-genome amplification
The GenomiPhi kit (Amersham Biosciences, Piscataway, New Jersey) was used to amplify genomic DNA from BuccalAmp DNA solutions and FTA hole-punches. Whole- genome amplification was performed with 1 µl of BuccalAmp DNA solution or one FTA hole-punch as recommended by the manufacturers. The WGA reaction mix was diluted 1:5 with DNase- and RNase-free distilled water (Invitrogen) before DNA quantification.

DNA yields from all BuccalAmp specimens were quantified both before and after WGA by real-time PCR for the beta-actin housekeeping gene. DNA from 1.2-mm FTA hole-punches was not quantified before WGA, as real-time quantification of DNA present on the same hole-punch prior to WGA can reduce the sensitivity of the WGA reaction, limit DNA yields, or result in allelic imbalance (failure to amplify both alleles) (35). DNA quantification prior to WGA on duplicate hole-punches was not feasible, as the amount of DNA present on any two punches from the same card can vary, because of the uneven distribution of DNA immobilized on the card (14).

Quantification of PCR inhibition
A subset of DNA samples collected with BuccalAmp swabs were examined for their potential inhibitory effect in quantitative real-time PCR analysis. Observed DNA levels quantified from a dilution series of BuccalAmp DNA were compared with those expected by extrapolating from the undiluted sample. PCR inhibition was further examined by quantifying a previously determined amount of placental DNA (50 ng) in the presence or absence of BuccalAmp DNA.

Statistical analysis
Wilcoxon's rank-sum tests were used to compare median DNA yields from cases and controls. Correlations between DNA yields before and after WGA or age at DNA collection were examined by use of Spearman's rank correlation tests. All results were analyzed with STATA, version 8.2, statistical software (StataCorp LP, College Station, Texas).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
DNA yields before and after WGA
DNA yields were analyzed in samples from 31 cases from Sydney Children's Hospital and 52 controls who provided buccal samples collected by both methods. On average, returned FTA cards from both cases and controls showed 40 percent coverage, providing approximately 160 hole-punches per card. Although the total DNA present on any one FTA card could not be accurately determined, we estimate that the DNA present on 160 punches would collectively allow for 160 or more molecular applications including multiplex PCR reactions or WGA. Table 1 shows median DNA yields obtained before and after WGA for samples collected with FTA and BuccalAmp techniques. Overall, total BuccalAmp DNA yields ranged from levels below detection (one case and three control samples) to 2.6 µg. Median DNA yields were significantly higher in BuccalAmp samples collected from cases compared with controls both before (79.1 vs. 26.3 ng; p = 0.007) and after (350.0 vs. 78.8 ng; p = 0.002) WGA, while no significant difference was observed for DNA yields obtained with FTA cards in cases and controls after WGA (240.0 vs. 257.0 ng; p = 0.443). Figure 1 shows the relation between the amount of BuccalAmp DNA in a single WGA reaction and the total DNA yield, where an increased DNA yield from WGA of BuccalAmp swabs was strongly correlated to a higher concentration of input DNA (p < 0.001). Figure 2 illustrates the absence of any significant correlation between age at collection and DNA yield from BuccalAmp swabs prior to WGA (p = 0.855).


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  		TABLE 1. DNA yields before and after whole-genome amplification in cases and controls who returned both BuccalAmp swabs* and FTA cards*, New South Wales, Australia, 2003–2004

 

Figure 1
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  		FIGURE 1. Correlation between BuccalAmp DNA yields obtained before and after whole-genome amplification in 79 children, New South Wales, Australia, 2003–2004. BuccalAmp (Epicenter BioTechnologies, Madison, Wisconsin).

 

Figure 2
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  		FIGURE 2. Correlation between age at collection and the BuccalAmp DNA yields obtained from 79 children, New South Wales, Australia, 2003–2004. BuccalAmp (Epicenter BioTechnologies, Madison, Wisconsin).

 
Multiplex PCR analyses showed variations in the quality of buccal DNA obtained with different methods. DNA from FTA cards produced clearer, well-defined PCR products before and after WGA compared with BuccalAmp samples, which appeared to yield more fragmented DNA (figure 3).


Figure 3
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  		FIGURE 3. Variation in DNA quality obtained before and after whole-genome amplification (WGA) in samples obtained from the same individuals by either BuccalAmp (A) or FTA card (B) technology, New South Wales, Australia, 2003–2004. MWT, molecular weight (base pairs). DNA yields from these samples are representative of those observed in our study. BuccalAmp (Epicenter BioTechnologies, Madison, Wisconsin); FTA cards (Whatman, Inc., Clifton, New Jersey).

 
Genotyping concordance in buccal samples before and after WGA
BuccalAmp specimens with detectable DNA (≥0.01 ng/µl) were genotyped for CYP3A4 A392G and SXR C25385T gene variants. One case and three controls with no detectable DNA prior to WGA were excluded from genotyping analyses as they were considered to have an inadequate template for the PCR reactions. Of the 79 BuccalAmp samples analyzed (30 cases and 49 controls), 1.3 percent failed to produce a real-time PCR product before WGA in either the CYP3A4 or SXR genotype analyses, while 6.3 percent of all samples failed to produce a real-time PCR product after WGA. No difference was observed in the failure rate between cases and controls, while 100 percent concordance was observed in all samples successfully genotyped. Genotype analyses carried out on 55 cases and 52 controls who supplied FTA cards showed that DNA collected with FTA cards consistently produced genotype results with 100 percent concordance before and after WGA.

Inhibition of real-time PCR analyses
Our investigations showed that quantitative real-time PCR analysis for the beta-actin gene was inhibited by DNA samples collected by use of BuccalAmp technology. As seen in figure 4, part A, the observed DNA concentration measured by real-time PCR analysis approached expected levels after the BuccalAmp sample was diluted twofold or more. Inhibition of real-time PCR analysis was also observed when a known quantity of placental DNA (50 ng) was amplified in the presence of buccal DNA (figure 4, part B). This investigation revealed that the amplification curve of the quantitative real-time PCR reaction peaked at a later cycle number in the presence of buccal DNA, compared with when the sample was analyzed without buccal DNA.


Figure 4
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  		FIGURE 4. DNA sample inhibition of real-time polymerase chain reaction in BuccalAmp samples from children, New South Wales, Australia, 2003–2004. A, DNA quantification after dilution of BuccalAmp sample. Expected DNA yield was extrapolated from DNA yield measured in the undiluted buccal specimen. B, real-time quantification of placental DNA in the presence or absence of the BuccalAmp specimen. BuccalAmp (Epicenter BioTechnologies, Madison, Wisconsin).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Results from our study show that the overall quality of buccal DNA collected with FTA cards was superior to that collected with BuccalAmp swabs and that FTA cards overcame the problems presented by PCR inhibitors in buccal specimens. Our investigations also demonstrated that yields of buccal DNA collected from children could be substantially improved with WGA and that this process did not generate genotyping errors in the real-time PCR assays examined. Nevertheless, successful PCR analyses both before and after WGA appeared to be dependent on the quality and quantity of buccal DNA.

Previous studies have reported variable yields of buccal DNA obtained from children, ranging from 1.7 µg to 28.3 µg (7, 11). To some extent, these variations are likely to reflect subtle differences in the methodologies used to collect and isolate DNA from buccal specimens, as well as the laboratory techniques used to quantify DNA. Although the DNA yields isolated from BuccalAmp swabs were lower than those previously reported, the use of quantitative real-time PCR analyses in our study provided a sensitive method to specifically measure human DNA compared with alternative methods, such as spectrophotometry or fluorometry, which have limited capacity to discriminate between human and bacterial DNA. Hence, the yields observed in our study accurately reflect the quantity of human genomic DNA collected.

Our investigations also showed that DNA yields from BuccalAmp swabs were substantially higher for cases than controls. This may have been because parents of children with cancer were more motivated to adhere to the instructions provided and to return their buccal samples as soon as possible compared with parents of control children. Alternatively, the delay between collection and processing of BuccalAmp samples from control families who did not attend the clinic may have resulted in an increased bacterial content or denatured DNA compared with the samples from cases that were predominantly returned in the clinic and processed within 12 hours of collection. Although it was not possible to measure the exact length of time from when the samples were collected and their arrival at the laboratory, five BuccalAmp swabs collected from control children did show visible evidence of microbial contamination on arrival in the laboratory, suggesting delayed transportation after sample collection. Hence, DNA degradation appears to be a more feasible explanation and is consistent with our observation of poorer quality DNA in samples collected with BuccalAmp swabs compared with FTA cards (figure 3). Although it is difficult to accurately assess the total DNA yield from FTA cards, we observed no visible difference in the level of sample coverage on FTA indicator cards returned by cases and controls. Moreover, DNA yields from FTA cards after WGA did not differ between cases and controls (table 1). This observation is consistent with previous reports that DNA in buccal samples immobilized on FTA cards remains stable for several years at room temperature (27, 40). Hence, DNA isolated from FTA cards was unlikely to be affected by any delay in returning samples to the laboratory.

Previous studies have reported that inhibitory factors in biologic specimens can influence DNA template amplification and PCR analyses with slight to moderate inhibition of target DNA resulting in underestimates of DNA concentration, while severe inhibition can lead to false negative results or assay failure unless additional purification steps are undertaken (7, 12, 41, 42). Over recent years, a wide range of inhibitors have been reported. However, the identification of specific inhibitors and their mode of action remain to be fully determined (43). In our study, the lack of PCR inhibition when the placental DNA was analyzed in the absence of buccal cells and saliva suggested that the observed PCR inhibition was caused by an unidentified contaminant in the BuccalAmp samples, possibly an unknown bacterial product or salivary enzymes. We hypothesize that the antimicrobial pretreatment of the FTA cards and repeated washes of each hole-punch prior to PCR amplification remove inhibitors from FTA cards. Despite the presence of PCR inhibitors in buccal samples collected with BuccalAmp technology, we were able to minimize PCR inhibition by diluting these samples prior to analyses (figure 4, part A). However, in some cases, dilution of the buccal specimen decreased the overall sensitivity of PCR assays by reducing the target sequence to levels below the detectable limit of the quantitative PCR assay.

Other investigators have shown that, when DNA template concentrations are low, stochastic fluctuations in PCR analyses can result in unequal sampling of heterozygous loci, leading to false inference of a homozygous genotype in samples with a low DNA template concentration (36, 44–46). Although we observed a higher failure rate in PCR analyses of samples collected with BuccalAmp swabs, 100 percent concordance was attained for all successful genotyping of DNA samples from buccal specimens collected by use of either FTA cards or BuccalAmp swabs, both before and after WGA.

Several studies have investigated the use of WGA for amplifying human genomic DNA in different types of biospecimens and have shown that the genomic coverage of the amplified products is generally good with a low rate of error (29, 32, 47, 48). Although WGA amplification has been reported to result in consistent DNA yields regardless of the starting DNA amounts (29), our results showed a positive correlation between increasing amounts of starting DNA template and higher DNA yields obtained after WGA. This finding is consistent with a previous report showing that increasing amounts of DNA template used in the WGA reaction can influence the yields from WGA (35). Moreover, trace amounts of bacterial contaminants or salivary enzymes in DNA samples derived from BuccalAmp swabs may also compete with the input template in WGA reaction, particularly when DNA template concentrations are low.

Prior investigations have also reported that the performance of WGA is dependent on the quality of DNA template used in the reaction and that poor DNA template can decrease the accuracy of genotyping (35, 49). In our study, multiplex PCR analyses showed that the quality of DNA collected with BuccalAmp swabs was somewhat poorer compared with the quality of DNA collected with FTA cards both before and after WGA (figure 3). This observation was further supported by the higher rate of genotype failure observed in specimens collected by use of BuccalAmp swabs compared with FTA cards. Nevertheless, this observation may have been limited to some extent by the small number of samples and gene variants examined in our study.

Overall, our investigations show that both FTA cards and BuccalAmp swabs provide useful tools for collecting buccal specimens from children and that the low yields of genomic DNA collected with these methods can be substantially improved after WGA. However, our findings show that FTA cards present a more robust method for collecting DNA samples for genetic epidemiologic studies, as they provide higher quality DNA and reduce potential inhibition of downstream investigations.


   ACKNOWLEDGMENTS
 
This project was partially supported by funding from the National Health and Medical Research Council, Australia (grant 350908). The Children's Cancer Institute Australia for Medical Research is affiliated with the University of New South Wales and Sydney Children's Hospital.

The authors would like to acknowledge the helpful advice provided by Professors Michelle Haber and Murray Norris on early versions of the manuscript and Stewart Smith for his technical advice.

Conflict of interest: none declared.


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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