PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jbtJBT IndexAssociation Homepage
 
J Biomol Tech. 2009 December; 20(5): 232–235.
PMCID: PMC2777348

DNA from Buccal Swabs Suitable for High-Throughput SNP Multiplex Analysis

Abstract

We sought a convenient and reliable method for collection of genetic material that is inexpensive and noninvasive and suitable for self-collection and mailing and a compatible, commercial DNA extraction protocol to meet quantitative and qualitative requirements for high-throughput single nucleotide polymorphism (SNP) multiplex analysis on an automated platform. Buccal swabs were collected from 34 individuals as part of a pilot study to test commercially available buccal swabs and DNA extraction kits. DNA was quantified on a spectrofluorometer with Picogreen dsDNA prior to testing the DNA integrity with predesigned SNP multiplex assays. Based on the pilot study results, the Catch-All swabs and Isohelix buccal DNA isolation kit were selected for our high-throughput application and extended to a further 1140 samples as part of a large cohort study. The average DNA yield in the pilot study (n=34) was 1.94 μg ± 0.54 with a 94% genotyping pass rate. For the high-throughput application (n=1140), the average DNA yield was 2.44 μg ± 1.74 with a ≥93% genotyping pass rate. The Catch-All buccal swabs are a convenient and cost-effective alternative to blood sampling. Combined with the Isohelix buccal DNA isolation kit, they provided DNA of sufficient quantity and quality for high-throughput SNP multiplex analysis.

Keywords: single nucleotide polymorphism

Traditionally, the collection of genetic material suitable for multiplex PCR-based genotyping assays has been derived from blood. However, blood sampling is invasive, time-consuming, and expensive, with limitations for larger studies, particularly where participants are dispersed geographically. Buccal swabs, on the other hand, are considered a convenient alternative for collecting genetic material, as they are relatively inexpensive and noninvasive and can be mailed for self-collection and return-mailed for testing.1,2 Buccal swabs collect epithelial cells, from which genomic DNA (gDNA) can be isolated for forensic applications, paternity testing,3 and determining susceptibility to disease and as a diagnostic tool for infectious4 or hereditary diseases.5

Single nucleotide polymorphisms (SNPs) are stable genetic variations within a DNA sequence, which may alter the biological function of a protein2 with the potential to influence the likelihood of developing a particular disease.6,7 SNP multiplex analysis, facilitated through automated genotyping platforms, such as the MassARRAY system, has the capacity to generate large quantities of genetic information. The resulting data can be used for linkage studies,8 fine mapping,9 and SNP studies.10 This application requires high-quality gDNA, which is not always recoverable from buccal swabs without compromising DNA quantity.11 Organic solvents, such as phenol, chloroform, and/or isoamyl alcohol, have been the benchmark for providing high-purity DNA.12 As a result of the hazardous nature of phenol, chloroform, and/or isoamyl alcohol, column-based methods were developed as a safer alternative (www.epicentre.com/lit/mpclit.htm). However, organic solvent and column-based methods are time-consuming, and spin columns are costly, so neither method is desirable for high-throughput genotyping. Previously, SNP multiplex analysis on a MassARRAY genotyping platform has used gDNA from blood samples. The objective of this study was to identify and use a commercial sample collection and DNA extraction protocol, which were low-cost and noninvasive and met quantitative and qualitative requirements suitable for a high-throughput SNP multiplex analysis on a MassARRAY system.

MATERIALS AND METHODS

This research was approved by the Research Ethics Committee of the Children, Youth and Women's Health Service.

Pilot Study

Buccal swabs were collected from 34 individuals from 8 to 65 years of age. Each participant was given a collection kit containing three Catch-All swabs (Epicentre Biotechnologies, Madison, WI, USA), two Isohelix DNA buccal swabs (Cell Projects, Kent, UK), and an instruction sheet detailing the manufacturers' instructions, with the exception of the collection time. To ensure adequate DNA collection, we recommended that the participant rub the inside of both cheeks firmly for a minimum of 1 min, rather than a minimum of 15 s or a predetermined number of rubs. For the purpose of this study, samples were returned to us by hand; however, to simulate likely postal times, we waited 48 h before processing the majority of samples. A subset of 24 samples was processed after 72 h (Table 1). All participants were nonidentifiable, and samples were tracked using a designated laboratory number.

TABLE 1
DNA yield and genotyping pass rate of commercially available buccal swabs and extraction kits

Four commercial DNA extraction methods were tested with one or both types of buccal swab for high-throughput SNP multiplexing: BuccalAmp DNA extraction kit QuickExtract (Epicentre Biotechnologies); Magnetic bead buccal cell gDNA extraction kit (Invitrogen, Carlsbad, CA, USA); MasterPure Complete DNA purification kit (Epicentre Biotechnologies); and Isohelix buccal DNA isolation kit (Cell Projects). Minor modifications (described later) were made to the Isohelix buccal DNA isolation kit; otherwise, the manufacturers' instructions were followed.

DNA was quantified on a spectrofluorometer (Gemini XS SPECTRAmax, Molecular Devises, Sunnyvale, CA, USA), courtesy of the Australian Genome Research Facility (AGRF; Adelaide Node, South Australia, Australia) using the Picogreen dsDNA quantification kit (Invitrogen). Samples that met quantitative requirements were sent to AGRF (Brisbane Node, Queensland, Australia) to assess if DNA integrity met multiplexing requirements. Three multiplex assays, consisting of 25, 11, and three SNPs, were designed by AGRF Laboratories (Brisbane Node), based on SNP sequences provided (data not shown). SNP genotyping was performed on a MassARRAY iPLEX Gold system (Sequenom, San Diego, CA, USA), which has the capacity to analyze up to 25 SNPs in a single multiplex assay.

High-Throughput Application

The Catch-All buccal swab and Isohelix buccal DNA extraction kit were selected for high-throughput application as a result of the pilot study (see Results).

Samples were collected from 1140 participants (570 mother and child pairs), who are currently part of an Australian study examining genetic susceptibility to Cerebral Palsy. Each participating family was mailed a collection kit containing an information sheet, consent form, health questionnaire, instruction sheet, and two labeled buccal swabs, one each for mother and child. The instruction sheet specified that samples were to be posted in the replied, paid envelope, provided on the day of collection. Written instructions were supplemented by a video presentation available at the study web site (www.adelaide.edu.au/cerebralpalsy/forms).

Upon receipt, buccal swabs were placed in a 1.5-ml microcentrifuge tube (Axygen, Union City, CA, USA), containing 500 μl lysis/storage buffer and 20 μl proteinase K, vortexed briefly, and then stored at room temperature for a minimum of 48 h and a maximum of 1 week until a suitable number of samples had been collected for processing. Samples were then incubated in a 60°C water bath for 90 min and vortexed briefly before removing the swab; 450 μl ice-cold Capture buffer was added to the remaining solution. Samples were mixed by inversion and allowed to sit for a further 5 min to maximize precipitation and then spun in a microcentrifuge at 20,000 g for 15 min. The supernatant was discarded immediately by turning the tubes upside down gently (carefully, so as not to dislodge the pellet) and leaving them to air-dry in this position before adding 75–100 μl rehydration buffer, depending on the pellet size. The tubes were left at room temperature for 1 h and then at 4°C overnight to aid in dissolution, prior to being dispensed into a 96-well plate format, which included one control.

For quality control, the first 176 extracted samples were quantified on a spectrofluorometer, prior to 12 96-well plates being transferred to AGRF (Brisbane Node) for genotyping.

RESULTS

Pilot Study

The samples from the 34 pilot study individuals were processed 48 h and 72 h after receipt to mimic postal delay. Based on spectrofluorometer results, no effect on DNA quantity was observed between these time-frames. The Catch-All swab and Isohelix DNA buccal swab were suitable for sample collection, providing similar DNA quantities (Table 1).

The BuccalAmp DNA extraction kit QuickExtract was the most simple and time-efficient method for DNA extraction, providing liberal DNA quantity, ranging from 0.9 to 11 μg, with an average yield of 4 μg. However, DNA quality did not meet multiplexing requirements. The DNA yield from the Magnetic bead gDNA extraction method ranged from 0.01 to 3 μg, with an average yield of 1.4 μg; this method was not as convenient for our high-throughput application and therefore, was not pursued any further. The MasterPure Complete DNA purification kit and Isohelix buccal DNA isolation kit provided sufficient DNA quantity and performed well under multiplexing conditions. DNA yield ranged from 0.1 to 2.1 μg (mean 0.44 μg) and 0.7 to 2.5 μg (mean 1.94 μg), respectively. Two of the 34 samples tested failed completely, and the overall pass rate was 94% (Table 1).

High-Throughput Application

The yield distribution for the 176 samples that were quantitated demonstrated a wider range in DNA quantity when compared with our pilot study; yields ranged from 0.15 to 13 μg, with a higher average yield of 2.44 μg (Table 1).

In total, 1140 DNA samples were assayed for 39 SNPs (44,460 genotypes) with a ≥93% pass rate. Of the 1140 samples assayed, 1058 samples had ≤10 failed SNPs/sample, 1017 samples had less than five failed SNPs/sample, and 739 samples had a 100% pass rate. Eighty-two samples (7%) had >10 failed SNPs. Thirty-four (41%) of the 82 failed samples were isolated in two of the 96-well plates. The remaining 48 (59%) failed samples were spread over 10 plates. The overall pass rate for these 10 plates was >95%. An average of 37/39 of our candidate SNPs was amplified successfully for every sample.

DISCUSSION

We sought a convenient and reliable method for collection of genetic material, which is inexpensive, noninvasive, and suitable for self-collection and mailing, and a compatible, commercial DNA extraction protocol to meet quantitative and qualitative requirements for high-throughput SNP multiplex analysis on an automated platform, specifically the MassARRAY iPLEX Gold system.

Blood samples provide generous amounts of good-quality DNA, but collection is invasive and expensive and requires patient attendance and staff for venepuncture. We have demonstrated that two commercially available systems can be used with little modification to meet the requirements for high-throughput SNP multiplex analysis. To the best of our knowledge, this is a novel approach using buccal swabs with a commercial extraction method that did not require columns or filtration for this particular SNP multiplex platform. The Catch-All swabs and the Isohelix swabs collected similar amounts of DNA, but the Catch-All swabs were packaged more conveniently for our labeling requirements. We were able to label the outer container of the buccal swab directly without removing any sealed packaging.

The BuccalAmp DNA extraction kit QuickExtract, although providing liberal DNA quantities, did not meet DNA qualitative requirements for our predesigned SNP multiplex assays. The Magnetic bead buccal cell gDNA extraction kit did not recover enough DNA efficiently from buccal swabs to warrant further investigation. The MasterPure DNA purification kit and Isohelix buccal DNA isolation kit met quantitative and qualitative requirements for multiplexing on a MassARRAY system. Both methods were cost-effective and suitable for high-throughput SNP multiplex analysis. We selected the Isohelix buccal DNA isolation kit, as opposed to the MasterPure DNA purification kit for our high-throughput application, as it was more time-efficient and did not require us to obtain any additional laboratory equipment; therefore, we were able to operate within our existing laboratory set-up.

It was important to have a clear set of sample collection instructions and resources to ensure the best possible outcome. For the best possible results, it was recommended to mail buccal swabs back to the laboratory on the same day as collection, whereupon they were processed immediately. The average DNA quantity for the high-throughput application was slightly higher when compared with the pilot study, when the preferred kit and technique were used, most likely as a result of increased power of sample size. However, a greater variability of DNA yield was observed in the high-throughput application. This may be a result of variation in participant collection techniques.

DNA yield varies according to individual collection techniques;(13) however, in most instances, even the minimum yield from the Isohelix buccal DNA isolation kit was sufficient for all three multiplex assays.

The pass rate of ≥93% was within the accepted range for PCR-based high-throughput SNP multiplex analysis. Two plates had a higher number of failed samples, which affected the overall genotyping success rate. Potentially important factors include participant compliance with DNA sampling (i.e., collection of enough epithelial cells) and speed of return. The convenience and affordability of buccal swabs as a collection method allow resampling if an assay fails.

CONCLUSION

Collection and extraction of gDNA from buccal swabs are convenient and cost-effective alternatives to blood sampling for participants and researchers. Buccal swabs are noninvasive and can be mailed out for self-collection, overcoming geographical impediments.

We described a novel method of collection and extraction, which was cost- and time-efficient, providing sufficient quantity and quality of DNA for high-throughput SNP multiplex analysis.

ACKNOWLEDGMENTS

This research was supported by the National Health and Medical Research Council, The Cerebral Palsy Foundation, The University of Adelaide, and The Channel 7 Children's Research Foundation. The supporting sources had no influences on the analysis, writing, or submission of the manuscript. We gratefully thank and appreciate all the technical help and support from the AGRF, in particular, John Stevens and David Hawkes, along with their staff from the Adelaide and Brisbane Nodes, respectively. We also thank the staff of the Department of Microbiology and Infectious Disease at the Women's and Children's Hospital for the use of their facilities.

REFERENCES

1. Freeman B, Powell J, Ball D, Hill L, Craig I, Plomin R. DNA by mail: an inexpensive and noninvasive method for collecting DNA samples from widely dispersed populations. Behav Genet May 1997; 27: 251– 257 [PubMed]
2. Richards B, Skoletsky J, Shuber AP, et al. Multiplex PCR amplification from the CFTR gene using DNA prepared from buccal brushes/swabs. Hum Mol Genet 1993; 2: 159– 163 [PubMed]
3. Rolf B, Keil W. Investigation of oral swabs taken from newborn infants after breastfeeding. Leg Med (Tokyo) 2002; 4: 52– 54 [PubMed]
4. Conyn-van Spaendonck MA, Reintjes R, Spanjaard L, van Kregten E, Kraaijeveld AG, Jacobs PH. Meningococcal carriage in relation to an outbreak of invasive disease due to Neisseria meningitidis serogroup C in the Netherlands. J Infect 1999; 39: 42– 48 [PubMed]
5. Sugata A, Fukushima K, Sugata K, et al. High-throughput screening for GJB2 mutations—its clinical application to genetic testing in prelingual deafness screening for GJB2 mutations. Auris Nasus Larynx 2002; 29: 231– 239 [PubMed]
6. Mengel-Jorgensen J, Sanchez JJ, Borsting C, Kirpekar F, Morling N. Typing of multiple single-nucleotide polymorphisms using ribonuclease cleavage of DNA/RNA chimeric single-base extension primers and detection by MALDI-TOF mass spectrometry. Anal Chem 2005; 77: 5229– 5235 [PubMed]
7. Sherry ST, Ward MH, Kholodov M, et al. dbSNP: the NCBI database of genetic variation. Nucleic Acids Res 2001; 29: 308– 311 [PMC free article] [PubMed]
8. Jurinke C, van den Boom D, Cantor CR, Koster H. Automated genotyping using the DNA MassArray technology. Methods Mol Biol 2002; 187: 179– 192 [PubMed]
9. Hinks A, Barton A, John S, Shephard N, Worthington J. Fine mapping of genes within the IDDM8 region in rheumatoid arthritis. Arthritis Res Ther 2006; 8: 145 [PMC free article] [PubMed]
10. Abel K, Reneland R, Kammerer S, et al. Genome-wide SNP association: identification of susceptibility alleles for osteoarthritis. Autoimmun Rev 2006; 5: 258– 263 [PubMed]
11. Min JL, Lakenberg N, Bakker-Verweij M, et al. High microsatellite and SNP genotyping success rates established in a large number of genomic DNA samples extracted from mouth swabs and genotypes. Twin Res Hum Genet 2006; 9: 501– 506 [PubMed]
12. Garcia-Closas M, Egan KM, Abruzzo J, et al. Collection of genomic DNA from adults in epidemiological studies by buccal cytobrush and mouthwash. Cancer Epidemiol Biomarkers Prev 2001; 10: 687– 696 [PubMed]
13. Freeman B, Smith N, Curtis C, Huckett L, Mill J, Craig IW. DNA from buccal swabs recruited by mail: evaluation of storage effects on long-term stability and suitability for multiplex polymerase chain reaction genotyping. Behav Genet 2003; 33: 67– 72 [PubMed]

Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities