Benchmarks processing time and an increase in the DNA elution from buccal cells number of PCR reactions per sample punch. stored on Whatman FTA Classic This revised method was tested in comparison with the standard Whatman FTA protocol for both buccal cells on Cards using a modified methanol Whatman FTA Classic Cards (stored for 0–1 years) and blood spots (stored for 2–5 fixation method years) on 1-mm Whatman BFC 180 filter 1 2 1 paper blood sample collection cards (Figure Helene C. Johanson , Valentine Hyland , Carol Wicking , and 1). Participants were sourced from a study Richard A. Sturm1 approved by the University of Queensland 1Institute for Molecular Bioscience, University of Queensland, Brisbane, Medical Research Ethics Committee in 2 accordance with the National Health and Queensland, Australia and Department of Molecular Genetics, The Royal Medical Research Council’s guidelines. Brisbane and Women’s Hospital, Brisbane, Australia Sample collection. A DNA self- collection kit was designed consisting of BioTechniques 46:309-311 (April 2009) doi 10.2144/000113077 a Whatman FTA Classic Card individ- Keywords: buccal swab; DNA; elution; methanol; FTA card ually sealed in a 9 × 15-cm transparent plastic zipper storage bag (Cat. no. 7233; We describe here a method for DNA elution from buccal cells and whole Glad Products Australia, Padstow, NSW, Australia) and then placed into a blood both collected onto Whatman FTA technology, using methanol fix- sealed envelope with a single Whatman ation followed by an elution PCR program. Extracted DNA is comparable sterile foam-tipped applicator. Partici- in quality to published Whatman FTA protocols, as judged by PCR-based pants collected the sample as directed [http://www.whatman.com/References/ genotyping. Elution of DNA from the dried sample is a known rate-limit- BUCCAL(1).pdf ] and all samples were ing step in the published Whatman FTA protocol; this method enables the dried, resealed in the plastic bag, then use of each 3-mm punch of sample for several PCR reactions instead of the stored at room temperature. Sample preparation. A single 3-mm standard, one PCR reaction per sample punch. This optimized protocol diameter circle punch was obtained from therefore extends the usefulness and cost effectiveness of each buccal swab each sample using a manual plier one-hole punch (Cat. no. 23517097; Fiskars Brands sample collected, when used for nucleic acid PCR and genotyping. Inc., Madison, WI, USA) from which the confetti trap was removed (http:// microscopy.tamu.edu/lab-protocols/ Whatman FTA Classic Cards (Cat. no. have the potential to generate more DNA protocols-FTA_paper_processing.pdf). WB120205; Whatman International product per volume than blood. With The paper punch was cleaned between Ltd, Piscataway, NJ, USA) combined improved stability and lack of discomfort samples by rigorous manual wiping with with sterile foam tipped applicators (Cat. during collection, they are becoming the 70% ethanol. It was then used to punch 3 no. WB100032) are used to painlessly sample of choice allowing easy collection holes from a separate clean, unused blood collect loose inner-cheek (buccal) cells, in any field situation. However, the time- sample collection card, prior to performing including those that are floating in saliva. consuming sample punch process is a a single sample punch. Sample spots were FTA technology prevents the degradation known rate-limiting step, which is exacer- then processed according to either the of genomic DNA at room temperature, bated if a particular analysis requires Whatman FTA protocol, or the alter- and PCR amplification of viable DNA multiple sample preparations (4) (http:// native method. after long-term archiving is possible (1). microscopy.tamu.edu/lab-protocols/ DNA elution – methanol fixation For analysis, a small disc is punched from protocols-FTA_paper_processing.pdf). method. The sample spot was fixed the dried sample area. Another issue with this is the relatively by overlaying with methanol (4 drops Whatman standard protocols require fast consumption of biological sample. delivered via Pasteur pipette; AJAX, the disc to be washed in FTA purification Methanol fixation during DNA extraction Taren Pt, NSW, Australia) three times reagent (Cat. no. WB120204), dried, has been used previously for blood samples and allowed to air-dry in between (the and the entire sample disc then incorpo- stored on Guthrie cards (5). Notably, the first time at room temperature for 20 min rated directly into the PCR amplification effects of different fixation techniques and the next two times incubated at 37°C reaction. The protocols do not adjust on chromatin ultrastructure and DNA for 40 min each). Genomic DNA was then reaction volume or PCR conditions to the extraction have shown cell morphology to eluted from the paper in a heat incubation presence of the disc (www.whatman.com/ be compromised with an increased yield step run on a MyCycler thermal cycler References/51613rerevised.pdf). of genomic DNA using a methanol-based (Bio-Rad, Hercules, CA, USA). Using It has been found that the use of saliva fixative (6,7). flat nosed forceps, dry sample spots were samples is a good alternative to blood We describe here for the first time transferred to a thin-walled 0.2-mL PCR samples (2) to obtain genomic DNA of DNA elution from buccal cells collected tube containing 5 μL 10× PCR buffer high quality, and that their use increases on Whatman FTA Classic Cards using (Invitrogen, Mt Waverley, VIC, Australia) the response rate considerably in epidemio- a modified methanol fixation method and 45 μL water. The tube was subjected logic studies (3). Theoretically, buccal cells (5,8). This enables a decrease in sample to one cycle of 60°C for 30 min, 99.9°C Vol. 46 | No. 4 | 2009 309 www.BioTechniques.com Benchmarks Figure 1. Comparative PCR amplifica- and the FTA method showed a marked tion of TYR exon 4 using DNA extracted decrease in yield for the blood spots on from dried buccal and blood spots via the Whatman FTA protocol and the methanol BFC 180 filter paper (hemolysed reaction fixation method. PCR conditions: Total noted). Both methods tested for buccal reaction volume 25 μL containing 2 μL cells yielded equal quantities of DNA per human genomic DNA template (2 μL PCR reaction. Readable sequence for the eluant or 3-mm treated sample disc), region of interest was able to be generated 0.25 μM both Tyr4f and Tyr4r primers from DNA extracted from both buccal (9), 200 μM each dNTP (Bioline, Alex- and blood samples via the methanol andria, NSW, Australia), 1× PCR buffer (Invitrogen), 2.0 mM MgCl2 (Invitrogen) elution method (Figure 2). and 1 U Taq polymerase (Invitrogen). We suggest the following alternative PCR products visualized on 1.5% aga- applications that add to the benefit of rose gel (Quantum Scientific Probiogen the methanol method for blood spots Biochemicals, Murarrie, QLD, Austra- and hair bulbs respectively, which have lia) in 1× Tris-acetate-EDTA (TAE) and stained with ethidium bromide (Sigma- been successfully used by V.H. (data not Aldrich, Sydney, NSW, Australia). Lanes shown). 1–12, samples from multiple subjects; Immediate methanol fixation. Fix Lane N, no DNA control; Lane M, mo- the entire collection card with methanol lecular size marker (GeneRuler 100-bp DNA Ladder Plus; Quantum Scientific Fermentas, Murarrie, immediately following collection in the QLD, Australia). Buccal spots were <1 month old and fresh-collected onto Whatman FTA Classic field and air-dry. Upon returning to the Cards. Blood spots were stored for 5.5 yrs on 1-mm Whatman BFC 180 filter paper. laboratory, fix the entire card once more by soaking in methanol. This could for 10 min, then cooled to 4°C. The FTA We have used the methanol method potentially increase the shelf life of the paper was left in the tube with the eluant to extract DNA from blood spots on sample and will also enable the fixation and stored at 4°C. PCR reactions were neonatal cards since 2003 and from step to be skipped when preparing sample performed using 2 μL extracted product. buccal spots on FTA Classic Cards since spots, thus decreasing processing time The quality of genomic DNA eluted 2007. Inclusion of samples from multiple even further. after methanol fixation was determined family groups has consistently shown Proteinase K step. To potentially through PCR amplification and sequen- expected Mendelian inheritance patterns. increase the DNA yield even further, add cing of human TYR gene exons 1–5 Best results were achieved when the Proteinase K (50 μg/mL; Roche, Castle (fragment range overall was 106–257 bp sample spot was completely dried before Hill, NSW, Australia) to the thermal in length). These fragments were amplified the thermal elution step. incubation elution step. The Proteinase using primer pairs Tyr1.1, Tyr1.2, Tyr2, This comparative study demon- K would be destroyed in the 99.9°C Tyr3, Tyr4, Tyr5 and the protocol as strates that amplimers of expected size incubation step. published (9) (Figure 1 and data not were able to be detected via both DNA The methanol DNA elution method shown). We did not attempt to sequence extraction methods for buccal and blood provides many advantages. The buccal the weak PCR fragments from blood samples collected on each of the two cell DNA entrapped on the Whatman samples purified via the FTA protocol: types of Whatman paper media (Figure FTA card can be eluted from each sample in our experience, yields of this level are 1). However, the methanol method was spot, enabling as little as 2 μL of product routinely unable to be sequenced. consistent for each biological sample to be used per PCR reaction instead of Tyrosinase exon 4 coding region B098TYREx4F/ Buccal swab, methanol method B067TYREx4F/ Buccal swab, FTA purification reagent B029TYREx4F/ Blood spot, methanol method Figure 2.