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© 2007 The Authors Doi: 10.1111/j.1742-7843.2007.00089.x Journal compilation © 2007 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 101, 132–137

DNA Cards: Determinants of DNA Yield and Quality in Collecting Genetic Samples for Pharmacogenetic Studies

Blackwell Publishing Ltd

Sergi Mas1, Anna Crescenti1, Patricia Gassó1, Jose M Vidal-Taboada2 and Amalia Lafuente1 1 Department of Pharmacology and Therapeutic Chemistry and 2Unit of Genetics, Department of Physioslogical Sciences I, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Faculty of Medicine, University of Barcelona, Barcelona, Spain

(Received February 9, 2007; Accepted April 9, 2007) Abstract: As pharmacogenetic studies frequently require establishment of DNA banks containing large cohorts with multi-centric designs, inexpensive methods for collecting and storing high-quality DNA are needed. The aims of this study were two-fold: to compare the amount and quality of DNA obtained from two different DNA cards (IsoCode Cards ® or FTA Classic Cards®, Whatman plc, Brentford, Middlesex, UK); and to evaluate the effects of time and storage temperature, as well as the influence of anticoagulant ethylenediaminetetraacetic acid on the DNA elution procedure. The samples were genotyped by several methods typically used in pharmacogenetic studies: multiplex PCR, PCR-restriction fragment length polymorphism, single nucleotide primer extension, and allelic discrimination assay. In addition, they were amplified by whole genome amplification to increase genomic DNA mass. Time, storage temperature and ethylenediaminetetraacetic acid had no significant effects on either DNA card. This study reveals the importance of drying blood spots prior to isolation to avoid haemoglobin interference. Moreover, our results demonstrate that re-isolation protocols could be applied to increase the amount of DNA recovered. The samples analysed were accurately genotyped with all the methods examined herein. In conclusion, our study shows that both DNA cards, IsoCode Cards ® and FTA Classic Cards®, facilitate genetic and pharmacogenetic testing for routine clinical practice.

Genetic and pharmacogenetic studies involve different centers, couriers and laboratory equipment. The collection and isolation of high-quality DNA from blood samples is the first and most important part of these studies [1,2]. Blood samples obtained by venipuncture (the standard DNA collection method) often cannot be used owing to lack of resources or of specially trained personnel or cultural factors. This method may also be too expensive for large-scale studies. In addition, it is laborious and invasive, and associated with potential security problems. The sample must be frozen rapidly, and management of liquid or frozen samples often results in high costs. Alternative methods are based on the use of DNA Cards that are easier to collect, transport and store. The methods are less invasive, and much cheaper than the usual venipuncture technique [3,4]. These methods use chemically treated paper cards, such as IsoCode Cards® (now marketed as FTA Elute®, Whatman plc, Middlesex, UK) or FTA Classic Cards® (Whatman plc). High-quality genomic DNA can be extracted from these samples providing suitable archival media in DNA banks for forensic [5] or genetic epidemiological studies [1]. The DNA card method has been used in several clinical studies on for instance malaria [2,6], staphylococcus [7], HIV [8], Gaucher’s disease [9] and cancer [1,10]. We recently used this method to perform a pharmacogenetic sub-study. DNA cards were used to obtain blood samples from a

Author for correspondence: Amalia Lafuente, Department of Therapeutic Pharmacology and Chemistry, Faculty of Medicine, University of Barcelona, Casanova 143, E-08036 Barcelona, Spain (fax + 34 934035881, e-mail [email protected])

multi-centre clinical trial (ITEMS) in which the response to tegaserod (Zelnorm®/Zelmac®, Novartis Pharmaceuticals AG, Basel, Switzerland), a selective 5-HT4 receptor partial agonist, was evaluated in patients with irritable bowel syndrome [11]. The aims of the present study were to assess and compare the amount and quality of DNA obtained from two different DNA cards (IsoCode Cards® or FTA Classic Cards®) and to evaluate the influence of time and storage temperature, as well as the presence of anticoagulants, on DNA collection. To ensure the quality of collection and isolation protocols, the samples were genotyped by several methods typically used in pharmacogenetic studies: multiplex PCR, PCR-restriction fragment length polymorphism (RFLP), single nucleotide primer extension and allelic discrimination assay. In addition, they were amplified by whole genome amplification (WGA) to increase genomic DNA mass.

Materials and Methods Thirty-six volunteers (25 females and 11 males, with an age-range of 20 –50 years) were recruited at the School of Medicine, University of Barcelona, Spain, and were asked to collect blood sample using these three different methods (venipuncture, IsoCode Cards® and FTA Classic Cards®). Only patients who were Caucasian and unrelated, were invited to participate in the study. Other ethnic groups were excluded. The study protocol was approved by the corresponding Ethical Committees. All patients gave their informed consent to participate in the study. Sample collection. To standardize the comparisons, blood spots were not collected directly from fingertip puncture. Instead, a drop of 10 µl of blood (collected by venipuncture) was placed on the

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DNA quantification. The elute containing the purified single-stranded DNA (ssDNA) from either IsoCode Cards® and FTA Classic Cards® was quantified and stored at –80°C until use. ssDNA yields were determined as instructed in the Oligreen ssDNA quantitation kit (Molecular Probes, Invitrogen, Carlsbad, CA, USA) using a spectrofluorometer (Hitachi F-4500, Hitachi, Tokyo, Japan). Haemoglobin concentration. Haemoglobin, the major contaminant in DNA samples purified from blood sources, was used as a marker to evaluate the purification protocols. Each DNA sample was diluted (1/10) and absorbance was read at 405 nm in a spectrophotometer (Cecil 2100, Tovatech, South Orange, NJ, USA) [12]. Fig. 1. Protocol for sample collection. *Standard conditions were stored at room temperature for two months without EDTA.

Genotyping methods. PCR-RFLP. This was used to genotype a polymorphism in the motilin gene (rs2281820) based on a previously described method [13].

fingertip to perform the blood spot. Four drops (10 µl each) were collected onto each DNA Card. First venipuncture blood was collected in tubes by ethylenediaminetetraacetic acid (EDTA) (Vacutainer, Becton Dickinson, Franklin Lakes, NJ, USA) to obtain the haematological profile (AVIA 2120, Hematology system, Bayer, Leverkusen, Germany). We then took different drops of 10 µl to obtain blood spots by EDTA in their corresponding cards (fig. 1). The same protocol was carried out using both types of DNA cards (IsoCode Cards® or FTA Classic Cards®). Once the cards were filled, the drops were dried at room temperature overnight, and stored in zip-sealed plastic bags with desiccant packs to avoid humidity and to protect them from light. Each volunteer filled several DNA cards for storage at various temperatures (room temperature, –20°C and –80°C) and at different times (1 week, 2 months and 1 year) (fig. 1). DNA elution. IsoCode Cards® DNA was extracted using a modification of the manufacturer’s instructions. Using a paper puncher 0.125 inch (3.2 mm) discs were punched from the complete card matrix region containing the dried blood. In between consecutive uses, the puncher was sterilized with both alcohol and flame before making several punches through a clean filter paper. Three discs containing dried blood were placed in a 1.5-ml tube and heated in an oven at 80°C for 20 min. To remove contaminants, the discs were washed twice with 500 µl of water and vortexed three times for 5 sec. The washed discs were transferred to a new tube and 40 µl of water was added. Before being stored on ice, the individual tubes were incubated at 100°C for 15 min. to elute the DNA from the card matrix. The samples were pulse-vortexed about 60 times and centrifuged for 1 min. at 13,000 ×g. The matrix discs were removed using plastic forceps, and squeezed on the side of the tube to remove excess water. The supernatant with the eluted DNA was stored at –20°C. The discs were dried and stored at room temperature for re-isolations: we dried the already isolated 3.2 mm discs, and then again proceeded with a new isolation.

Multiplex PCR. The GSTM1 and GSTT1 polymorphisms (gene deletions) were assayed using a multiplex PCR based on a previously described method [14]. Multiplex PCR and single nucleotide primer extension (SNuPE). Three polymorphisms of the CYP2C9 (CYP2C9*2, *3 and 5 ′flanking region C-1189T polymorphisms) were genotyped simultaneously based on a previously published method [15] using ABI Prism® SNaPshotTM Multiplex kit (Applied Biosystems, Foster City, CA, USA). Allelic discrimination assay (TaqMan ®). Here, we followed the specifications for Genotyping Assays (Applied Biosystems) to genotype two polymorphisms of the serotonin receptor 4: rs1345697 (Applied Biosystems c_11267705_10) and rs 980062 (Applied Biosystems c_7505278_10). Whole genome amplification (WGA). To amplify genomic DNA, we followed the manufacturer’s instruction (GenomiPhi DNA Amplification kit, Amersham Biosciences, Buckinghamshire, UK). Statistics. The percentage of DNA recovered was calculated, taking into account the haematological profile values (white blood cell count) and assuming 3 µl of blood per disc, 6 pg DNA per white blood cell, and 100% DNA yield [12]. Multiple parameters of the two cards were compared using one-way anova (analysis of variance). P ≤ 0.05 was considered statistically significant. For comparative purposes we defined as standard conditions storage at room temperature for two months without EDTA. Statistical analysis was performed using SPSS version 11.0 (Chicago, IL, USA).

Results FTA Classic Cards®. Three 3.2-mm discs were obtained using the same protocol as that for the IsoCode Cards®. DNA was extracted according to the manufacturer’s instructions with some modification. Three discs containing dried blood were placed in a 1.5-ml tube and heated in an oven at 80°C for 15–20 min. Three washes in 200 µl each of FTA purification reagent were carried out and the samples were then incubated at room temperature for 5 min. Two additional washes in TE-1 buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) were made before the discs were allowed to dry at room temperature for 1 hr or 10 min. at 60°C. To elute the DNA, 102 µl of pH 13 solution (0.1 N NaOH, 0.3 mM EDTA) was added and incubated for 5 min. Subsequently, 198 µl of pH 7 solution (0.1 M Tris-HCl) was added and the tube was inverted several times to keep the discs immersed in the solution. The sample was vortexed for 5 min. and then left at room temperature for 10 min. The supernatant was transferred to a new sterile tube and stored at –20°C. The discs were dried and stored at room temperature for re-isolations.

The amount of DNA obtained using the two DNA cards proved similar under each storage condition (table 1). Time, temperature and EDTA had no significant effects upon IsoCode Cards® or FTA Classic Cards®. Although the differences were not significant, the DNA yields in the FTA Classic Cards® appeared to decrease markedly in the absence of EDTA. In the IsoCode Cards®, the drying time of blood samples proved extremely important in ensuring high DNA quality. As shown in fig. 2, when the isolation was made during the first week, the haemoglobin concentration in the eluted DNA was very high. Indeed, at this haemoglobin concentration, the DNA amplification procedures were inhibited (data not shown). To avoid this situation, blood spots must be dried for no less

© 2007 The Authors Journal compilation © 2007 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 101, 132–137

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SERGI MAS ET AL. Table 1.

Mean and standard deviations for total amounts of DNA (ng) obtained using each storage protocol and the percentage of recovery in the re-elution protocol. IsoCode Card®

FTA Classic Card®

Temperature ng DNA (percentage of recovery)* Tª Ambient 131.6 ± 74.2 (25.7) 110.2 ± 77.3 (22.3) –20°C 162.7 ± 71.4 (34.1) 120.2 ± 65.1 (23.5) –80°C 177.6 ± 83.2 (45.1) 157.4 ± 74.2 (34.2) Time ng DNA (percentage of recovery)† 1 week 138.9 ± 62.7 (29.6) 148.2 ± 80.5 (30.5) 2 months 168.2 ± 82.8 (34.7) 200.2 ± 111.3 (41.3) 1 year 159.5 ± 65.3 (34.3) 175.3 ± 100.6 (45.3) EDTA ng DNA (percentage of recovery)‡ Yes 164.3 ± 86.1 (34.7) 170.6 ± 120.8 (35.3) No 144.4 ± 71.4 (30.1) 102.7 ± 66.2 (32.3) Re-elution protocol (percentage of recovery)§ 1st elution 34.7 24.3 2nd elution 31.1 45.1 Total 65.9 69.4 *Samples stored for 2 months without EDTA. † Samples stored at room temperature without EDTA. ‡ Samples stored at room temperatures for 2 months. § Samples stored at room temperatures for 2 months without EDTA.

than 1 week. Additionally, the haemoglobin concentration should be decreased if two washes are performed instead of one (i.e. at the first step of the elution procedure; fig. 3). Moreover, to ensure that blood spots are thoroughly dried, we heated the DNA cards prior to elution. This problem was not encountered with FTA Classic Cards®, although the oven pre-elution step was similarly carried out. To increase the amount of DNA, we tested the re-isolation. As shown in table 1, the second elution procedure allowed for the isolation of more DNA, leading to recovery values close to 65%. When the IsoCode Card® was re-isolated, the same efficiency was observed during recovery, whereas with the FTA Classic Cards® the second isolation proved more efficient that the first. All samples were successfully genotyped for the selected polymorphisms with the different methods and amplified with the WGA. As shown in table 2, differences between the two types of DNA cards involved the amount of DNA needed for each method.

Fig. 2. Concentration of haemoglobin (Hb) in the eluted DNA during the first week after the collection procedure with IsoCode Card®.

Fig. 3. Concentration of haemoglobin (Hb) in the eluted DNA after performing one or two washes using IsoCode Card® as part of the elute protocol.

Finally, a comparison of the time and cost of each DNA cards and a traditional method of whole blood isolation is shown in table 3. Discussion The aims of this study are two-fold: to evaluate different protocols and systems for DNA collection from DNA cards (IsoCode Cards® or FTA Classic Cards®) and to examine the influence of time and storage temperature, as well as the presence of anticoagulants on DNA elution. As commented in the Materials and Methods section, to standardize the comparisons, blood spots were not collected directly from fingertip puncture. Difference in blood counts in venous and fingertip blood has long been a controversial issue. Recently, a study comparing blood counts and their measurement variation in venous, fingertip and arterial blood, concluded that the major differences between fingertip and venous blood were leucocyte counts that were higher in the fingertip blood than in venous blood [16]. No differences in the haemoglobin or haematocrit values were observed [16]. In the present study, these differences could lead to a major DNA recovery. However, in a previous pharmacogenetic study by our group, using DNA cards filled with fingertip blood, the total amount of DNA was similar (1.06 ± 0.5 µg) to those obtained here [11]. Our results encompass two important technical findings. The first involves the importance of avoiding haemoglobin interference, which can be achieved by observing the following steps: drying the blood spots at room temperature for 1 week; performing a pre-elution step in the oven; and twice washing the discs when IsoCode Cards® are used. The second is related to the possibility of increasing the amount of DNA that could be isolated from a DNA card by reeluting the discs. That time and temperature exert no influence upon the elution procedure is consistent with previous studies, which all reported high DNA stability in dried blood samples for those DNA cards stored at room temperature for up to 16 months [4,5]. Other authors have described successful PCR


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DNA CARDS IN PHARMACOGENETIC STUDIES Table 2. Amount of DNA needed in each genotyping technique. Multiplex (ng)

RFLP (ng)

SNuPE (ng)

TaqMan (ng)

WGA (µl)

25 5

10 5

25 5

15 5

1 1

®

IsoCode Card FTA Classic Card®

Abbreviations: RFLP, restriction fragment length polymorphism; SNuPE, single nucleotide primer extension; TaqMan, allelic discrimination assay; WGA, whole genome amplification.

analyses after storage for 4 or 8.5 years [17,18]. In fact, the manufacturer says that it is possible to obtain PCR amplifications after 14 years of DNA storage (Whatman brochure). This stability offers a great advantage over other methods, as it simplifies trial logistics, management and costs. Transport, storage and DNA extraction can all be performed at room temperature, making it easier to move such samples from the various collection centres to the central DNA bank or laboratory. This procedure not only drastically reduces the work necessary for sample management, but also lowers the overall costs involved. Examples include the following: fingertip puncture is less labour-intensive than venipuncture; freezer or refrigerator management and handling of frozen samples is not needed, and low-temperature packaging and specialized couriers are not required. Additionally, the duration of storage after blood collection (before the sample can be transported) is much longer than is possible with fresh blood samples, allowing several samples to be transported together, thus reducing both the total number of parcels transported and the associated control cost. This increased storage time also removes time constraints on receipt of samples at the central laboratory, as blood samples do not require immediate processing after delivery. In addition, transport by standard mail or courier is inexpensive (cards are compact and lightweight), the cards are economical and

Table 3. Time and cost of collection and isolation of DNA cards and traditional isolation of whole blood samples. IsoCode Card® Time Before storage (minimum) Elution protocol Protocol steps Costs DNA card Elution reagents Total† DNA Total amount (µg)‡

Whole FTA Card® blood*

1 week 24 hr 0 45–70 min. 60 –90 min. 45–60 min. 6 20 19 3$ – 3$

3.92 $ 0.97 $ 4.89 $

– 3.5 $ 3.5 $

0.96 ± 0.4

1.01 ± 0.5

10.0 ± 5.0

*Traditional isolation of 300 µl whole blood using PureGenetTM (Gentra Systems, Minneapolis, MN, USA). † Total cost after eluting the DNA from all of the blood spots on one DNA card, or 300 µl of whole blood. ‡ Total DNA amount after eluting the DNA from all of the blood spots on one DNA card, or 300 µl of whole blood.

require little storage space [19], and the working time required for sample management by the investigators and study monitors is shorter than with conventional methods that involve liquid or frozen sample handling. Based on our experience, we estimate that using DNA cards will result in a 56% savings in sample collection and management (material and reagent costs, freezers, control and sample management, transport of specimens and purification of DNA) compared to the alternative use of frozen samples [11]. Consequently, DNA cards are increasingly gaining acceptance as a cost-minimizing method for conducting genetics analyses, as other authors have suggested [3,4,19]. Additionally, we had previously noted that the DNA cards were well received by both investigators and patients in a multicentre clinical trail, and were in fact preferred to conventional methods due to their easy use and safety [11]. The main disadvantage of the DNA cards method is the quantity of DNA recovered compared to the yields obtained from fresh venous blood samples [20]. However, by utilizing re-isolation protocol described here, the amount of DNA could be easily increased. This observation can be partly explained by the fact that 40% or more of the DNA is retained on the card matrix [3]. In addition, the amount of DNA is highly variable between patient specimens, due to intrinsic factors such as the proportion of white blood cells in the blood [4]. Primarily, however, this variability stems from the quantity of blood actually deposited on the card matrix. This last factor is influenced by the investigator, who should be aware of the importance of completely staining the sample collection area of the card [11]. While the amount of DNA purified from the DNA cards could prove problematic for molecular analyses requiring quantities in micrograms, such as Southern blot or genomic cloning, it is entirely suitable for use in SNP analyses. As shown in our study, the DNA eluted from DNA cards could be used not only for conducting WGA but also for genotyping the selected polymorphisms with the methods described. The amount of DNA obtained potentially allows anywhere from dozens to hundreds of polymorphisms to be processed using these multiplex PCR analyses, especially if all of the blood spots on the DNA card are used. This capacity for polymorphism analysis is usually sufficient for the objectives sought in pharmacogenetic studies. If additional polymorphism analyses are required, investigators will need to use the WGA to obtain a sufficient quantity of genomic DNA, a method that has been proven feasible in our study. This is also true of cases in which an investigator must recover lower quantities of DNA from patients.


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There are traditional, non-invasive alternatives to blood collection, such as the use of buccal mucus sampling. Mouthwash from expectoration provides genomic DNA of high molecular weight, and represents an easy way of sample collection for adults. However, this is not an option for infants and small children, with whom a cytobrush is required. While this method has been used in several large epidemiologic studies, the DNA yields between specimens are highly variable and bacterial contamination must be addressed [21]. The stability of DNA remains good for 5 days, thereby allowing samples to be transported fresh. To increase the stability of buccal cell DNA over time, DNA cards have been used to store such DNA at room temperature, reportedly for years. The major advantage of collecting saliva with DNA cards is its non-invasive nature compared to finger puncture. However, a disadvantage is that the DNA yields from such specimens are more variable than blood specimens. In addition, it is sometimes necessary to repeat the procedure in those patients from whom low recoveries were initially made [3,19]. Moreover, there is the possibility that different genomes might co-exist in the mouth, which could interfere with correct genotype assignments in those persons. The weight of a sample collected with either a cytobrush or mouth-wash is higher than with DNA cards, which therefore increases the cost of mailing [19]. Finally, when comparing the attendant time and costs associated with the two DNA cards, the methods were similar. The main difference between the cards is the amount of DNA needed for genotyping, which ensures 200 reactions with the FTA Classic Cards® and no more than 100 reactions with IsoCode Cards®. However, this difference is minimized by the use of WGA with both DNA cards. The use of FTA Classic Cards® requires more time when performing the elution protocol. However, not only is the amount of DNA needed for genotyping less, but also drying of the blood spots and the presence of haemoglobin appears to be insignificant. IsoCode Cards® protocol, on the other hand, proved more rapid in isolation of the samples, albeit with the inconvenience of haemoglobin contamination. However, this can be avoided if the blood spots are dried for 1 week and if a pre-oven elution step and a second wash is added to the manufacturer’s protocol. In conclusion, taking into account the main limitation of our design (using venipuncture blood instead of fingertip puncture), our study shows that both DNA cards, IsoCode Cards ® or FTA Classic Cards ®, facilitate genetic and pharmacogenetic testing for routine clinical practice. Acknowledgements We wish to thank the volunteers who participated in this study and the Language Advisory Service at the University of Barcelona, Spain, for editing the manuscript. This study was supported by the Spanish Ministry of Health, Instituto Carlos III, RETICS; Red de Enfermedades Mentales (REM-TAP NetworkRD06/0011/06-05) and DURSI GRC2005SGR00039.

References 1 Struewing JP, Hartge P, Wacholder S et al. The risk of cancer associated with specific mutations of BRCA1 and BRCA2 among Ashkenazi Jews. N Engl J Med 1997;336:1401– 8. 2 Henning L, Felger I, Beck HP. Rapid DNA extraction for molecular epidemiological studies of malaria. Acta Trop 1999;72:149–55. 3 Myers RL, Burghoff RL, English DW, Wenzel AZ, Harvey AH. Evaluation of IsoCode and Other Novel Collection Matrices as Nucleic Acid Storage and Processing Devices for Clinical Samples [Abstract #774]. 49th AAAC Annual Meeting, Atlanta, July 20 –24, 1997. 4 Harty LC, Garcia-Closas M, Rothman N, Reid YA, Tucker MA, Hartge P. Collection of buccal cell DNA using treated cards. Cancer Epidemiol Biomarkers Prev 2000;9:501– 6. 5 Kline MC, Duewer DL, Redman JW, Butler JM, Boyer DA. Polymerase chain reaction amplification of DNA from aged blood stains: quantitative evaluation of the ‘suitability for purpose’ of four filter papers as archival media. Anal Chem 2002;74:1863 –9. 6 Zhong KJ, Salas CJ, Shafer R et al. Comparison of IsoCode STIX and FTA Gene Guard collection matrices as whole-blood storage and processing devices for diagnosis of malaria by PCR. J Clin Microbiol 2001;39:1195 – 6. 7 Rogers C, Burgoyne L. Bacterial typing: storing and processing of stabilized reference bacteria for polymerase chain reaction without preparing DNA – an example of an automatable procedure. Anal Biochem 1997;247:223 –7. 8 Panteleef DD, John G, Nduati R et al. Rapid method for screening dried blood samples on filter paper for human immunodeficiency virus type 1 DNA. J Clin Microbiol 1999;37:350–3. 9 Devost NC, Choy FY. Mutation analysis of Gaucher disease using dot-blood samples on FTA filter paper. Am J Med Genet 2000;94:417–20. 10 Dobbs LJ, Madigan MN, Carter AB, Earls L. Use of FTA gene guard filter paper for the storage and transportation of tumor cells for molecular testing. Arch Pathol Lab Med 2002;126:56 – 63. 11 Vidal JM, Cucala M, Mas S, Lafuente A, Cobos A. Satisfaction survey with DNA cards method to collect genetic sample for pharmacogenetic studies. BMC Med Genet 2006;7:45. 12 Heath EM, O’Brien DP, Banas R, Naylor EW, Dobrowolski S. Optimization of an automated DNA purification protocol for neonatal screening. Arch Pathol Lab Med 1999;123:1154– 60. 13 Annese V, Piepoli A, Andriulli A et al. Polymorphism of motilin gene in patients with crohn’s disease. Dig Dis Sci 1998;43:715–9. 14 Laso N, Lafuente MJ, Mas S et al. Glutathione S-transferase (GSTM1 and GSTT1)-dependent risk for colorectal cancer. Anticancer Res 2002;22:3399– 403. 15 Mas S, Crescenti A, Vidal-Taboada JM et al. Simultaneous genotyping of CYP2C9*2, *3, and 5′-flanking region (C-1189T) polymorphisms in a Spanish population through a new minisequencing multiplex single-base extension analysis. Eur J Clin Pharmacol 2005;61:635 – 41. 16 Yang Z-W, Yang S-H, Chen L, Qu J, Zhu J, Tang Z. Comparison of blood counts in venous, fingertip and arterial blood and their measurement variation. Clin Lab Haematol 2001;23:155– 9. 17 Smith LM, Burgoyne LA. Collecting, archiving and processing DNA from wildlife samples using FTA databasing paper. BMC Ecol 2004;4:4. 18 Burgoyne L, Kijas J, Hallsworth P, Turner J. Safe Collection, Storage and Analysis of DNA from Blood. Proceedings of the 5th


DNA CARDS IN PHARMACOGENETIC STUDIES International Symposium on Human Identification, Scottsdale, AZ, October 8–11,1994. 19 Mulot C, Stucker I, Clavel J, Beaune P, Loriot MA. Collection of human genomic DNA from buccal cells for genetics studies: comparison between cytobrush, mouthwash, and treated card. J Biomed Biotechnol 2005;3:291– 6.

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20 Ausubel FM, Brent R, Kingston RE et al. Current Protocols in Molecular Biology. John Wiley & Sons, New York, 1997. 21 Steinberg K, Beck J, Nickerson D et al. DNA banking for epidemiologic studies a review of current practices. Epidemiology 2002;13:246 –5.
