International Journal of Laboratory Hematology

An assessment of 3 non-commercial DNA extraction

methods from dried blood spots for beta-thalassaemia
mutation identification.
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Journal: International Journal of Laboratory Hematology

Manuscript ID: IJLH-11-10-0260

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Manuscript Type: Technical Report

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Date Submitted by the

15-Nov-2010
Author:

Complete List of Authors: Lai, Mei I; Faculty of Medicine and Health Science, Department of
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Pathology
Karthipan, Sharon; Universiti Putra Malaysia, Department of
Pathology
George, Elizabeth; Universiti Putra Malaysia, Department of
Pathology
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Sathar, Jameela; Hospital Ampang, Department of Haematology

Lim, Wai Feng; Universiti Putra Malaysia, Department of Pathology
Teh, Lai Kuan; Universiti Putra Malaysia, Department of Pathology
Lee, Tze Yan; Universiti Putra Malaysia, Department of Pathology
Chin, Voon Kin; Universiti Putra Malaysia, Department of Pathology
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beta-thalassaemia, dried blood spots, DNA extraction, non-

Keywords:
commercial, assessment
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International Journal of Laboratory Hematology

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5 1. Title Page
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8 An assessment of 3 non-commercial DNA extraction methods from dried blood
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10 spots for beta-thalassaemia mutation identification.
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17 S.N. Karthipan1, E. George1, S. Jameela2, W.F. Lim1, L.K. Teh1, T.Y. Lee1, V.K. Chin1,
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19 M.I. Lai1
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Department of Pathology, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia;
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Department of Hematology, Ampang Hospital, 68000 Ampang, Kuala Lumpur,
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27 Malaysia.
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33 Correspondence: Mei I Lai, Department of Pathology, Faculty of Medicine and Health

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36 Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia. Tel: +603-8947
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38 2494. Fax: +603-8941 2787. Email: laimeii@medic.upm.edu.my

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51 Short running title: Non-commercial DNA extraction from dried blood spots
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7 2. Abstract
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10 Introduction. Dried blood spots (DBS) are currently the recommended sample
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12 collection method for newborn screening programs in America. Early diagnosis of beta-
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15 thalassaemia screening is essential as it provides an added advantage especially in sickle
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17 cell disease. Beta-thalassaemia frequency is high in many poor countries and the cost of
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using commercial DNA extraction kits can be prohibitive. Our study assessed 3
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22 methods which uses minimal reagents and materials to extract DNA from dried blood
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24 spots for beta-thalassaemia identification.
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27 Methods. The methods assessed in this study are Tris-EDTA (TE) buffer-based method
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30 by Bereczky S. et al., 2005, NaCL/NaOH/SDS method by Huang S. et al., 1990 and

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32 NaOH method by Zhou H. et al., 2006. Extracted DNA was amplified for 3 common
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beta-thalassaemia mutations in Malaysia.
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38 Results. Amplicons derived from TE buffer-based method was very faint and almost
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40 non-existent while the NaCl/NaOH/SDS method did not produce any visible amplicons.
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The amplicons using NaOH method produced visible bands that were comparable to the
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45 standard method using extraction kit.
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48 Conclusion. The NaOH method is a simple method that uses minimal equipments and
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50 reagents which makes it labour and cost-effective. This method could be adopted by
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53 poorer countries to extract DNA for beta-thalassaemia mutation characterization.
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International Journal of Laboratory Hematology

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5 Keywords: beta-thalassaemia, dried blood spots, DNA extraction, non-commercial,
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7 assessment
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8 3.0 Introduction
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11 Collecting dried blood samples for downstream disease identification was first
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13 introduced by Guthrie and Susi in 1963 as an alternative blood collection method to
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16 venipuncture (Guthrie & Susi, 1963). This method has been found to be particularly
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18 useful when venipuncture, transportation and storage conditions were not favourable.
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For example, finger pricks and heel pricks are less invasive and less traumatic compared
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23 to venipuncture for young patients or neonates. The volume collected is minimal and
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25 do not require a highly trained staff for the procedure and this is advantageous when
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28 experienced human resources are limited. DBS samples do not require much space and
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30 can be stored at room temperature - important factors to be considered when samples

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32 had to be collected from areas like the interior rural areas of Sabah and Sarawak in
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35 Malaysia which are accessible only by helicopters or small boats. The costs involved in
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37 obtaining DBS samples are relatively low as this collection method does not require
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39 vacutainers, butterfly needles or syringes (Knudsen et al., 1993; Lakshmy R., 2008).
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43 Studies have shown that the quality of DBS-extracted DNA was not greatly
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45 compromised and the results were reproducible even when samples have been stored for
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47 periods up to 5 years at room temperature or when subjected to heat or humidity (Cassol
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50 et al., 1992; Zhou, Hickford & Fang, 2006). The drying process has been shown to
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52 reduce the risks of infections as most viruses lose their infectivity when dried and DBS
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sample collection is more convenient for mass screening purposes (Parker and Cubitt,
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57 1999; Bhatti et al., 2009).
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5 The use of filter papers to collect samples is not limited only to blood samples but can
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7 be used to collect plasma samples and proteins too (Ayele W et al., 2007). Dried blood
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9 spots have been used to screen for many diseases including haemoglobinopathies,
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12 phenylketonuria, malaria, cystic fibrosis, congenital hypothyroidism, dengue and human
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14 immunodeficiency virus-1 (Chen et al., 1993; Ainoon et al., 1995; Bhardwaj, Zhang &
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16 McCabe, 2003; Bereczky et al., 2005; Maeno et al., 2008; Raskin et al., 1992; Prado et
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19 al., 2005; Cassol et al., 1992)
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22 The distribution of β-thalassaemia is generally coincides with the malarious regions. It
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24 is prevalent in the Mediterranean, parts of North and West Africa, through the Middle
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27 East and Indian subcontinent to South East Asia including Yugoslavia, Romania,
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29 southern parts of USSR and China. Coinciding with the migration patterns, β-
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thalassaemia is now distributed to North America and Europe (Flint et al., 1998).
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34 Newborn screening for β-thalassaemia is essential especially for sickle cell anaemia.
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36 Upon diagnosis of sickle cell disease, affected infants may receive penicillin
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prophylaxis which would reduce the incidence of infection by 85%. Affected infants are
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41 now recommended to receive penicillin prophylaxis prior to 4 months of age (Bhardwaj
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43 et al., 2003; Wethers et al., 1987).
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Even though the prevalence of β-thalassaemia ranges between 2% to 30% generally,
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49 many of these affected countries are poor and the use commercially available DNA
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51 extraction kits may be prohibitive due to the costs involving these kits. Not only that,
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54 usually these commercial kits require a centrifuge to be used and again the capability to
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56 acquire such equipments may not be an option. Protocols that are easy to optimize and
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58 does not require costly reagents and materials are important to increase the level of
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diagnosis for β-thalassaemia in these poor countries.

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5 In this paper, we have assessed 3 different non-commercial DNA extraction methods
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7 from dried blood spots which use minimal reagents and materials.
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13 Methodology
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17 Subjects. Subjects were suspected β-thalassaemia individuals from Hospital Ampang
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19 Thalassaemia Clinic. The study was approved by the Medical Research and Ethics
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21 Committee, Ministry of Health Malaysia (KMM/NIHSEC/08/0804/P09-341), and

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24 Medical Research Ethics Committee, Faculty of Medicine and Health Sciences,
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26 Universiti Putra Malaysia (UPM/FPSK/PADS/T7-
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MJKEtikaPer/F01(LECT_JUN(08)10). Informed consent was given by the subjects
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31 prior to blood collection and all data were anonymised with numerical identification
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33 throughout the study.

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36 Sample preparation. Peripheral blood samples were spotted on Whatman 903®
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39 Specimen collection paper before the remaining amount was stored in BD Vacutainer®
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41 spray-dried K2EDTA tubes. The blood spots were left at room temperature to
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completely dry before storage in Glassine envelopes. The dried blood spots were kept in
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46 room temperature and away from direct heat or sunlight while the EDTA-peripheral
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48 blood was kept at 4ºC before DNA extraction.
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DNA extraction.
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55 Tris-EDTA (TE) buffer-based extraction. This rapid detection method was introduced
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57 by Bereckzy et al. (2005). Briefly, a 3 mm-sized disc was punched out using a metal
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59 hole puncher from a completely dried blood spot. The hole-puncher was sterilized using
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5 70% ethanol and dried between each sample to avoid contamination. The disc was then
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7 placed in a 0.5 ml microcentrifuge tube an added with 65 µl TE buffer. The tube was
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then incubated at 50ºC for 15 minutes. Intermittently, the disc was pressed gently at the
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12 bottom of the tube several times with a pipette tip. After the incubation, the tube was
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14 heated to 97ºC for 15 minutes to elute the DNA. A quick spin was performed and the
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extract was stored at 4ºC before ARMS PCR.
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20 Sodium Hydroxide (NaOH)/Sodium Chloride (NaCl)/Sodium dodecyl sulphate (SDS)
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22 solution method. This method was described by Huang et al. (1990). Briefly, a 3 mm-
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sized disc was punched out and placed into a 0.5 ml microcentrifuge tube. The disc was
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27 washed with 100 µl saline solution and centrifuged for 1 minute at 3000 rpm. The
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supernatant was discarded and the washing step was repeated. After discarding the
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32 supernatant, 100 µl of pre-prepared 0.1 N NaOH/0.1 M NaCl/5% SDS solution was
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34 added and the disc was incubated either overnight at room temperature or for 1 hour at
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37 37ºC. Then 10 µl of the supernatant was used for ARMS PCR.
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40 Sodium Hydroxide (NaOH) method. This method was published by Zhou et al. (2006),
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42 which they have been using for genotyping blood samples from sheep, goats, and cattle
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45 since 2003. This method is a simple 2-step technique. Briefly, a 1.5 mm disc was
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47 punched out. The disc was then placed in a 0.5 ml microcentrifuge tube and 200 µl
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50 NaOH solution was added prior to incubation at room temperature for 30 minutes. The
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52 tube was inverted occasionally. After incubation, the solution was discarded and 200 µl
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Tris-EDTA (TE) buffer, pH 8.0, was added prior to 2 minutes incubation with
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5 overnight at room temperature. Once the disc was dried, it was either used immediately
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QIAamp DNA Midi Kit method. This commercially produced kit was used as a control
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13 to extract DNA from EDTA peripheral blood samples. DNA was extracted according to
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15 manufacturer’s protocol.
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19 DNA amplification. ARMS PCR was used to diagnose and confirm the β-thalassaemia
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21 mutations of each samples using DNA template from each extraction method. Primer
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sequences and PCR protocol were modified from Old et al., 1990. The 3 mutations that
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26 were tested were CD 26 (Hb E), IVS I-5 (G-C) and IVS I-1 (G-T), which were the most
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28 common β-thalassaemia mutations found in the Malays of Malaysia (George et al.,
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31 2001). Beta actin was used as the internal control.
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34 Results
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38 Tris-EDTA (TE) buffer-based extraction. The PCR results were very faint compared to
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amplified despite several attempts of optimization (Figure 1).
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46 Sodium Hydroxide (NaOH)/Sodium Chloride (NaCl)/Sodium dodecyl sulphate (SDS)
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48 solution method. The amplicons looked degraded and did not amplify (Figure 1).
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52 Sodium Hydroxide (NaOH) method. Initially, we could not amplify the internal control
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54 when we used 3 mm-sized disc for amplification. However, after reducing the disc to
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5 52 out of 71 beta-thalassaemia individuals were successfully screened for the 3
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7 mutations in our study (Figure 1 and 2). From the pool, there were 44 Hb E alleles, 22
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9 IVS I-5 alleles and 9 IVS 1-1 alleles.
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13 Discussion
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The introduction of dried blood spots as a means of blood sample collection has its boon
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19 and banes. During the period in which the study was conducted, several significant
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21 advantages were seen. The first of these was the general response of patients or
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respondents towards dried blood spots. They were more co-operative or willing to
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26 volunteer in this study when filter paper was used instead of EDTA vacutainers for
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28 sample collection and storage although some were wary of the effectiveness of the
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31 method since the amount of blood collected seemed minimal.
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34 Another advantage would be the ease of storage, transportation and handling. DBS took
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36 up little storage space and posed no risk of contamination during handling or transport.
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39 The DNA was stable although stored in room temperature and for long periods (nearly 4
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41 months for our study) and have been proven to be able to last up to 5 years (Zhou et al.,
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43 2006). In terms of handling, there were less risks of exposure to liquid blood and
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46 contamination which could be hazardous to the staff involved if proper care was not
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48 taken.
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51 One of the disadvantages of DBS was the amount of DNA could not be quantified
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54 unlike DNA extracted from liquid blood. Thus the amount of DNA per reaction could
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56 not be standardized. Older spots have been observed to contain more debris compared to
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58 newer spots. The extent of interference from this debris on PCR could not be
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5 determined. Further tests needs to be carried out to eliminate the possibility of
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7 inaccuracy due to presence of excessive debris.
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10 In assessing the 3 non-commercial DNA extraction methods from DBS, DNA
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quantification using the supernatant from the TE buffer-based method showed that there
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15 was DNA in the supernatant but subsequent PCR gave very faint bands and almost non-
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17 existent. The inability to amplify could be due to the high temperatures used for heating
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20 which could have degraded most of the DNA, leaving insufficient amounts for
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22 successful PCR especially for older spots. The NaOH/NaCl/SDS solution gave the least
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24 successful amplifications which could be attributed to the lack of information regarding
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27 the extraction method. Optimizations on different temperatures and time periods were
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32 From out study, the 2-step NaOH method was the most successful, simple and required
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35 the least reagents and materials. We were able to screen all our subjects with this
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37 method and the results were comparable to the results from conventional kit-extracted
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39 DNA. Initially when we used 3 mm-sized disc, the internal control could not be
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42 amplified but once we replaced it with a smaller disc, the internal control was amplified.
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44 This large size of the initial disc could be a hindrance for an efficient amplification. The
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only disadvantage we find with this method was the amount of time needed to allow for
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49 the spot to dry after extraction was much longer compared to a kit method. This method
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51 was proven to work with blood samples collected on domestic kitchen paper towels and
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54 other absorbable papers (Zhou et al., 2006). For countries where commercial extraction
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5 4. Acknowledgements
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8 This work was supported by the Fundamental Research Grant Scheme, Ministry of
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10 Higher Education, Malaysia (03-1-07-324FR) to M.I. Lai. Authors would like to thank
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the Malaysian Ministry of Health for their great support in making this study a success.
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5 23. Zhou H., Hickford J.G.H. & Fang Q. (2006) A two-step procedure for extracting
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9 reaction amplification. Analytical Biochemistry 354, 159-161.
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 International Journal of Laboratory Hematology Page 16 of 17

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30 Figure 1. ARMS PCR of Codon 26 (G>A) using DNA extracted from A) NaOH/NaCl/SDS solution, and
31 B) TE buffer-based method (Lane 1); DNA extraction kit method (Lanes 2, 3 and 4) and NaOH
32 method (Lanes 5 and 6). L denotes the DNA ladder; B denotes the blank; WT denotes the wild type
allele for Codon 26 (G); and M denotes the mutant allele for Codon 26 (A).
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33 333x226mm (72 x 72 DPI)

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Page 17 of 17 International Journal of Laboratory Hematology

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31 Figure 2. ARMS PCR of Codon 26 (G>A) (Lanes 1 – 4); IVS 1-5 (G>C) (Lanes 5 – 8) and IVS 1-1
32 (G>T) (Lanes 9 – 12) using DNA extracted from DNA extraction kit method and NaOH method. L
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33 denotes the DNA ladder; B denotes the blank; WT denotes the wild type allele; and M denotes the
34 mutant allele. Lanes 1-4 was from a sample with homozygous Hb E mutation; Lanes 5 – 8 was from
a sample with homozygous IVS 1-5 mutation; and Lanes 9 – 12 was from a sample with
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homozygous IVS 1-1 mutation. Lane 1: Kit extraction WT allele; Lane 2: Kit extraction mutant
36 allele; Lane 3: NaOH method WT allele; Lane 4: NaOH method mutant allele. Lane 5: Kit extraction
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37 WT allele; Lane 6: Kit extraction mutant allele; Lane 7: NaOH method WT allele; Lane 8: NaOH
38 method mutant allele. Lane 9: Kit extraction WT allele; Lane 10: Kit extraction mutant allele; Lane
39 11: NaOH method WT allele; Lane 12: NaOH method mutant allele.
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