International Hemato Journal

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An assessment of 3 non-commercial DNA extractionmethods from dried blood spots for beta-thalassaemia

mutation identification.

Journal: International Journal of Laboratory Hematology

Manuscript ID: IJLH-11-10-0260

Manuscript Type: Technical Report

Date Submitted by theAuthor: 15-Nov-2010

Complete List of Authors: Lai, Mei I; Faculty of Medicine and Health Science, Department of PathologyKarthipan, Sharon; Universiti Putra Malaysia, Department of PathologyGeorge, Elizabeth; Universiti Putra Malaysia, Department of PathologySathar, Jameela; Hospital Ampang, Department of HaematologyLim, Wai Feng; Universiti Putra Malaysia, Department of PathologyTeh, Lai Kuan; Universiti Putra Malaysia, Department of Pathology

Lee, Tze Yan; Universiti Putra Malaysia, Department of PathologyChin, Voon Kin; Universiti Putra Malaysia, Department of Pathology

Keywords: beta-thalassaemia, dried blood spots, DNA extraction, non-commercial, assessment

International Journal of Laboratory Hematology




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1. Title Page

An assessment of 3 non-commercial DNA extraction methods from dried blood

spots for beta-thalassaemia mutation identification.

S.N. Karthipan 1, E. George 1, S. Jameela 2, W.F. Lim 1, L.K. Teh 1, T.Y. Lee 1, V.K. Chin 1,

M.I. Lai 1

1Department of Pathology, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia;2Department of Hematology, Ampang Hospital, 68000 Ampang, Kuala Lumpur,

Malaysia.

Correspondence: Mei I Lai, Department of Pathology, Faculty of Medicine and Health

Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia. Tel: +603-8947

2494. Fax: +603-8941 2787. Email: [email protected]

Short running title: Non-commercial DNA extraction from dried blood spots

e 1 of 17 International Journal of Laboratory Hematology

mailto:[email protected]






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2. Abstract

Introduction. Dried blood spots (DBS) are currently the recommended sample

collection method for newborn screening programs in America. Early diagnosis of beta-

thalassaemia screening is essential as it provides an added advantage especially in sickle

cell disease. Beta-thalassaemia frequency is high in many poor countries and the cost of

using commercial DNA extraction kits can be prohibitive. Our study assessed 3

methods which uses minimal reagents and materials to extract DNA from dried blood

spots for beta-thalassaemia identification.

Methods. The methods assessed in this study are Tris-EDTA (TE) buffer-based method

by Bereczky S. et al ., 2005, NaCL/NaOH/SDS method by Huang S. et al. , 1990 and

NaOH method by Zhou H. et al. , 2006. Extracted DNA was amplified for 3 common

beta-thalassaemia mutations in Malaysia.

Results. Amplicons derived from TE buffer-based method was very faint and almost

non-existent while the NaCl/NaOH/SDS method did not produce any visible amplicons.

The amplicons using NaOH method produced visible bands that were comparable to the

standard method using extraction kit.

Conclusion. The NaOH method is a simple method that uses minimal equipments and

reagents which makes it labour and cost-effective. This method could be adopted by

poorer countries to extract DNA for beta-thalassaemia mutation characterization.

Page 2 International Journal of Laboratory Hematology




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Keywords: beta-thalassaemia, dried blood spots, DNA extraction, non-commercial,

assessment





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3.0 Introduction

Collecting dried blood samples for downstream disease identification was first

introduced by Guthrie and Susi in 1963 as an alternative blood collection method to

venipuncture (Guthrie & Susi, 1963). This method has been found to be particularly

useful when venipuncture, transportation and storage conditions were not favourable.

For example, finger pricks and heel pricks are less invasive and less traumatic compared

to venipuncture for young patients or neonates. The volume collected is minimal and

do not require a highly trained staff for the procedure and this is advantageous when

experienced human resources are limited. DBS samples do not require much space and

can be stored at room temperature - important factors to be considered when samples

had to be collected from areas like the interior rural areas of Sabah and Sarawak in

Malaysia which are accessible only by helicopters or small boats. The costs involved in

obtaining DBS samples are relatively low as this collection method does not require

vacutainers, butterfly needles or syringes (Knudsen et al., 1993; Lakshmy R., 2008).

Studies have shown that the quality of DBS-extracted DNA was not greatly

compromised and the results were reproducible even when samples have been stored for

periods up to 5 years at room temperature or when subjected to heat or humidity (Cassol

et al. , 1992; Zhou, Hickford & Fang, 2006). The drying process has been shown to

reduce the risks of infections as most viruses lose their infectivity when dried and DBS

sample collection is more convenient for mass screening purposes (Parker and Cubitt,

1999; Bhatti et al., 2009).





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The use of filter papers to collect samples is not limited only to blood samples but can

be used to collect plasma samples and proteins too (Ayele W et al. , 2007). Dried blood

spots have been used to screen for many diseases including haemoglobinopathies,

phenylketonuria, malaria, cystic fibrosis, congenital hypothyroidism, dengue and human

immunodeficiency virus-1 (Chen et al., 1993; Ainoon et al., 1995; Bhardwaj, Zhang &

McCabe, 2003; Bereczky et al. , 2005; Maeno et al., 2008; Raskin et al., 1992; Prado et

al., 2005; Cassol et al., 1992)

The distribution of β-thalassaemia is generally coincides with the malarious regions. It

is prevalent in the Mediterranean, parts of North and West Africa, through the Middle

East and Indian subcontinent to South East Asia including Yugoslavia, Romania,

southern parts of USSR and China. Coinciding with the migration patterns, β-

thalassaemia is now distributed to North America and Europe (Flint et al., 1998).

Newborn screening for β-thalassaemia is essential especially for sickle cell anaemia.

Upon diagnosis of sickle cell disease, affected infants may receive penicillin

prophylaxis which would reduce the incidence of infection by 85%. Affected infants are

now recommended to receive penicillin prophylaxis prior to 4 months of age (Bhardwaj

et al., 2003; Wethers et al., 1987).

Even though the prevalence of β-thalassaemia ranges between 2% to 30% generally,

many of these affected countries are poor and the use commercially available DNA

extraction kits may be prohibitive due to the costs involving these kits. Not only that,

usually these commercial kits require a centrifuge to be used and again the capability to

acquire such equipments may not be an option. Protocols that are easy to optimize and

does not require costly reagents and materials are important to increase the level of

diagnosis for β-thalassaemia in these poor countries.





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In this paper, we have assessed 3 different non-commercial DNA extraction methods

from dried blood spots which use minimal reagents and materials.

Methodology

Subjects. Subjects were suspected β-thalassaemia individuals from Hospital Ampang

Thalassaemia Clinic. The study was approved by the Medical Research and Ethics

Committee, Ministry of Health Malaysia (KMM/NIHSEC/08/0804/P09-341), and

Medical Research Ethics Committee, Faculty of Medicine and Health Sciences,

Universiti Putra Malaysia (UPM/FPSK/PADS/T7-

MJKEtikaPer/F01(LECT_JUN(08)10). Informed consent was given by the subjects

prior to blood collection and all data were anonymised with numerical identification

throughout the study.

Sample preparation. Peripheral blood samples were spotted on Whatman 903 ®

Specimen collection paper before the remaining amount was stored in BD Vacutainer ®

spray-dried K 2EDTA tubes. The blood spots were left at room temperature to

completely dry before storage in Glassine envelopes. The dried blood spots were kept in

room temperature and away from direct heat or sunlight while the EDTA-peripheral

blood was kept at 4ºC before DNA extraction.

DNA extraction.

Tris-EDTA (TE) buffer-based extraction . This rapid detection method was introduced

by Bereckzy et al. (2005). Briefly, a 3 mm-sized disc was punched out using a metal

hole puncher from a completely dried blood spot. The hole-puncher was sterilized using





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70% ethanol and dried between each sample to avoid contamination. The disc was then

placed in a 0.5 ml microcentrifuge tube an added with 65 µ l TE buffer. The tube was

then incubated at 50ºC for 15 minutes. Intermittently, the disc was pressed gently at the

bottom of the tube several times with a pipette tip. After the incubation, the tube was

heated to 97ºC for 15 minutes to elute the DNA. A quick spin was performed and the

extract was stored at 4ºC before ARMS PCR.

Sodium Hydroxide (NaOH)/Sodium Chloride (NaCl)/Sodium dodecyl sulphate (SDS)

solution method. This method was described by Huang et al. (1990). Briefly, a 3 mm-

sized disc was punched out and placed into a 0.5 ml microcentrifuge tube. The disc was

washed with 100 µ l saline solution and centrifuged for 1 minute at 3000 rpm. The

supernatant was discarded and the washing step was repeated. After discarding the

supernatant, 100 µ l of pre-prepared 0.1 N NaOH/0.1 M NaCl/5% SDS solution was

added and the disc was incubated either overnight at room temperature or for 1 hour at

37ºC. Then 10 µ l of the supernatant was used for ARMS PCR.

Sodium Hydroxide (NaOH) method. This method was published by Zhou et al. (2006),

which they have been using for genotyping blood samples from sheep, goats, and cattle

since 2003. This method is a simple 2-step technique. Briefly, a 1.5 mm disc was

punched out. The disc was then placed in a 0.5 ml microcentrifuge tube and 200 µ l

NaOH solution was added prior to incubation at room temperature for 30 minutes. The

tube was inverted occasionally. After incubation, the solution was discarded and 200 µ l

Tris-EDTA (TE) buffer, pH 8.0, was added prior to 2 minutes incubation with

occasional inversion. The solution was then discarded and the disc was left to dry





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overnight at room temperature. Once the disc was dried, it was either used immediately

for ARMS PCR or stored in 4ºC.

QIAamp DNA Midi Kit method. This commercially produced kit was used as a control

to extract DNA from EDTA peripheral blood samples. DNA was extracted according to

manufacturer’s protocol.

DNA amplification. ARMS PCR was used to diagnose and confirm the β-thalassaemia

mutations of each samples using DNA template from each extraction method. Primer

sequences and PCR protocol were modified from Old et al. , 1990. The 3 mutations that

were tested were CD 26 (Hb E), IVS I-5 (G-C) and IVS I-1 (G-T), which were the most

common β-thalassaemia mutations found in the Malays of Malaysia (George et al.,

2001). Beta actin was used as the internal control.

Results

Tris-EDTA (TE) buffer-based extraction . The PCR results were very faint compared to

the commercially extracted DNA and NaOH method. Internal control could not be

amplified despite several attempts of optimization (Figure 1).

Sodium Hydroxide (NaOH)/Sodium Chloride (NaCl)/Sodium dodecyl sulphate (SDS)

solution method. The amplicons looked degraded and did not amplify (Figure 1).

Sodium Hydroxide (NaOH) method. Initially, we could not amplify the internal control

when we used 3 mm-sized disc for amplification. However, after reducing the disc to

1.5 mm, the results were similar to the results using extraction kits.





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52 out of 71 beta-thalassaemia individuals were successfully screened for the 3

mutations in our study (Figure 1 and 2). From the pool, there were 44 Hb E alleles, 22

IVS I-5 alleles and 9 IVS 1-1 alleles.

Discussion

The introduction of dried blood spots as a means of blood sample collection has its boon

and banes. During the period in which the study was conducted, several significant

advantages were seen. The first of these was the general response of patients or

respondents towards dried blood spots. They were more co-operative or willing to

volunteer in this study when filter paper was used instead of EDTA vacutainers for

sample collection and storage although some were wary of the effectiveness of the

method since the amount of blood collected seemed minimal.

Another advantage would be the ease of storage, transportation and handling. DBS took

up little storage space and posed no risk of contamination during handling or transport.

The DNA was stable although stored in room temperature and for long periods (nearly 4

months for our study) and have been proven to be able to last up to 5 years (Zhou et al .,

2006). In terms of handling, there were less risks of exposure to liquid blood and

contamination which could be hazardous to the staff involved if proper care was not

taken.

One of the disadvantages of DBS was the amount of DNA could not be quantified

unlike DNA extracted from liquid blood. Thus the amount of DNA per reaction could

not be standardized. Older spots have been observed to contain more debris compared to

newer spots. The extent of interference from this debris on PCR could not be





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determined. Further tests needs to be carried out to eliminate the possibility of

inaccuracy due to presence of excessive debris.

In assessing the 3 non-commercial DNA extraction methods from DBS, DNA

quantification using the supernatant from the TE buffer-based method showed that there

was DNA in the supernatant but subsequent PCR gave very faint bands and almost non-

existent. The inability to amplify could be due to the high temperatures used for heating

which could have degraded most of the DNA, leaving insufficient amounts for

successful PCR especially for older spots. The NaOH/NaCl/SDS solution gave the least

successful amplifications which could be attributed to the lack of information regarding

the extraction method. Optimizations on different temperatures and time periods were

carried out but the results were unsuccessful.

From out study, the 2-step NaOH method was the most successful, simple and required

the least reagents and materials. We were able to screen all our subjects with this

method and the results were comparable to the results from conventional kit-extracted

DNA. Initially when we used 3 mm-sized disc, the internal control could not be

amplified but once we replaced it with a smaller disc, the internal control was amplified.

This large size of the initial disc could be a hindrance for an efficient amplification. The

only disadvantage we find with this method was the amount of time needed to allow for

the spot to dry after extraction was much longer compared to a kit method. This method

was proven to work with blood samples collected on domestic kitchen paper towels and

other absorbable papers (Zhou et al., 2006). For countries where commercial extraction

kits are not an option, this method could be used to lower the cost of mass screening and

mutation identification.

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4. Acknowledgements

This work was supported by the Fundamental Research Grant Scheme, Ministry of

Higher Education, Malaysia (03-1-07-324FR) to M.I. Lai. Authors would like to thank

the Malaysian Ministry of Health for their great support in making this study a success.





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Figure 1. ARMS PCR of Codon 26 (G>A) using DNA extracted from A) NaOH/NaCl/SDS solution, andB) TE buffer-based method (Lane 1); DNA extraction kit method (Lanes 2, 3 and 4) and NaOH

method (Lanes 5 and 6). L denotes the DNA ladder; B denotes the blank; WT denotes the wild typeallele for Codon 26 (G); and M denotes the mutant allele for Codon 26 (A).

333x226mm (72 x 72 DPI)

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Figure 2. ARMS PCR of Codon 26 (G>A) (Lanes 1 – 4); IVS 1-5 (G>C) (Lanes 5 – 8) and IVS 1-1(G>T) (Lanes 9 – 12) using DNA extracted from DNA extraction kit method and NaOH method. L

denotes the DNA ladder; B denotes the blank; WT denotes the wild type allele; and M denotes themutant 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 withhomozygous IVS 1-1 mutation. Lane 1: Kit extraction WT allele; Lane 2: Kit extraction mutant

allele; Lane 3: NaOH method WT allele; Lane 4: NaOH method mutant allele. Lane 5: Kit extractionWT allele; Lane 6: Kit extraction mutant allele; Lane 7: NaOH method WT allele; Lane 8: NaOH

method mutant allele. Lane 9: Kit extraction WT allele; Lane 10: Kit extraction mutant allele; Lane11: NaOH method WT allele; Lane 12: NaOH method mutant allele.

333x226mm (72 x 72 DPI)


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