Clinical and Experimental Medical Sciences, Vol. 4, 2016, no. 1, 25 - 32
HIKARI Ltd, www.m-hikari.com
http://dx.doi.org/10.12988/cems.2016.654
Donors Left Over Blood as a Source of
Genomic DNA for Genetic Studies
Siti Nazihahasma Hassan1*, Wan Suriana Wan Ab Rahman1,
Wan Muhamad Amir W Ahmad1, Suharni Mohamad1,
Rosline Hassan2 and Selamah Ghazali2
1
2
School of Dental Sciences, Universiti Sains Malaysia (USM)
16150 Kubang Kerian, Kelantan, Malaysia
School of Medical Sciences, Universiti Sains Malaysia (USM)
16150 Kubang Kerian, Kelantan, Malaysia
Copyright © 2016 Siti Nazihahasma Hassan et al. This article is distributed under the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Abstract
Screening a mutation, polymorphism or genetic studies in a population involving a
large sample size and costing. Left over blood from donors can be a genomic DNA
(gDNA) source without collection cost. Thus, this article report the mean
concentration, yield and purity of gDNA extracted from donors left over blood and
the association of gDNA yield with age, gender and blood type of donors. Sample
size was calculated using PS software (Dupont & Plumber, 1997). Eighty donors
N  80  were taken randomly from each blood type for this analysis which
consist of nBlood A  20, nBlood B  20, nBlood AB  20 and nBlood O  20 . The bloods
were subjected for the genomic DNA extraction using NucleoSpin® Blood L
(Macherey-Nagel, Duren, Germany). Total concentration and purity of extracted
gDNA was measured by NanoDropND-1000 spectrophotometer (Nano-Drop
Technologies, USA). Mean for gDNA concentration (183.10 ± 47.43), yield (36.62
± 9.49) and purity (A260/280 = 1.90 ± 0.02 O.D; A260/230 = 2.54 ± 0.20 O.D)
respectively showed good quantity and quality of gDNA. There is no correlation
between age, gender and blood type with DNA yield. Donors left over blood can
be considered a reliable gDNA resource. A DNA biobank could be established from
the donors left over blood suitable either for preliminary as well as for complete
genetic epidemiological study.
Keywords: Donors blood, left over blood, genomic DNA
26
Siti Nazihahasma Hassan et al.
Introduction
Deoxyribonucleic acid (DNA) is the genetic material located in the cell nucleus of
majority of all well-defined organisms. DNA is the largest human chromosome [1].
DNA isolation from bloods is the first and the foremost step involved in molecular
biology as its serve high yield and quality [2]. Blood is the main source of DNA for
genetic studies in humans [3-4]. DNA typing is a powerful, convenient tool used
for various applications, including DNA profiling [5], deoxyribozyme [6],
bioinformatics [7], DNA nanotechnology [8], history and anthropology [9], and
information storage [10].
Blood bank laboratory often possess donors left over blood that may represent
an excellent and economic source of gDNA. Current efforts in molecular medicine
and genetic epidemiology increasingly rely on partnerships with available DNA
biobank as the study base. In this respect, the recovery and amplification of nucleic
acids from different sources such dried blood spots, frozen serum or plasma are
growing fields in retrospective genetic studies [11-12]. In the present work, we
propose donors left over blood as gDNA resource without any risk of disease
transmission. This study also assesses the correlation of age, gender and blood type
of donors with gDNA yield. However, the main advantage in using donors left over
blood is the elimination of expensive and time-consuming sample collection and
classification of the subjects.
Materials and Methods
Eighty donors  N  80  were randomly selected for this analysis which consist of
nBlood A  20, nBlood B  20, nBlood AB  20 and nBlood O  20 . An approximately two
ml of blood in ethylenedinitrilotetraacetic acid (EDTA) tubes (BD Vacutainer®,
USA) were used for gDNA extraction using NucleoSpin® Blood L (MachereyNagel, Duren, Germany). The bloods were left over from routine hematology tests
and have been kept at room temperature for 24 hour before the gDNA being
extracted. All the procedures were in accordance with the ethical guideline of
Human Research Ethics Committee of USM (JEPeM).
DNA extraction
DNA was extracted according to the manufacturer’s instructions as follow: 2.0 ml
of blood was added with 150 µl of Proteinase K (Macherey-Nagel, Duren, Germany)
and 2.0 ml of buffer BQ1, followed by vigorously vortexed for 10 seconds. The
samples were incubated at 56ºC for 20 minutes and vortexed once during incubation.
The samples were let to cool down to room temperature before the addition of 2.0
ml of absolute ethanol, and later mixed by inverted the tubes for 10 times. The lysate
samples were let to cool down to room temperature before loading it into the column.
Three ml of lysate samples were loaded into the NucleoSpin®Blood L column loca-
Donors left over blood as a source of genomic DNA
27
ted in a collection tube and centrifuged for three minutes at 4,500 xg. All the
remaining lysate samples were loaded into the NucleoSpin®Blood L column and
were centrifuged for another five minutes at 4,500 xg. The columns were then
inserted into a new 15 ml collection tube. Two ml of buffer BQ2 was added to the
columns and were centrifuged for two minutes at 4,500 xg. For a second wash,
another 2.0 ml of buffer BQ2 was added to the columns and were centrifuged for
10 minutes at 4,500 xg. The columns were then inserted into a new 15 ml collection
tube and 200 µl of preheated buffer BE at 70°C was directly applied to the center
of the silica membrane-based columns. The columns were incubated for five
minutes at room temperature, followed by centrifugation for two minutes at 4,500
xg.
DNA quantification and purity
A NanoDrop ND-1000 spectrophotometer (Nano-Drop Technologies, USA) was
used to measure the total concentration and purity of extracted gDNA. Absorbance
was measured at wavelengths of 230, 260 and 280 (A230, A260 and A280, respectively)
nm. The absorbance quotients (OD260/OD230) and (OD260/OD280) provide an
estimation of DNA purity.
Statistical analysis
The data were presented as mean ± standard deviation (SD). The normally
distributed data were analyzed using parametric Independent T and Analysis of
Variance (ANOVA) tests. The associations of gDNA yield with age, gender and
blood type was assessed using Biserial correlation. The significant value was set as
P<0.05. A mentioned statistical analysis was conducted using Statistical Product
and Service Solutions (SPSS) software, version 22 and Statistical Analysis
Software (SAS) version 9.3.
Results and Discussion
Demographic profile
A total of 80 donors aged from 20 to 50 years were enrolled into this study. Most
donors were aged between 20 to 29 years with 51 (63.75%), 16 (20.00%) aged
between 30 to 39 years, 11 (13.75%) aged between 40 to 49 years and only 2
(2.50%) of the donors aged 50 years (Table 1). There was no huge gap between
male, 38 (47.50%) and female, 42 (52.50%) donors. Most of the donors in Blood
Bank of Hospital USM was Malay with 70 (87.50%), Chinese and Indian were 5
(6.25%) respectively.
28
Siti Nazihahasma Hassan et al.
Table 1. Demographic profile of donors
Profile
Demographic
Gender
Age
Blood type
Race
Category
Frequency
Percentage (%)
Male
Female
20-29 years old
30-39 years old
40-49 years old
50 years old
A
B
O
AB
Malay
Chinese
Indian
38
42
51
16
11
2
20
20
20
20
70
5
5
47.50
52.50
63.75
20.00
13.75
2.50
25.00
25.00
25.00
25.00
87.50
6.25
6.25
Concentration, yield and purity of gDNA
Mean concentration, yield and purity of gDNA extracted from donors left over
blood showed in Table 2. By measuring the 260/230 and 260/280 nm absorbance
ratios, the gDNA purity was determined. The gDNA yield was assessed for the
associations with age, gender and blood type using Biserial correlation (Table 3).
Table 2. Spectrophotometric data of the gDNA
Mean ± SD
(N=80)
DNA
concentration
(ng/ul)
DNA
Yield
(µg)
A260/A280
ratio
A260/A230
ratio
183.10±47.43
36.62±9.49
1.90±0.02
2.54±0.20
Donors left over blood as a source of genomic DNA
29
Table 3. Biserial correlation between gDNA yield with age, gender and blood type
Mean±SD
gDNA yield
r
P-value*
Gender
Male
Female
35.86 ± 10.04
37.31 ± 9.02
0.08
0.50
Age
20-29 years old
30-39 years old
40-49 years old
50 years old
37.88 ± 9.60
32.55 ± 6.23
34.61 ± 10.60
38.03 ± 11.75
-0.02
0.90
-0.11
0.34
Blood type
A
37.87 ± 11.25
B
37.05 ± 8.11
AB
35.01 ± 9.02
O
36.56 ± 9.79
*Biserial correlation
We examined a quantity and quality of gDNA extracted from donors left over
blood. Genomic DNA was quantified using spectrophotometer and the purity was
determined based on the absorbance ratio of A260/280 and A260/230. Mean for gDNA
concentration (183.10 ± 47.43) and yield (36.62 ± 9.49) showed sufficient DNA
quantity to be used for downstream amplification. Usually only 1 to 2 µl of gDNA
template with 100 ng concentration was used in the polymerase chain reaction
(PCR). Good quality of gDNA was obtained with mean ratios of A260/280 (1.90 ±
0.02 O.D) and A260/230 (2.54 ± 0.20 O.D) respectively. A ratio of 260/280 OD above
1.8 was accepted as pure DNA [13] while the ratio of 260/230 OD above 2.0 was
accepted as free from phenols, aromatic compounds, peptides and carbohydrates
contaminants [14]. We found no significant influences of demographic
characteristic on gDNA yield (Table 3). There is no significant associations among
age r  0.02; p  0.90, gender r  0.08; p  0.50  and blood type
r  0.11; p  0.34 of donors on gDNA yield. One study found a correlation
between age and gender on DNA yield [15]. While, the increasing age has led to a
significant reduction in DNA yield [16]. This decline in DNA yield may represent
the known decline in total leukocyte and lymphocyte count that occurs between
birth and adulthood. In this article, molecular test result was not included. However,
the gDNA extracted from donors left over blood was successfully used for
Miltenberger blood group typing [17]. Actually, we are working to reduce or
eliminate sample collection cost. Thus, the use of left over blood from donors was
reliable to be used for large scale epidemiological study.
30
Siti Nazihahasma Hassan et al.
Summary and Conclusion
Left over blood in EDTA tubes kept at room temperature for 24 hour were red in
color, and visual inspection of supernatant plasma did not suggest significant
hemolysis. No evidence existed of clotting in any of the tubes, even over extended
time period to five days. Genomic DNA isolated from donors left over blood
yielded sufficient DNA quantity as well as quality to be used for molecular study.
As compare to RNA, DNA is quite stable for long-term storage. A medical DNA
database and research DNA database could be established from the donors left over
blood.
Acknowledgements. The authors would like to express their gratitude to Universiti
Sains Malaysia for providing the research funding (Grant no.304/PPSG/61312118),
the support of USM fellowship, My Brain15 (Ministry of Higher Education
Malaysia) and Hematology Department staff, School of Medical Sciences, USM.
Conflict of Interest
The authors declare there is no conflict of interest regarding the publication of this
paper.
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Received: June 1, 2016; Published: July 5, 2016