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Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 24-28
agarose gel. Sigma Gel Loading Solution Type I was control samples such as Hb AS and AC. The 250-350
used as the loading buffer. It contains 0.25 % (w/v) V was applied for 20 minutes or until visible and clear
Bromophenol blue, 0.25 % (w/v) Xylene cyanole FF separation was obtained [10].
and 40 % (w/v) sucrose in water.
Statistical Analysis: Paired ‘t’ test was used for the
The size of DNA product (203 bp for Hb A and S; analysis of difference between the results. P < 0.01
216bp for Hb C) were read against the 100 bp DNA indicates significance
ladder and genotype results were obtained.
RESULTS AND DISCUSSION:
Cellulose acetate paper electrophoresis: Lysate of
Visible bands were seen at 203 bp regions for both
each sample was made by washing the sample four
HbA and HbS alleles and at 216 bp regions for HbC
times in saline, and haemolysed in carbon
alleles and the hemoglobin phenotypes obtained as
tetrachloride. The haemolysate of each sample was
shown in Figure 1.
loaded on the cellulose acetate paper along with
203 bp
500 bp
100
bp
1A 1S 2A 2S 3A 3S 4A 4S 5A 5S 6A 6S 7A 7S SM
500 bp
216 bp
100 bp
1A 2A 3A 4A 5A NC SM 1C 2C 3C 4C 5 NC SM
Lanes 1,2,3,4,5 are for HbA on the left side of size marker while Lanes 1,2,3,4 on the right side of SM are identified as
th
HbC. There is no amplification on the 5 lane. NC; negative control
SM –size marker.
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Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 24-28
Table 1: Comparison of AS - PCR and blindly run conventional cellulose acetate paper hemoglobin electrophoresis
on adult control samples
Hb genotype AS-PCR Acetate paper Students t-test P- value
electrophoresis
AC 09 9 >0.01
AA 53 53 >0.01
SC 05 5 >0.01
SS 08 8 >0.01
AS 25 25 >0.01
Values are number of Hb genotypes per method; n = 100.
Results also revealed that hemoglobins A, S and C new in the country could be applicable to pre-natal
identified from adult blood samples using allele- diagnosis of sickle cell anaemia to assist in early
specific PCR correlated well with those obtained detection and onset of interventory measures.
using conventional hemoglobin electrophoresis. However, the prospect of abortion as a solution to
There was no statistical difference (t- test; p > 0.01) homozygous ‘S’ detection, remains a strong issue of
between the two methods (Table 1). The study thus religious and ethical concern in Nigeria.
demonstrates the accuracy and precision of allele-
ACKNOWLEDGEMENT: The molecular biology
specific PCR for the diagnosis of sickle cell anaemia
works were all carried out in the Molecular Biology
and other forms of hemoglobinopathies in the study
Laboratory, Department of Biomedical Sciences,
population. The method is convenient and also safe
Ladoke Akintola University of Technology, Mercyland
since the result is obtained without the use of
Campus, Osogbo. We would like to thank the Ex-Vice
radioactivity. AS-PCR technique could therefore be
Chancellor (Professor B.B.A. Adeleke) and
conveniently applicable to prenatal diagnosis of
Management of Ladoke Akintola University of
hemoglobinopathy and screening of neonates since
Technology for the establishment of the Molecular
patients’ or clients’ DNA is required for analysis
Biology Laboratory. The authors are grateful to the
unlike the conventional cellulose acetate paper
International Society for Laboratory Hematology
electrophoresis method that requires erythrocytes’
(ISLH) for the Berend Houwen travel award granted
hemoglobin.
this work at the ISLH 2011 conference in New
Following successful determination of accuracy and Orleans, USA. We also acknowledge Mr Oyenike for
precision of the AS-PCR method in identification of his technical assistance.
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