ISSN (Online) 2581-9429

IJARSCT
International Journal of Advanced Research in Science,, Communication and Technology (IJARSCT)

Volume 3, Issue 2, March 2023

Impact Factor: 7.301

Prevalence of Alpha Thalassemia Gene Deletion by

PCR Technique in Baiga Tribe of Anuppur
District (M.P)
Dr. Mamta Saha
Assistant Professor, Bio-Chemistry,
Dr. Ambedkar College of
o Arts, Commerce & Science, Chandrapur, Maharashtra
Maharashtra, India
.Corresponding
Corresponding Author: E-mail: mamtasaha14@gmail.com

Abstract: Analysis of Alpha Thalassemia and several phenotypically resembling alleles at the beta globin
gene cluster such as co-inherited
inherited delta and beta thalassemia or β-β thalassemia is a critical step in genetic
counseling. In this paper we report our experience in alpha globin gene disorder using by polymerase chain
reaction
eaction and feasibility and simplification of screening for thalassemia using this type of approach
approach.

Keywords: Baiga Tribe, cbc,α- thalassemia,

thalassemia PCR

I. INTRODUCTION
Haemoglobin is a negatively charged protein at alkaline pH and migrates toward the anode (+) in an electric field.
During electrophoresis, haemoglobin variants separate at different rates due to differences in their surface electrical
charge as determined by their amino
ino acid structure (Chanarin, 1989).
District Anuppur is situated in South Eastern part of Madhya Pradesh. Anuppur district has been formed from the
district of Shahdol. The Baiga population is one of the crowd tribal population of Madhya Pradesh and Chha
Chhattisgarh.

II. MATERIALS AND METHOD

Sample Collection: Whole blood was collected in EDTA to prevent clotting. Typically 200μL of fresh blood was used
for DNA isolation. 400μL of lysis solution was taken in 1.5 mL micro centrifuge tube to it was added 200 μμL of whole
blood and mixed thoroughly. Then the tube was incubated at 65o C for 10 minutes in a water bath. After the incubation,
600 μL of chloroform was added and gently emulsified by inversion (3-5(3 5 time). The mixture was then centrifuged at
10,000 rpm for 5 minutes. The upper aqueous phase containing DNA was been transferred to a fresh tube containing
800 μL of freshly prepared precipitation solution and then mixed gently by inversions at room temperature for 11-2 min
and centrifuge at 10,000 rpm for 5 minutes.
minutes. Then the supernatant discarded and the DNA pette
pettel so obtained was
resuspended in100 μL of NaCl solution by gently vortexing. Then 300 μL of cold ethanol was added and followed the
DNA precipitate (10 min at 20o C) and spun down at 10,000 rmp for 5 minutes. The ethanol was removed and DNA
was finally dissolved in 100 μL of sterile deionizedwater, DNA concentration was measured spectrophotometrically.

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 ISSN (Online) 2581-9429
IJARSCT
International Journal of Advanced Research in Science,, Communication and Technology (IJARSCT)

Volume 3, Issue 2, March 2023

Impact Factor: 7.301

Description of the kit: The Genomic DNA Purification kit is a simple and rapid system for high quality genomic DNA
purification from various including whole blood serum, cell line, bacterial cells, plant and mammalian tissues. The kit is
based on selective detergent-mediated
mediated DNA precipitation from lysate. The entire procedure
rocedure is rapid – only 20-25 min –
with a typical yield of 2-10
10 μg genomic DNA from 0.2 mL of blood. High molecular weight genomic DNA purified
with the kit is suitable for direct use I all common molecule biology applications: PCR, Thermo Scientific Fast Digest
and conventional restriction digestion, cloning, DNA sequencing and Southern blot analysis.

Detection of –α3.7 and –α4.2 deletions:

The α1and α2 gene are embedded within two highly homologous 4-Kb 4 duplicated
ed segments on chromosome 16. The
3.7
chromosome with a hybrid α2 / α1 gene (–α ( deletion) result from the reciprocal recombination between ZZ segments
which are 3.7 Kb apart and recombination between homologous X segments (4.2 Kb apart) results in a single α1 gene,
thus deleting the entire α2 gene (-α42 deletion). Gene deletions responsible for α2 – thalassaemia are detected by Gap-
PCR using two primers complimentary to the sense and antisense strand in the DNA regions that flank the deletion,
Demonstration of presence of – α42 deletions were done by PCR (Baysal&Huisman, 1994).
Location of primers for the amplification of DNA fragments from chromosomes
with –α3.7 and –α4.2 deletions

A3.7kbB

C α2
Ψα1 α1
F 4.2kb
-α3.7 deletion
G -α4.2 deletion
DE
I II III

X2 Y2 Z2 X1 Y1 Z1

Primer used for the demonstration of – α3.7and – α4.2deletions are shown in Table
Table-1
Table 1: Primers used in detection of α-thalassaemia
A 5’ CTTTCCCTACCCAGAGCCAGGTT-‘
5’-
B 5’ CCCATGCTGGCACGTTTCTGAGG-3 ‘
5’-
C 5’
5’-CCATTGTTGGCACATTCCGGGACA-3’
E 5’
5’-CCCTGGGTGTCCAGGAGCAAGCC-3’

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F 5’-GGCACATTCCGGGACAGAGAGAA-3’
G 5’-CCGGTTTACCCATGTGGTGCCTC-3’

To Show the presence of the – α3.7 genotype, two separate reaction were run simultaneously for each DNA sample
(Table-2). One reaction is to amplify the abnormal chromosome carrying the deletion and the other is to amplify the
normal chromosome (Normal). One reactio0n contains the pair of primers (A+B) that amplify a segment of the
chromosome with the deletion consisting of sequence 5’ an d3’ to the deletion and the other pair of primer (A+C)
amplifies a segment of the normal gene. Since the expected abnormal fragment size for the detection of the – α3.7
deletion is the same as the normal fragment were run in separate tubes. To demonstrate the presence of the – α4.2
genotype, one reaction was run with G+E+F primer with their concentrations os 20, 15 and 6 pico moles (Per reaction)
respectively.
Table 2: Combination of primers used in PCR rection for the diagnosis of α–thalassaemia
Genotype Primers Combination Size of amplified Fragment
3.7
–α For Mutant A+B 1.8Kb
For Mutant A+C 1.8Kb
– α4.2 G+E+F 227bp (Normal)
1.8Kb(Mutant)

 2X AMPLIFICATION BUFFER: Thiry five milliliter of deoinized water was taken in a 50mL centrifuge
tube. To it was added 67mM Tris (0.812/50mL) and 16.6mM (NH4)2So4 (0.219g/50mL and dissolved. Then
pH has been adjusted to 8.8 by adding concentrated HCL (appx. 140μL). Then BSA (10mg/50mL).10mM β-
mercaptoethanol (70 μL/50mL) and 70% DMSO – (7.5 mL/50ml) have benn added. Then volume was made
upto to 50mL with sterilized deoinized water.
 IX TEB BUFFER FOR AGAROSE ELECTROPHORESIS: 10.8g of Tris base, 5.5g of Boric acid and
4mL of 0.5M EDTA was added in 100mL of solution.
 ETHIDIUM BROMIDE:10mg of ethidium bromide (Sigma, USA) was added in 1 mL of Sterile distilled
water. Then 2μl of this stock was added to 30mL of TEB buffer and used for the gel preparation.
 GEL LOADING DYE:1% Bromophenol blue solution (BBS) was prepared by adding 1 g of Bromophenol
blue (Sigma, USA) in 100mL of sterile distilled water. This stock was used to prepare working loading dye by
adding 100μ of 1% BBS and 400 μL of Glycerol in 500 μL sterile water.

PREPARATION OF REACTION MIXTURE:

For –α3.7 deletion (For 50 μL Reaction mixture )
NORMAL MUTANT
Amplification buffer 25 μL 25 μL
Mg Cl2(25mM) 4 μL 4 μL
dNTP mix (25mM) 200 μM 0.4 μL 0.4 μL
Primer A 25 Pico moles Volume adjusted accordingly Volume adjusted accordingly
Primer B 25 Pico moles Volume adjusted accordingly Volume adjusted accordingly
Primer C 25 Pico moles Volume adjusted accordingly Volume adjusted accordingly
Taq DNA polymerase 1.5 Units Volume adjusted accordingly Volume adjusted accordingly
Sterile distilled water Added to adjust the volume Added to adjust the volume up
up to 50μL to 50μL
DNA (100ng) 2μL 2μL

For – α4.2 deletion (For 50μL Reaction mixture)

Amplification buffer 25 μL
Mg Cl2(25mM) 4 μL
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 ISSN (Online) 2581-9429
IJARSCT
International Journal of Advanced Research in Science, Communication and Technology (IJARSCT)

Volume 3, Issue 2, March 2023

Impact Factor: 7.301
DNTP mix (25mM) 2004 μM 0.4μL
Primer G 20 Pico moles Volume adjusted accordingly
Primer G 15 Pico moles Volume adjusted accordingly
Primer G 6 Pico moles Volume adjusted accordingly
Tap DNA polymerase 1.5 Units Volume adjusted accordingly
Sterile distilled water Added to adjust the volume up to 50μL
DNA (100ng) 2μL

Table 3: Haematological Parameters Of Anaemic Population of District Anuppur

Group N Hb Hct TRBC MCV MCH MCHC HbF HbF2( WBC PLT
(g/dl) (%) (x106/µI) (fl) (pg) (g/dl) (%) %) (x103/µI) (x103/
µI)
Male 15 11.5 33.8 4.6 72.6 25.4 35.1 1.7 2.9 6.9 209.3
±2.0 ±4.8 ±0.9 ±6.3 ±2.1 ±0.9 ±4.2 ±0.5 ±1.8 ±54.1

Female 20 10.4 29.4 4.3 68.6 24.1 35.2 0.7 2.4 6.5 168.7
±1.2 ±2.9 ±0.5 ±7.1 ±2.9 ±1.3 ±0.3 ±0.5 ±2.0 ±41.2

Childr 6 11.2 31.2 4.8 68.2 23.3 35.9 0.8 2.7 7.6 186.5
en ±0.6 ±1.6 ±0.4 ±7.5 ±2.6 ±0.8 ±0.5 ±0.6 ±2.4 ±36.1

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 ISSN (Online) 2581-9429
IJARSCT
International Journal of Advanced Research in Science, Communication and Technology (IJARSCT)

Volume 3, Issue 2, March 2023

Impact Factor: 7.301
Table 4: Haematological Parameters Of Normal Haemoglobin Level Population of District Anuppur
Group N Hb Hct TRBC MCV MCH MCHC HbF HbF2 WBC PLT
6
(g/dl) (%) (x10 /µI) (fl) (pg) (g/dl) (%) (%) (x103/µI) (x103/µI)

Male 11 14.1 39.1 5.5 72.5 26.1 36.1 0.9 2.8 7.7 199.2
±0.5 ±1.7 ±0.6 ±7.4 ±3.1 ±1.1 ±0.4 ±0.5 ±2.9 ±62.3
Female 14 12.7 35.2 4.9 72.7 26.2 36 0.6 3.0 8.1 202.6.
±0.5 ±2.9 ±0.4 ±4.9 ±2.3 ±1.0 ±0.4 ±0.7 ±1.9 ±56.5
Children 4 12.8 35.6 4.8 74.9 26.8 35.8 0.6 2.8 10 262.2
±0.1 ±1.0 ±0.4 ±5.8 ±2.8 ±1.1 ±0.2 ±0.5 ±2.4 ±53.7

III. RESULT AND DISCUSSION

Mean haematological parameters of anaemic population in shown in Table -3Results shown that anaemic population
has lower values for MCH in all the three groups. This is the indicative of microcytosis and iron deficiency.
Identification of Iron deficiency is not done in the studied population. The mean haemoglobin level for the adult male is
11.5±2.0 g/dl. It is 10.4±1.2 g/dl for female and 11.2±0.6 g/dl for the children. Children have relatively lowindices.
The mean value of MCV is 68.2±7.5 fl in children group and it is 72.6±6.3 for adult male and (68.6±7.1) female. Lower
values are observed for MCH in all the three groups. Mean MCH value for adult male and female is 25.4±2.1 pg and
24.1±2.9 pg respectively and it is observed as 23.3±2.6 pg for children. The mean value of MCHC for the adult male is
35.1±0.9 g/dl and the mean MCHC values of 35.2±1.3 g/dl are observed in adult female group. The mean value of
MCHC for children anaemic group is 35.9±0.8 g/dl. Mean fetal haemoglobin levels are ranging from 0.7% in females
to 0.8% in children and 1.7% in males. More or less same mean levels of haemoglobin A2 (≈2.5%) were observed in all
three groups. The haematological parameters for all normal haemoglobin level individuals are given in Table – 2. The
mean haemoglobin was 14.1±0.5 g/dl and 12.7±0.5 g/dl for adult male and female group respectively and in children it
is 12.8±0.1. Mean values for MCV is 72.5±7.4 fl for adult males, 72.7±4.9 fl is for females and 74.9±5.8 fl for children.
Low mean values of MCH in all groups indicate the microcytosis. The mean value for MCHC is in normal range for all
three groups i.e. male (36.1±1.1pg), female (36. ±1.0pg) and children (35.8±1.1). The mean values for WBC is
7.7±2.9X103/μl among male, 8.1±1.9 X 103/μl for female and 10±2.4 for children. The mean HbA2 level in all male
(2.8±0.5), female (3.0±0.7) and children (2.8±0.5) is in normal range. The mean Hb F levels are observed as 0.9±0.4 in
males, 0.6±0.4 among female group and 0.6±0.2 in children.

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 ISSN (Online) 2581-9429
IJARSCT
International Journal of Advanced Research in Science, Communication and Technology (IJARSCT)

Volume 3, Issue 2, March 2023

Impact Factor: 7.301
REFERENCES
[1]. Chanarin I (1989). Laboratory Haematology: An account of Laboratory techniques. 1st edition Pub. by
Churchill Livingstone, London.
[2]. Baysal E, Huisrnan THJ (1994). Detection of common deletional a-thalassaemia-2 terminants by PCR. Am J
Haematol. 46(3): 208-213.
[3]. Agrawal MB (2005). The Burden of Haemoglobinopathies in India-Time to Wake Up? JAPI 53: 1017-1018.
[4]. Aguilar C, Vichinsky E, Neumayr L (2005). Bone and joint disease in sickle cell disease. Hematol. Oncol.
Clin North Am. 19(5):929-941.

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