MATERIALS of biochemistry Abdul Wali Khan University, Mardan (AWKUM).

MATERIALS AND METHODS

Ethical
Consideration

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The design study
was approved by ASRB, the ethical committee of department of biochemistry Abdul
Wali Khan University, Mardan (AWKUM).

Sampling
 Area

 The current research was arranged keeping in attention
to the country of Pakistan, a malaria epidemic country located within South
asia cover an area of 796,096 km² having five provinces and Federally Administrated
Tribal Area (FATA). According to 2017 sensus Pakistan population of was 207,774,520 (Khilji et al., 2017). Congested regions were focused of the south
of Khyber Pakhtunkhwa (KP) such as Kohat
region 2,545 km² with population  993,874,
and Bannu region 1,227 km² with population 1.168 million. The above regions
having warm and temperate climate creates fertile and productive environment
for mosquitoes.

 Blood
sampling

 Blood was collected from different falciparum
positive patients in the selected regions of south of KP for conducting
research work. The present study was organized from July, 2017 to feb, 2018 in Molecular
laboratory of Biochemistry of the department of Abdul Wali Khan University,
Mardan. A number of P.falciparum positive
samples done based on assistance and accessibility of the donor’s personnel
in  private and hospital’s lab during the
study.

Malaria
Prevalence and Data collection

Data was collected
from different regional hospitals of south of KP provides full assistance in
data collection, and malaria prevalenc during visit of District Headquarter
Hospitals (DHQs) and other clinical laboratories nearby. Different laboratory
technicians and hospital personells helped us in collection of three years of
data from 2016 to 2018.

 

Collection
of Blood Samples

From malaria positive patients blood samples were collected
using 5 ml syringes and saved in sterile vacutainer tubes
enclose an anticoagulant agent Ethylenediamine
tetra acetic acid (EDTA).
These samples were attained from south of KP including  Hangu, Kohat, Bannu and Dera Ismail Khan .Collection
of blood samples was design in period ( i.e. march 2017  to 
September 2017 ,  during which
malaria show epidemicity with high rate). These samples were preserved at -20°C in the
Molecular lab of biochemistry department AWKUM.

DNA Extraction from Human Blood

DNA
was extracted from human blood using a phenol chloroform method to study and
differentiate various genotypes of P.falcipaum (Loparev et
al., 1991)

Table 2.2 Compositions
of solutions used in DNA extraction

 

 

 

 

 

 

S.No.

SOLUTIONS

Composition of chemical

 

 

 

 

 

 

 

1.

Solution A

0.3 M sucrose

 

 

 

 

10 mM
Tris pH 7.5

 

 

 

 

5 mM
MgCl2

 

 

 

 

1%
(v/v) Triton X-100

 

 

 

 

 

 

 

2.

Solution B

10 mM Tris pH 7.5

 

 

 

 

400mM
NaCl

 

 

 

 

2 mM
EDTA pH 8.0

 

 

 

 

 

 

 

3.

Solution C

Saturated Phenol

 

 

 

 

 

 

 

4.

Solution D

Chloroform 24% (v/v)

 

 

 

 

Isoamyl
alcohol 1% (v/v)

 

 

 

 

 

 

 

 

 

Procedure

1. 700 µl of  of positive blood sample and 700 µl of sol A
were taken in 2ml of  Eppendorf tube for
RBC lysis.

2. Blood sample was mixed with sol A in
the tubes by inverting 4-6 times and incubate at room temperature for 5-10
mins.

3. The tube
was  Centrifuge at 13000 rpm for 1 min.

4.The supernatant
was discarded again and 400 µl of Sol.A was added in pellet and dissolved it by
vortexing.

5. Centrifugation
was followed again for 1 min at 13000 rpm. The supernatant was discarded and
re-suspend the nuclear pellet in 400 µl of Sol.B, 17 µl of 20% SDS and 8 µl of
Proteinase K.

6. Overnight incubation
was done at 37C or °65?
for 3 hours.

7. 500 µl of fresh
Sol C and Sol D mixture of equal volume were added to the sample after
incubation which separate layer. Invert the mixture 2-3 times and centrifuge
13000 rpm for 10 min.

8. The aqueous phase
(Upper layer) was transfer to a new eppendorf tube. 55 µl of Sodium Acetate
(3M, PH 6) and equal volume of 500 µl of Isopropanol were added and several
time mix  the tubes for DNA precipitation.

9. The tubes were
then centrifuge at 13000 rpm for 10 mins, the supernatant was discarded. 200 µl
of 70% ethanol to DNA pellet was added for washing, and Centrifugation was
followed at 13000 rpm for 7 min and the ethanol should be discarded.

10. For half an
hour DNA was dried by keeping the tubes for for 30 mins at 37?.

11. The precipitated
DNA was dissolved in 200 µl of TE and stored at -20?.

 

 

Gel
Electrophoresis

Extracted DNA was confirmed
by prepared 1% agarose gel from both qualitative and quantitative point of view
and was seen by using Ethidium bromide. Visualization of extracted DNA was done
using ultraviolet illuminator, at a wavelength of 254 nm using Gel
documentation system.

Nested Polymerase Chain Reaction

Nested PCR is carried to amplify the target DNA and inhance the
specificity of target region of DNA.  Some time the primer bind other than the
target sequence and creates problem in the desired results. In Nested PCR two
sets of primers are apply. First role of primer set was possible to get
attached to the outside sequences of the target region of DNA, standard PCR is
the same, but it can also get bound to other region of the DNA template. The
target region of  DNA amplified by first
set of primer was subjected to the second set of primer, to amplify the region
within first amplification. Thus to the products of the first PCR reaction, the
primers of second set gets attached and amplified the DNA region.

Primer set Used in Nested
PCR

During the current
study, P. falciparum merozoites surface protein specific primers pfmsp1, Pfmsp2 and Pf GLURP types
specific primers such as K1, RO33 and MAD20 allelic family of pfmsp1, 3D7/IC1 and FC27 allelic family
of pfmsp2 and polymorphic allele
GLURP R2 repeat region were designed by eurofinsm wg/operon. The sequence of
these primers is given in Table No 0.0

 

 

 

 

 

Table
1: Primers sequences used for Nested PCR
analysis

 
Primer

 
Sequence

 
Pf mspl
(N1)

 
F:
5′-CTAGAAGCTTTAGAAGATGCAGTATTG-3′
R:
5′-CTTAAATAGTATTCTAATTCAAGTGGATCA-3′
 

 
Pfmsp-2
(N1)
 

 
F: 5
‘-ATGAAGGTAATTAAAACATTGTCTATTATA-3,
R:
5′-CTTTGTTACCATCGGTACATTCTT-3′
 

 
Pf GLURP (N1)

 
F: 5?-TGAATTTGAAGATGTTCACACTGAAC-3?;
R:5’GTGGAATTGCTTTTTCTTCAACACTAA-3?
 

 
K1 family
(N2)

 
F:
5′-AAATGAAGAAGAAATTACTACAAAAGGTGC-3′
R:
5′-GCTTGCATCAGCTGGAGGGCTTGCACCAGA-3’
 

 
R033
family(N2)
 

 
F: 5
‘-TAAAGGATGGAGCAAATACTCAAGTTGTTG-3 ‘
R: 5
‘-CATCTGAAGGATTTGCAGCACCTGGAGATC-3 ‘
 

 
MAD20family
(N2)

 
F:
5’AAATGAAGGAACAAGTGGAACAGCTGTTAC-3′ R:5’ATCTGAAGGATTTGTACGTCTTGAATTACC-3′
 

 
FC27family
(N2)

 
F:
5′-AATACTAAGAGTGTAGGTGCARATGCTCCA-3’
R: 5
‘-TTITATTTGGTGCATTGCCAGAACTTGAAC-3
 

 
3D7/I
C1family (N2)

 
F: 5
‘-AGAAGTATGGCAGAAAGTAAKCCTYCTACT-3 ‘
R: 5
‘-GATTGTAATTCGGGGGATTCAGTTTGTTCG-3 ‘
 

 
Pf GLURP
(N2)

 
F: 5?-TGTTCACACTGAACAATTAGATTTAGATCA-3’
R:5’GTGGAATTGCTTTTTCTTCAACACTAA-3

 

                                                                                      

 

 

PCR Analysis

Initial PCR

For initial PCR, a 25µl of total volume
prepared in PCR tube .One PCR tube contain 16.2µl PCR water, 1.8µl Magnecium
chloride, 2.5 µl Taq buffer,0.5 µl dNTPs, 0.5µl of forward and reversed primer
of pfmsp1, pfmsp2
or glurp, 0.5µl Taq polymerase and
1.5µl of gDNA was added, each tube has the same components and equal volune.
Master mix prepapation took place on ice because Taq pol is temperature
sensitive.  

The PCR run was optimized using the following program:

The initial Denaturation at 95°C for 5 minutes and 30 cycles of,
denaturation at 95°C for 50 seconds, annealing at 58°C for 50 seconds and
extension at 72°C for 1 minute and 45 seconds. Final, extension at 72°C for 5
minutes was performed. PCR was run and initial PCR products were obtained.

2nd PCR

Initial PCR products were taken for which five sets of PCR
tubes  were labelled for each product of pfmsp1 and pfmsp2 and a separate PCR tube
for GLURP R2 nested according to the desire. A total of 25µl volume
of  product prepared for each, to which 16.2
µl PCR water, 1.8 µl Magnecium chloride, 2.5
µl Taq buffer, 0.5 µl dNTPs, 0.5 µl forward and reversed primers of family of pfmsp1 and pfmsp2 and Pf Glurp R2
repeat nested, 0.5 µl Taq pol and 2.5 µl initial PCR product added to each tube.

The PCR run was again optimized using the following program:

 The initial
denaturation at 95°C for 5 minutes and 30 cycles of, denaturation at 95°C for 45
seconds, annealing at 58°C for 50 seconds and extension at 72°C for 1 minute
and 45 seconds. Final extension at 72°C for 5 minutes was performed. PCR was
run and final PCR products were preserved at -20Co.

Visualization of PCR
Products by Gel Electrophoresis

A
2 % agarose gel was prepared  to study
the amplified DNA samples using agarose gel electrophoresis technique, and were
seen by staining with Ethidium bromide. Visualization
of the amplified products were done under ultraviolet illuminator at a wave
length of 254 nm using Gel documentation system.