Bio-Genetics Journal

Vol. 1, No. 1 (2013): 15-17

Research Article
Open Access

PCR analysis of prothrombin polymorphism in

selected patients
Rushinadha Rao Kakara*
Central Institute of Fisheries Technology, Indian Council of Agricultural Research, AU Po; Visakhapatnam, Andhra Pradesh, India

* Corresponding author: Rushinadha Rao Kakara; e-mail: kakararushi@gmail.com

Received: 20 October 2013 Accepted: 10 November 2013 Online: 20 November 2013

ABSTRACT
An attempt was made to study the prothrombin polymorphism using simple PCR and followed by Restriction
Fragment Length Polymorphism. 10ml EDTA Blood sample from 10 healthy volunteers of age group 20 to 35,
DNA was isolated using salting out method. The isolated DNA was subjected to Single Polymerase Chain
Reaction. Then the resultant amplicons were subjected to Restriction Fragment Analysis using Hindlll enzyme.
From the analysis, it was observed that the samples analyzed were homozygote wild type.

Keywords: prothrombin, polymorphism, amplicon

INTRODUCTION started and that, in some circumstances, it may run out

Prothrombin is a blood-clotting protein. Injury to a
blood vessel produces a signal which triggers the
conversion of prothrombin to thrombin. Thrombin is a
protein which plays a central role in provoking the
assembly of other proteins to form the blood clot.
Prothrombin (F2) is a vitamin K-dependent clotting
factor [1]. It is synthesized in the liver as a single-chain
glycoprotein composed of 579 amino acid residues
with a molecular weight of 72,000 [1, 2]. The primary
structure [3] the cDNA sequence [4] the genomic DNA
sequence of human prothrombin containing 14 exons
in a total length of 21 kb [5] and chromosomal mapping
to chromosome 11p11-q12 [6] have been reported.
The nucleotide sequences of cDNA (Degen et al.1983)
and genomic DNA [5] for human prothrombin have
been reported and this gene has been assigned to the
chromosomal region 11p11-q12 [6]. Its polymorphisms
have also been described [7].
Mechanism of Action of Prothrombin Gene
Mutation
Prothrombin is the precursor to thrombin in the
coagulation cascade Thrombin is required in order to
convert fibrinogen into fibrin, which is the primary goal
of the coagulation cascade. The gene has a mutation at
position 20210, hence the disorder being referred to as
prothrombin mutation 20210. It is thought that the
increased amount of circulating prothrombin provides
a springboard upon which the clotting cascade can get
of control because of that springboard potential [8].
Epidemiology of the Prothrombin Gene Mutation
The prothrombin gene mutation is seen more
commonly in the Caucasian population. About 1-2% of
the general population is heterozygous (one copy) for
the prothrombin gene mutation. The Prothrombin gene
mutation is relatively uncommon in the native
populations of India, Korea, Africa and North America.
In contrast, in Spain rates of 6% have been reported
[9].
Diseases related to prothrombin
Mutations in the gene for prothrombin (F2 20210A)
and factor V (F5 1691A, factor V Leiden) are
established risk factors for deep venous thrombosis
(DVT). Genetic variant in the 38-untranslated region of
the prothrombin (factor II) gene (G20210A), it is
apparent that this mutation confers a moderate risk for
venous Thrombosis Recently, a mutation in the gene for
factor XIII (F13 100T) leading to a Valine–Leucine
exchange at amino acid position 34 has been reported
to be protective against DVT [10].
The results shown by the early study say that in
general, the prevalence of the 20210A allele among
Europeans ranges from 1.7% to 2%, and it was found
that this polymorphism is very rare (1%) in individuals
of Asian and African descent. Detection of prothrombin

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gene polymorphism was done according to method RESULTS AND DISCUSSION

described by Poort et al. Presence of 20210 G-A allele
was screened by PCR followed by Hind III enzyme
digestion. Forward and reverse primers used for exon
14 were 5'TCTAGAAACAGTTGCCTGGC-3'and 5'
ATAGCACTGGGAGCATTGAAGC-3'. Wild type allele
(20210G) lacks the restriction site and therefore
remains undigested (345bp). Positive controls for both
the mutant allele were taken from recurrent
spontaneous abortion [11]. Human studies also showed
an influence of the intronic 19911A>G polymorphism
on prothrombin activity [1]. However, the lack of
restriction site in normal individuals, genotype G/G
(not carrying the prothrombin 20210A allele, wild
type), will generate only the 345-bp fragment [5].
MATERIALS AND METHODS
Sample collection and DNA extraction by Salting out
method
Peripheral blood samples were collected by vein
puncture. 125ul of whole blood was taken and 125ulof
TKM-1 buffer was added along with 2-3 drops of
Triton-X 100 for removing RBS by centrifugation at
2700rpm till white pellet was obtained. To the pellet
80ul of TKM-2 buffer and 10% 12.5ul of SDS was added
and incubated at 550C for dissolving the pellet for 15
min.
HindIII is a type II site-specific deoxyribonuclease
restriction enzyme isolated from Haemophilus
influenzae that cleaves the palindromic DNA sequence
AAGCTT in the presence of the cofactor Mg2+ via
hydrolysis. The cleavage of this sequence between the
AA's results in 5' overhangs on the DNA called sticky
ends:
5'-A |A G C T T-3'
3'-T T C G A| A-5'
Undigested product of Prothrombin when amplified
with specific primers the size is 507bp when amplicon
is digested with hind III enzyme normal homozygote
produces 407 and 99bp if there is a polymorphism as G
at 20210 the resulting RFLP pattern will produce 384,
99, 23bp where as in case of heterozygote 407,384,99
and 23 bp bands will be resulted. In table 1 the
expected RFLP banding pattern is depicted in Table 1.
Table 1. Prothrobin Hindlll RFLP pattern
Allelic type Banding pattern
Normal homozygote 407bp, 99bp
Homozygote polymorphic 384bp, 99bp, 23bp
Heterozygote 407bp, 384bp, 99bp,
23bp

After dissolution of the pellet freshly prepared 35ul of Based on the banding patterns observed in the gel
5M NaCl was added and incubated for 10min. After image 1 and gel image 2 samples are interpreted as
centrifugation at 1200 rpm DNA from supernatant was Homozygote dominant, Homozygote recessive and
collected into a fresh tube and 100% ethanol was Heterozygous.
added. DNA threads or lumps obtained after
centrifugation was finally washed with 70% alcohol.
Pellet thus obtained was further dissolved in pre
heated 100uL TE buffer at 650C. Complete procedure
was carried in bio safety cabinet.

Preparation of 2% Agarose gel for Electrophoresis

2% agarose gel was prepared in 1XTBE buffer. Samples
along with the ladder were loaded in the gel and after
completion of the run; the gel slab was removed form
the buffer tank and bands were visualized under UV
Transilluminator. This was followed by PCR
amplification.

PCR Setup Gel Image 1. Prothrombin gene amplification and RFLP by

The PCR is set up with 20pM of forward and reverse Hind lll. The lanes 1,3.5,7,10,12,14 are undigested PCR
primers. The reaction is setup in 20ul of reaction products of Prothrombin gene amplicon. 2, 4,6,8,11,13 and 15
volume with final concentration of 50mM KCl, 10mM are digested products using enzyme HindIII of the same.
Tris, and 200mM dNTP’s and 1.5mM MgCl2. Reaction
Table 2. Selected population were Homozygote polymorphic
were setup under the cycling parameters 940 C for
S. Sample ID Band size Results
4min, 940 C for 1min, 550 C for 1min, 720 C for 1min, No detected(bp)
720 C for 10 min. The number of PCR cycles was 35. 1 SD3 166r 407 and 99 Normal Homozygote
2 SD2 157r 407 and 99 Normal Homozygote
The resultant PCR product was further loaded in 2% 3 SD3 294r 407 and 99 Normal Homozygote
4 SD 4304r 407 and 99 Normal Homozygote
Agarose gel and the expected band size was 500bp 5 SD 2 166r 407 and 99 Normal Homozygote
which was run along the DNA ladder. To the resultant 6 SD2 274r 407 and 99 Normal Homozygote
PCR product (5ul) Hind III restriction enzyme was 7 SD2 56r 407 and 99 Normal Homozygote
added. Reaction was set up to 20ul with final 8 Sd2 226r 407 and 99 Normal Homozygote
concentration of 10mM Tris-HCl and 0.4ug/100ul. The
expected band size after digestion is 384bp and 99bp.

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 Rushinadha Rao Kakara / Bio-Genetics J. 2013, 1(1): 15-17

myocardial infarction weaker than in venous

thrombosis (2-fold increase in risk), without reaching
statistical significance; in a more recent report, the
heterozygous mutant factor II genotype was found
associated with a significant increased risk (4-fold) for
myocardial infarction in young women.6 The
investigations so far conducted on unselected patients
with cerebral ischemia did not show any increase in
risk associated with the mutant.

CONCLUSION
All the samples analyzed were homozygote wild type.
No polymorphisms which reported to involve in
thrombosis are not present in coastal Andhra
population. But the sample size is limited. More
number of samples to be analyzed for confirmed
results. However, the studies from other parts of India
also reported lack of this polymorphism in India.

REFERENCES
Gel image 2. Prothrombin gene amplification and RFLP by
Hind lll. The lanes 1 is undigested PCR products of
Prothrombin gene amplicon and 2 is digested products using
enzyme HindIII of the same
Thrombophilia is a group of conditions which are
associated with an increased predisposition to
thrombosis. Venous Thrombo Embolism is a relatively
common disorder the true incidence of which is not
known. However, VTE is known to increase
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with high morbidity and mortality. For these reasons
the area has generated considerable interest especially
with regard to identification of risk factors and role of
thromboprophylaxis. Predisposition to thrombosis may
be inherited or acquired. The increase in inherited risk
factors includes disfibrinigenemia, protein c and
protein s deficiency, factor V leaden mutations,
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association of the mutant factor II allele with arterial
thrombosis has been reported in patients with
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