PCR, HEPARIN SODIUM NOTES FOR MPHARM IST SEM

1. DEPARTMENT OF PHARMACEUTICAL ANALYSIS
PRESENTATION ON :-PCR AND HEPARIN
SODIUM
SUBMITTED TO :-
DR. C. SREEDHAR
H.O.D.
ANALYSIS DEPT.
SUBMITTED BY
SARFARAJ A KHAN
M.PHARM 1ST SEM

2. Contents:
Heparin sodium
PCR (Polymerase chain reaction)

3. Heparin Sodium:
Introduction:
Heparin is a highly-sulfated glycosaminoglycan of natural
origin.
It is also one of the oldest drugs still in widespread use –
Heparin, along with vitamin K antagonists, have been the main
anticoagulant drugs for more than 70 years, as it has been
used since the 1930s.

4. Mechanism of action:

5. Biological activity:
Heparin can interact and regulate the activities of a wide range
of proteins that are essentials to important biological processes
such as
– blood clotting
– pathogen infection
– cell differentiation
– cell growth and migration
– inflammation

6. PRINCIPLE;
 The potency of heparin sodium is determined in vitro by
comparing the concentration necessary to prevent the clotting
of sheep or goat or human plasma with the concentration of
the standard preparation of heparin sodium. •Its is necessary
to give the same effect under the same conditions of the
method of assay.

7. Bioassay of Heparin
Prepared plasma:
 Collect the blood from sheep or goat or human in vessel
containing 8% w/v of sodium citrate (The ratio of sodium
citrate and blood is 1:19)
 Mix gently and centrifuge to pool out plasma Clean test
tube
 In one ml of pooled plasma, add 0.2 ml of 1% w/v of
calcium chloride solution.

8. Solution of standard preparation:
The potency of standard heparin is determined in relation to
the International Standard stated by the World Health
Organization.
Test solution:
Weigh accurately about 25 mg of the test sample Dissolve in
sufficient saline to give the concentration of 1 mg/ml.

9. Method:
 In clean test tube, add graded amount of the solution of
standard preparation (the largest dose not exceed 0.8 ml)
 Add sufficient volume of saline to make total volume of 0.8ml
and add 1 ml of prepared plasma to each test tube.
 Add 0.2 ml of 1 % w/v solution of calcium chloride, note the
time.
 Mix the content properly so entire inner surface of the tube is
wet.

10. Continuation…
 In same manner setup a series of test preparation (complete the
entire process within 20 min after addition of prepared plasma)
 After 1 hr the addition of calcium chloride solution, determine the
extent of clotting in each test tube
 Recognize three grades between zero and full clotting.
 Dilution of the test solution which contains same concentration as
that of standard shows same degree of clotting.

11. Cont….
 If the degree of clotting in dilution of the standard preparation
lies between that observed in 2 of the dilution of test
preparation, the potency of later is estimated
 If there is no correspondence between the degree of clotting by
standard and test, new dilution prepared and assay is repeated
 Calculate the estimated potency of the preparation by
combining the result of assay with standard statistical method

12. Polymerase chain reaction (PCR):
PCR is a technique that takes specific sequence of
DNA of small amount and amplifies it to be used for
further testing.
• In vitro technique

14. Principle of PCR:
•Purpose
•Condition
•Components
Purpose:
• To amplify a lot of double-stranded DNA molecules (fragments)
with same (identical) size and sequence by enzymatic method
and cycling condition.

15. Condition:
1. Denaturation of ds DNA template
2. Annealing of primers
3. Extension of ds DNA molecules

22. Chemical Components:
• Magnesium chloride: .5-2.5mM
• Buffer: pH 8.3-8.8• dNTPs: 20-200µM
• Primers: 0.1-0.5µM
• DNA Polymerase: 1-2.5 units
• Target DNA: ≤ 1 µg

23. Example of PCR programme:
 Initial denaturation 95C for 5 mins
 Denaturation : 95C for 30 secs
 Annealing : 55C for 30 secs
Extension : 72C for 45 secs
 Final extension 72C for 5 mins

24. Advantage:
1.Small amount of DNA is required per test.
2. Result obtained more quickly - usually within 1 day for PCR
3. PCR can be used to detect point mutations.

25. Refrences:
www.heparinscience .com
www.drug.com
www.labtest .com