Polymarase chain reaction EUSL Assaignment

1. POLYMERASE CHAIN
REACTIONS
GROUP 3
Eastern University . Sri Lanka
Faculty of Health-Care Sciences

2. POLYMERASE CHAIN REACTION?
• Polymerase chain reaction is laboratory techniques
used to make multiple copies of segment of DNA.

3. INTRODUCTION
• PCR targets and amplifies a specific region of a
DNA strand.
• PCR permits the synthesis of millions of copies
of a specific nucleotide sequence in a few hours.
• This method can be used to amplify DNA
sequences from any sources.
Eg :- Viral , Bacterial , Plant , Animals

4. HISTORY OF PCR
• Great mind behind this PCR :Kary Banks Mullis
• -1983—Kary Mullis, a scientist working for the Cetus Corporation was driving
along US Route 101 in northern California when he came up with the idea for
the polymerase chain reaction.
• Developed PCR in 1985 and was awarded Nobel prize in 1993.
• -1985—the polymerase chain reaction was introduced to the scientific
community at a conference in October .Cetus rewarded Kary Mullis with a
$10,000 bonus for his invention
• -Later, during a corporate reorganization, Cetus sold the patent for the PCR
process to a pharmaceutical company Hoffmann-LaRoche for $300 million.

5. introduction cont…..
• PCR cycle consists of three steps.
1. Denaturing (Strand separation upon heating)
2. Hybridization of primers (Annealing)
3. DNA synthesis (Extending)

6. COMPONENTS OF PCR
• PCR is carried out by adding the following components to a solution containing
the target sequence.
1. The DNA template is that particular DNA sequence which you want to copy.
2. A pair of primers that hydrolase with flanking sequence of the targets.
3. All for deoxyribonucleoside triphosphate (dNTPs)
4. Heat stable DNA polymerase.
5. Finally a reaction buffer is used to provide a stable pH.

7. PROCEDURE
• PCR uses DNA polymerase to repetitively targeted portions of
genomic or DNA.
• Each cycle of amplification doubles the amount of DNA in the
sample.
• The DNA products can be separated by gel electrophoresis ,
detected by southern blotting and hybridization and
sequenced.

8. • In PCR method it is not necessary to know the nucleotide
sequence of target DNA.
• But it is necessary to know the nucleotide sequence of short
segments on each side of target DNA.
• These stretches called as Franking sequences.
1) Constructing Primers

9. • Flanking regions are used to construct two single stranded
oligonucleotides.
• Usually 20-35 nucleotides long.
• The 3’ hydroxy end of each oligonucleotide points
• This oligonucleotide complementary to the receptive flanking
sequences.
• This synthetic oligonucleotide act as primers
constructing primers cont…..

10. • The target DNA to be amplified heated to 95℃ to separate DNA
into single strand.
2) Denaturing DNA

11. • The separated strands are cooled to 50℃.
• Then the two primers anneal to complementary sequence on the
SSDNA
3) Annealing DNA

12. • DNA pol and dNTP are added to the mixture.
• The temperature is shifted to 72℃.
• DNA pol and nucleotides to the 3’ hydroxyl end of the
primer, and strand growth in the 5’ 3’ direction
3) Extending Primers

13. • At the completion of one cycle , the mixture is heated again.
• Each strand by a complementary primer.
• Typically 20 - 30 cycles run during this process amplifying the
DNA by a million fold to a billion fold.
Extending primers cont…..

14. New automated PCR Old PCR

15. ADVANTAGES OF PCR
• As a mechanism for amplifying a specific DNA sequence are sensitivity and speed.
• DNA sequences present in only trace amount can be amplified to become the
predominant sequence.
• PCR is so sensitive that DNA sequence present in individuals cell can be amplified
and studied.
• Isolating and amplifying a specific DNA sequence by PCR is faster and less
technically difficult.

16. APPLICATIONS OF PCR
• PCR has become a very common tool in research , forensics and
clinical diagnostics.
1. Comparison of a normal gene to it’s mutant form.
2. Forensic analysis of DNA samples.
3. Detection of low-abundance nucleic acid sequences.
4. Prenatal diagnosis and carrier detection of cystic fibrosis.

17. • PCR allows the synthesis of mutant DNA .
• For that sufficient quantities for a sequencing protocol.
• it’s not laborious biologic cloning of DNA
1)Comparison of a normal gene to it’s mutant form

18. • PCR may also be used for genetic finger printing.
• A forensic technique used to identify a person by comparing
experimental use DNA markers.
• DNA isolated form ,
 A single human hair.
 A tiny spot of blood.
 A sample of semen.
2) Forensic analysis of DNA samples.

19. • Viruses have a long latency period.
• Such as HIV (Human immunodeficiency virus).
• There for difficult to detect the early stage of infection conventional
methods.
• PCR rapid and sensitive method for detecting viral DNA when a small
proportion of viral DNA.
3) Detection of low-abundance nucleic acid sequences.

20. • Cystic fibrosis is an autosomal – recessive genetic disease.
• Resulting of mutations in the gene for the FTCR (Fibrosis
transmembrane conductance regulator) protein.
• Most common mutation is a three base deletion.
• Result of that the loss of a phenylalanine residue from FTCR protein.
• Therefore the mutant allele is shorter than normal allele
• The results of PCR test identify,
 Homozygous Normal
 Heterozygous Carriers
 Homozygous mutant affected
4) Prenatal diagnosis and carrier detection of cystic fibrosis.

22. Thank You
Eastern University . Sri Lanka
Faculty of Health-Care Sciences
FROM - GROUP 3