GENE AMPLIFICATION IN VITRO TECHNIQUE.

1. POLYMERASE CHAIN
REACTION.
(PCR)
BY
MISS.SNEHA A.SONAR.
(M.Sc. B.Ed. PET.)

2. Who is the founder?
Kary Banks Mullis (December 28, 1944 –
August 7, 2019) was an American
biochemist
• He is known for his invention of the
polymerase chain reaction (PCR)
technique (In,1983).
• He shared the Nobel Prize in,
Chemistry with Michael Smith and was
awarded the Japan Prize in the 1993.
• His invention became a central
technique in biochemistry and
molecular biology,

3. DEFINITION
• Polymerase chain reaction
(PCR) or gene amplification is a
fast, inexpensive laboratory
technique (In vitro), which is
used to amplify or to generate
billions of copies of the small
segments of DNA.

4. REQUIRMENTS
• DNA segments (100-35, 000bp in length)
to be amplified.
• DNA primers(Forward and reverse).
• Free nucleotides called dNTPs,
• DNA polymerase i.e Taq polymerase
isolated from a bacterium called as
Thermus aquaticus. This enzyme can
withstand and remain functional upto
94°C.

6. PRINCIPLE OF PCR.
• This method combines the
principles of complementary
nucleic acid hybridization with
those of nucleic acid replication
applied repeatedly through
numerous thermal cycles.

7. MECHANISM
• STEPS
1. DENATURATION
2. ANNEALING
3. POLYMERIZATION/EX
TENSION.

8. STEPS.
Step 1: Denaturation. As in DNA replication, the
two strands in the DNA double helix need to be
separated.
Step 2: Annealing. Primers bind to the target
DNA sequences and initiate polymerisation. .
Step 3: Extension. New strands of DNA are
made using the original strands as templates.

9. DENATURATION
• The reaction mixture (DNA) is heated to
95°C for a short time period (about 15–
30 sec) to denature the target DNA into
single strands that can act as templates
for DNA synthesis.
• dsDNA cnverts into ssDNA.

10. ANNEALING
• The mixture of DNA is rapidly cooled to a
defined temperature about 55°C which
allows the two primers to bind to the
sequences on each of two strands flanking
the target DNA.
• Simply it is a pairing of primers to the ssDNA
segments.

11. PRIMERS
Are short, single-stranded sequences
of nucleic acid (i.e.oligonucleotides
usually 20 to 30 nucleotides long)
selected to specifically hybridize
(anneal) to a particular nucleic acid
target, essentially functioning like
probes.

12. EXTENSION/POLYMERIZA
TION
The temperature is raised to 72°C and
the Taq polymerase adds dNTPs behind
the primer of ssDNA to form two
dsDNAs.

14. AMPLIFICATION
PCR steps of denaturation, annealing, and
extension are repeated (or “cycled”) many
times to amplify the target DNA. The
number of cycles is usually carried out 25–
35 times but may vary upon the amount of
DNA input and the desired yield of PCR
product.
Each cycle of PCR takes about 3 - 5 mins.

15. APPLICATIONS
PCR is used in many research labs, and it also
has practical applications in forensics, genetic
testing, and diagnostics. For instance, PCR is
used to amplify genes associated with genetic
disorders from the DNA of patients (or from
fetal DNA, in the case of prenatal testing).

16. THANK YOU!