PCR & it's Modifications. This presentation contains basic PCR process and it's 4 important modifications.

1. •Hot Start PCR
•Q-PCR
•RT-PCR
•Nested PCR
Kary Mullis

2. Polymerase Chain Reaction
 PCR is technology in molecular biology
used to amplify a single or a few copies of
DNA across several orders generating
thousands and millions copies of a
particular sequence.
 PCR takes a specific sequence of DNA of
small amounts and amplifies it to be used
for further testing.
 It relies on thermal cycling consisting of
cycles of repeated heating & cooling of the

3. Processes
1. Denaturation - It is the first step where DNA
stands are separated by heating At 950 C.
2. Annealing - It is the process in which two
stands of DNA are allowed to form hydrogen
bonds. (550 C – 650 C)
3. Extension- The process in which the
nucleotides are added to primer by Taq
polymerase. (720 C)

4. The basic protocol—what’s in the tube
Target DNA
5’ 3’
3’ 5’
Primers
(0.1-o.5uM)
A B
Free
Nucleotides
(20-200uM)
Taq DNA
Polymerase
1-2.5units
Mg2+
Mg2+
Mg2+
Mg2+
Mg2+
Mg2+
Buffer
containing
magnesium
(MgCl2) 0.5-2.5mM

5. The basic protocol--denaturation
Target DNA
95oC
5’ 3
3’ 5
5’ 3’
3’ 5

6. The basic protocol--annealing
~55oC
5 3’
3’ 5’
5’ 3
3’ 5’
5’
5’
Target DNA
A
B
primers
A
B

7. The basic protocol--extension
72oC
5’ 3
3’ 5’
5
’
3’ 5
5’
5’
Target DNA
Taq polymerase
3’

8. The basic protocol--extension
72oC
’5
3’ 5
5’ 3
3 5’
5’
5’
Target DNA
3’

9. MODIFICATIONS OF PCR
 HOT-START PCR
 QUANTITATIVE PCR
 REVERSE TRANSCRIPTION PCR
 NESTED PCR

10. HOT-START PCR
 It is a modified form of PCR which avoids non-specific
amplification of DNA by inactivating Taq polymerase
at lower temperature
 In the second step in addition to primer and Taq
polymerase we add specific antibodies to block Taq
polymerase from annealing.
 When temperature raises for amplification at 72o C,
the specific antibodies detaches from Taq polymerase
& amplification begins with greater specificity.

11. HOT-START PCR

12. Q-PCR (Quantitative-PCR)
 It is also known as Real Time PCR
 Real Time PCR is a laboratory technique of molecular
biology based on the PCR, which is used to amplify &
simultaneously detect or quantify targeted DNA molecules.
 This is the new approach compared to std. PCR where the
product of the reaction is detected at it’s end.

13. Methods
1. Non specific fluorescent dyes that intercalate with
any dsDNA.
2. Sequence specific DNA probes consisting of
oligonucleotides that are labelled with a fluorescent
with a fluorescent reporters which permits detection
only after hybridization of probe with it’s
complimentary sequence to quantify mRNA or non
coding RNA in cells or tissue.

14. TaqMan Probe

15. Fluorescent dyes

16. Reverse Transcription PCR
 In RT PCR, the RNA templates first converted to
complementary DNA (cDNA) using reverse
transcriptase.
 The cDNA is used as template for amplification of
DNA by PCR
 The amplification of RNA can be done with the help of
ONE STEP PCR & TWO STEP PCR

19. NESTED PCR
 Nested polymerase chain reaction (PCR) is a
modification of PCR intended to reduce the
contamination in products due to the amplification of
unexpected primer binding sites.
 Nested PCR utilizes two different sets of primers
during a two-step amplification.
 The PCR reaction is run using the "outer primers“
during a first cycle of amplification which is
immediately followed by a second cycle of
amplification carried out with the "inner primers".

20. NESTED PCR
• NESTED PCR is a variation of the PCR, using two pairs
of primers to amplify fragments of DNA.
• The first pair primer amplify a fragment similar to
standard PCR.
• The second pair of primers called nested primers
(because they are in the first PCR amplification of an
internal fragment) incorporated inside the first PCR
product, so that the second PCR amplified fragment is
shorter than the first amplification.