it may useful for science students to study molecular biology techniques

1. Aneesha .A
Ist MSc Botany
University College

3. PCR
• Polymerase chain reaction is a cell free DNA
cloning techniques in which millions of copies of
target DNA sequences can be produced.
• Developed by Kary Mullis-1983

4. REQUIREMENTS OF PCR
• DNA Template
• Primers
• Taq polymerase
• dNTPs
• Buffer solution
• Divalent cations(eg.Mg2

5. STEPS INVOLVED
1. Denaturation
2. Annealing
3. Extension

6. STEPS INVOLVED
1) DENATURATION:.
o The DNA strands are separated by heating to
95°C.
o The Hydrogen bonds between the two strands
breaks down and the two strands separates.

7. DENATURATION
92C
3’5’
3’ 5’
+
5’3’
5’ 3’

8. ANNEALING
o Reaction temperature is lowered to 50–65°C
o Primer anneals to the complementary bases on
flanking region of template DNA

9. ANNEALING
5’
3’
5’ 3’
Forward primer Reverse primer

10. EXTENSION
• The temperature is now shifted to 72° C which is
ideal for polymerase.
• DNA polymerase synthesizes new DNA strand
complementary to the DNA template strand by
adding dNTPs.

11. EXTENSION
Taq
5’
3’
Taq5’

12. PRIMERS
• Primer is an oligonucleotide sequence – 18-26 bp in length
provides free 3’OH for the attachment of nucleotide bases
by Polymerase.
• Forward primer –
• Reverse primer
• Ta must be set 5oC below the Tm of your primers.
• It should not have secondary structures
• Complementarity between two primers are not allowed

13. DNA POLYMERASE
1)Taq polymerase
• Thermophilic bacterium - Thermus aquaticus
• Optimum temperature - 70–80°C
• Half-life - 40 minutes at 95°C
• Lacks 3’-5’ proofreading activity.

14. 2) pfu DNA Polymerase
• Isolated from Pyrococcus furiosus.
• 3’-5’ & 5’-3’ exonuclease activity
• Fidelity of enzyme is 12 fold higher.
• Half life at 95°C – 2 hours

15. 3) Pwo DNA Polymerase
• Isolated from Pyrococcus woesei
• Possess 3’-5’ exonuclease proof reading activity.
• Half life – 2 hours & 5 minutes at 100°C
• Fidelity of enzyme is 10 fold higher than Taq
polymerase.
• Processevity rate is same as Taq polymerase.

16. 4) Tth DNA polymerase
• Isolated from Thermus thermophilus.
• Does not possess a proofreading activity.
• Possess reverse transcriptase (RT) activity.
• RT activity is dependent upon presence of
manganese ions while the DNA polymerase
functions best in the presence of magnesium
ions.
• Optimum temperature is 70°C

17. DIVALENT CATIONS
• All polymerases require free divalent cations –
usually Mg 2+ for activity. Some require Mn 2+
(Tth for RT action).
• Cofactor in the catalytic addition of
deoxynucleoside monophosphates to the 3’ end
of the growing DNA chain
• Taq DNA polymerase shows its highest activity
around 1.2–1.3 mM free Mg2+.

18. APPLICATIONS
In clinical diagnosis
In DNA sequencing
In forensic medicine
In gene manipulation and expression studies
In comparative study of genomics
In comparison with gene cloning

19. PROBLEMS WITH PCR
• PCR is used only when the sequence of the
gene to be amplified is known
• PCR is error prone

20. TYPES
1. Quantitative real time PCR (Q-RT PCR)
2. Reverse Transcriptase PCR (RT-PCR)
3. Multiplex PCR
4. Nested PCR
5. Long-range PCR
6. Single-cell PCR
7. Fast-cycling PCR
8. Methylation-specific PCR (MSP)
9. Hot start PCR

21. 10. High-fidelity PCR
11. In situ PCR
12. Variable Number of Tandem Repeats (VNTR) PCR
13. Asymmetric PCR
14. Repetitive sequence-based PCR
15. Overlap extension PCR
16. Assemble PCR
17. Intersequence-specific PCR(ISSR)
18. Ligation-mediated PCR
19. Methylation –specific PCR
20. Miniprimer PCR
21. Solid phase PCR
22. Touch down PCR, etc

22. REVERSE TRANSCRIPTASE PCR
• Based on the process of reverse transcription,
which reverse transcribes RNA into DNA and was
initially isolated from retroviruses.
• Allows the detection of even rare or low copy
mRNA sequences by amplifying its
complementary DNA.

23. PRINCIPLE
• In RT-PCR, the RNA template is first converted
into a complementary DNA (cDNA) using
a reverse transcriptase. The cDNA is then used as
a template for exponential amplification using
PCR.

24. PROTOCOL:
 Two step RT-PCR
Step one
• Combine template RNA, primer, dNTP mix, in a
PCR tube.
• Add RNase inhibitor and reverse transcriptase to the
PCR tube.
• Place PCR tube in thermal cycler for one cycle that
includes annealing, extending and then inactivating
reverse transcriptase.
• Proceed directly to PCR or store on ice until PCR can
be performed.

25. Step two
• Add a master mix (containing buffer, dNTP mix,
MgCl2, Taq polymerase ) to each PCR tube.
• Add appropriate primer.
• Place PCR tubes in thermal cycler for 30 cycles of the
amplification program, which includes three steps:
• denaturation
• annealing
• Elongation
• The RT-PCR products can then be analyzed with gel
electrophoresis.

27. APPLICATIONS
•Used in detection of RNA viruses e.g. HIV viral
load
•To study mRNA expression levels in cells,
tissues.
•To study for the presence of active infection
e.g. TB

28. REAL TIME PCR
• In RT-PCR, process of amplification of DNA is
monitored in real time.
• PCR with an added probe or dye to generate a
fluorescent signal from the product.
• Detection of signal in real time allows quantification of
starting material.
• Performed in specialized thermal cyclers with
fluorescent detection systems.

29. General Description of Instruments
1. PCR cycler
2. Fluorescence
emission & detection :
1. Fluorometer
2. CCD camera
3. Excitation source:
xenon, halogen,
laser
3. Fluorescent Dye
Labeling of:
1. Oligonucleotides

30. REAL
TIME PCR
Using DNA
dyes
eg:- SYBR Green
I,Eva Green
Using
fluorescent
reporter probe
eg:-Taqman
probe,
Molecular
Beacon,

31. 1)USING DNA DYES
• PCR mixture is prepared
along with dsDNA binding
dye.
• Double stranded DNA
binding dyes which emit
fluorescence only when
bound to dsDNA.
• It will binds to minor groove
(dsDNA)

32. • Increased quantity of dsDNA = Increased
binding of SYBR green = Increased
fluorescence.
• After each cycle, the levels of fluorescence are
measured.
• Machine has fluorescence detectors. Which
captures the signals and convert them to graphical
representation on the screen.

33. 2)USING FLUORESCENT REPORTER
PROBE
What is a Taqman probe?
Probe having a fluorescent tag (F) at 5’ end and a
quenching tag (Q) at its 3’-end.

34. How it works in Real Time PCR?
• On annealing Taqman probe bind to its
complementary base sequence within the
template DNA
• During extension,Taq polymerase polymerise
the ss DNA while it encounters the Taqman
probe due to its 5’-3’ exonuclease activity

35. Continue….
• Fluorescence tag detaches from the quenching tag
and emitting fluorescence
• In each cycle following , the level of fluorescence
emitted increases and it can be monitored with a
computerised system

37. PCR
• More time consuming as it
uses gel electrophoresis to
analyze the amplified PCR
products.
• Conventional PCR is highly
sophisticated and labor
intensive
• uses fluorescent dye
system to detect the
products
REAL TIME PCR
• Less time consuming as it
can detect amplifications
during the early phases of
the reaction.
• More sensitive
• Uses ethidium bromide
and UV light to visualize
bands in the agarose gel
medium.

38. Reference
• David P Clark ,Nanette J.Pazdernik; Biotechnology –Applying the
genetic revolution;2009;British library cataloguing in publication
data
• Benjamin A.Pierce;Genetics A conceptual
approach;2008;W.H.Freeman and company Newyork
• D.Peter Snustard and Michael J Simmons; Genetics;
2008;W.H.Freeman and company Newyork
• John M . Walker and Ralph Rapley;Molecular biology and
biotechnology2002;The royal society of chemistry
• Channarayappa; Molecular Biotechnology: Principles and Practices;
2007; Taylor & Francis
• Keya Chaudhuri;Recombinant DNA technology;2013;TERI
• Desmond S.T. Nicholl; An Introduction to Genetic
Engineering;2008;Cambridge University Press.