Polymerase chain reaction (PCR) is a technique used to amplify a specific segment of DNA. It involves repeated cycles of heating and cooling of the DNA sample to separate the DNA strands and allow primers to anneal. This allows the targeted DNA sequence to be exponentially amplified. PCR was invented by Kary Mullis in 1983 and has numerous applications, including detection of genetic diseases. It is a critical technique used in research and clinical diagnostics.

1. By
Maria Syed

2.  It’s a means of selectively amplifying a
particular segment of DNA
 Each cycle of amplification doubles the
amount of DNA in the sample
 Source of DNA could be any- bacterial,
viral, plant and animal
Dr. Sumbul Fatma

3.  PCR allows the DNA in a single cell, hair
follicle, or spermatozoan to be amplified
and analyzed
 DNA sequences as short as 50-100bp
and as long as 10kb can be amplified
 As few as 20 cycles would yield ~106
times the amount of target DNA initially
present
Dr. Sumbul Fatma

4.  Invented by Kary B
Mullis in 1983
 First published
account appeared in
1985
 Awarded Nobel Prize
for Chemistry in 1993
Dr. Sumbul Fatma

5.  Components of PCRComponents of PCR
 Template DNA
 primers
 dNTPs (dATP, dTTP, dCTP & dGTP)
 Taq DNA polymerase
 MgCl2
 PCR buffer, pH 8

6. DNA polymerase- to repetitively amplify targeted
portion of DNA
Nucleotide triphosphates- ATP, GTP, CTP and
TTP
Primers- two single stranded oligonucleotides (20-
25ntds long), which are complimentary to the
flanking sequences that bracket the target DNA
sequence
Dr. Sumbul Fatma

7. THERMAL CYCLER
 PCR cyclers are
available from many
suppliers
 Reactions are done in
tubes or 96 well
microtitre plates
Dr. Sumbul Fatma

10.  Three major phases in PCR:Three major phases in PCR:
 Denaturing (94ºC)
 Annealing (55ºC)
 Extension (72ºC)
 The total time to perform a standard PCRThe total time to perform a standard PCR
is approximately 4 hours.is approximately 4 hours.

11.  Primer construction- it is synthetic
oligonucleotide complimentary to the short
nucleotide segments on each side of the
target DNA
Dr. Sumbul Fatma

12.  Denature the DNA- The DNA to be
amplified is heated to separate the double
stranded target DNA into single strands(1
min. 940
C )
Dr. Sumbul Fatma

13.  Annealing of primers to ssDNA- the
separated strands are cooled and allowed
to anneal to the two primers (one for each
strand)
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45 sec, 540
C
Forward and reverse primers

14.  Chain extension- the DNA polymerase adds
nucleotides to the 3’-hydroxyl end of the
primer, and strand growth extends across the
target DNA, making complimentary copies of
the target (2 min 720
C)
 At the completion of one cycle of replication,
the reaction mixture is heated again to
denature the DNA strand (of which there are
now 4)
Dr. Sumbul Fatma

18.  Denaturation - 940
C
 Annealing - 550
C
 Extension - 720
C
 Denaturation again………….
 20-30 cycles
 The amplified target sequence is called
amplicons
 With each cycle there is an exponential
increase in the amount of target DNA, hence
the name “Polymerase Chain Reaction”
Dr. Sumbul Fatma

19. Dr. Sumbul Fatma
Heat stable DNA
polymerase is vital to
the ease of the
process ……

20. Dr. Sumbul Fatma
Thermus aquaticus, a thermophilic bacteria that lives and
replicates at 70-800
C is the source of Taq DNA polymerase
used in PCR reactions.

21. Dr. Sumbul Fatma

22. the RNA must be enzymatically converted
to DNA
Reverse Transcriptase- are the RNA
directed DNA polymerases
Reverse Transcriptase
RNA cDNA
cDNA is then amplified by PCR
This process is termed as RT-PCR
Dr. Sumbul Fatma

23.  Amplicons can be analyzed by gel
electrophoresis and Southern Blot-
qualitative analysis
 Quantitative PCR- used to measure the
viral loads in HIV and Hep C-infected
patients
 These numbers allow physicians to
determine disease status and evaluate
efficacy of antiviral treatment.
Dr. Sumbul Fatma

24.  Quality of template DNA
 Concentration of template DNA
 Primers
 Concentration of MgCl2
 Annealing temperature

25. Quality of template DNAQuality of template DNA
- should be free of proteases that could
degrade the DNA polymerase.
- template DNA with high levels of
proteins or salts should be diluted or
cleaned up to reduce inhibition of DNA
polymerase activity.

26. Concentration of template DNAConcentration of template DNA
- highly concentrated template DNA may
yield nonspecific product or inhibit the
reaction.
- it is rare that template DNA concentration
is too low.

27. PrimersPrimers
- select primers with a random base
distribution and GC content similar to
template DNA being amplified.
- avoid sequences with secondary
structure, especially at the 3’ end.
- check primers for complementary and
avoid primers with 3’ overlaps to reduce
primer-dimer artifacts.
- design so the base at the 3’ end of the
primer is a G or C to enhance specificity.

28. Concentration of MgClConcentration of MgCl22
- MgCl2 concentration is very important.
- excess Mg2+
promotes production of
nonspecific product and primer-dimer
artifacts.
- insufficient Mg2+
reduces yield.

29. Annealing temperatureAnnealing temperature
- annealing temperature depends on length
and GC content of primers (55ºC good for
primers 20 nucleotides long; 50%).
- Higher annealing temperatures may be
needed to increase primer specificity.

30.  Does not measure the amount of end
product of PCR but its production or
accumulation in real time
 Two common methods of quantification are-
1. the use of fluorescent dyes that intercalate
with double-stranded DNA e.g. SYBR
green
2. modified DNA oligonucleotide probes that
fluoresce when hybridized with a
complementary DNA (Taqman probes,
molecular beacons and scorpion primers)
Dr. Sumbul Fatma

31.  Fluorescent dyes like SYBR green-
A DNA-binding dye binds to all dsDNA in
PCR, causing fluorescence of the dye. An
increase in DNA product during PCR
therefore leads to an increase in
fluorescence intensity and is measured at
each cycle, thus allowing DNA
concentrations to be quantified
Dr. Sumbul Fatma

32. Dr. Sumbul Fatma
Unhybridized probe has donor fluorophore and non-
fluorophore acceptor molecule (quenchers) in close proximity –
no signal
• Upon hybridization to the target, the fluorophore and
quencher become separated through either
•Conformational change- molecular beacons, scorpion
primers
•Enzymatic cleavage of the fluorophore from the quencher
as a result of 5’ to 3’ nuclease activity of the Taq DNA
polymerase- Taqman probes

33.  Comparison of a normal cloned gene with
an uncloned mutant form of the gene
 Detection of low abundance nucleic acid
sequences e.g. viruses, mRNA in cells or
tissue
 Forensic analysis of DNA sample
 Prenatal diagnosis and carrier detection of
Cystic Fibrosis
Dr. Sumbul Fatma

34. It is an autosomal recessive
disorder
Results from mutations in the
cystic fibrosis transmembrane
conductance regulator gene
The most common mutation is
loss of Phe residue from the
protein
Distinguished by difference in
size of the mutated PCR
product
Dr. Sumbul Fatma

35.  Lippincott ‘s Illustrated Reviews, 4th
Edition
 Clinical Chemistry: Principles, Procedures,
Correlations by Michael L Bishop
Dr. Sumbul Fatma