Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction at glance

1. Polymerase Chain Reaction
Mr. Lodha Rahul K.
Department of Biotechnology,
PVP College, Loni

2. It is hard to exaggerate the impact of the
polymerase chain reaction. PCR, the quick,
easy method for generating unlimited
copies of any fragment of DNA, is one of
those scientific developments that
actually deserves timeworn superlatives
like "revolutionary" and "breakthrough."
-Tabitha M. Powledge

3. 1966, Thomas Brock discovers Thermus
Aquaticus, a thermostable bacteria in the hot
springs of Yellowstone National Park
1983, Kary Mullis postulated the concept of PCR
( Nobel Prize in 1993)
1985, Saiki publishes the first application of PCR
( beta-Globin)
1985, Cetus Corp. Scientists isolate Thermostable
Taq Polymerase (from T.Aquaticus), which
revolutionized PCR
HISTORY

4. Introduction
PCR, polymerase chain reaction, is an in-
vitro technique for amplification of a region
of DNA whose sequence is known or which
lies between two regions of known
sequence
Before PCR, DNA of interest could only be
amplified by over-expression in cells and
this with limited yield

5. Purpose of PCR
PCR is an in vitrotechnique for the amplification of a
region of DNA which lies between two regions of known
sequence i.e.Amplify specific nucleic acids in vitro
(“Xeroxing” DNA)
PCR will allow a short stretch of DNA (usually fewer
than 3000 base pairs) to be amplified to about a million
fold
This amplified sample then allows for size determination
and nucleotide sequencing
Introduced in 1985 by Kary Mullis
Millions of copies of a segment of DNA can be made
within a few hours.

6. DNA Replication vs. PCR
PCR is a laboratory version of DNA
Replication in cells
The laboratory version is commonly called “in
vitro” since it occurs in a test tube while “in vivo”
signifies occurring in a living cell.

7. DNA Replication in Cells (in vivo)
DNA replication is the copying
of DNA
It typically takes a cell just a
few hours to copy all of its
DNA
DNA replication is semi-
conservative (i.e. one strand of
the DNA is used as the template
for the growth of a new DNA
strand)
This process occurs with very
few errors (on average there is
one error per 1 billion
nucleotides copied)
More than a dozen enzymes and
proteins participate in DNA
replication

8. Key enzymes involved in DNA
Replication
DNA Polymerase
DNA Ligase
Primase
Helicase
Topoisomerase
Single strand binding protein

9. DNA Replication enzymes:
DNA Polymerase
Catalyzes the elongation of DNA by adding
nucleoside triphosphates to the 3’ end of the
growing strand
A nucleotide triphosphate is a 1 sugar + 1 base + 3 phosphates
When a nucleoside triphosphate joins the DNA strand, two
phosphates are removed.
DNA polymerase can onlyadd nucleotides to 3’
end of growing strand

10. Complementary Base-Pairing in DNA
DNA is a double helix, made up of nucleotides, with a sugar-
phosphate backbone on the outside of the helix.
Note: a nucleotide is a sugar + phosphate + nitrogenous
base
The two strands of DNA are held together by pairs of
nitrogenous bases that are attached to each other via hydrogen
bonds.
The nitrogenous base adenine will only pair with thymine
The nitrogenous base guanine will only pair with cytosine
During replication, once the DNA strands are separated, DNA
polymerase uses each strand as a template to synthesize new
strands of DNA with the precise, complementary order of
nucleotides.

11. DNA Replication enzymes:
DNA Ligase
The two strands of DNA in a double helix are antiparallel
(i.e. they are oriented in opposite directions with one
strand oriented from 5’ to 3’ and the other strand oriented
from 3’ to 5’
5’ and 3’ refer to the numbers assigned to the carbons in the 5
carbon sugar
Given the antiparallel nature of DNA and the fact that
DNA ploymerases can only add nucleotides to the 3’ end,
one strand (referred to as the leading strand) of DNA is
synthesized continuously and the other strand (referred to
as the lagging strand) in synthesized in fragments (called
Okazaki fragments) that are joined together by DNA
ligase.

12. DNA Replication enzymes:
Primase
DNA Polymerase cannotinitiate the synthesis of
DNA
Remember that DNA polymerase can onlyadd nucleotides to
3’ end of an already existing strand of DNA
In humans, primaseis the enzyme that can start
an RNA chain from scratch and it creates a
primer (a short stretch RNA with an available
3’ end) that DNA polymerase can add nucleotides
to during replication.
Note that the RNA primer is subsequently replaced with DNA

13. DNA Replication enzymes:
Helicase, Topoisomerase and Single-strand binding
protein
Helicaseuntwists the two parallel DNA strands
Topoisomeraserelieves the stress of this twisting
Single-strand binding proteinbinds to and
stabilizes the unpaired DNA strands

14. PCR: the in vitroversion of DNA
Replication
The following components are needed to perform PCR
in the laboratory:
1)DNA (your DNA of interest that contains the target
sequence you wish to copy)
2)A heat-stable DNA Polymerase (like Taq Polymerase)
3)All four nucleotide triphosphates
4)Buffers
5)Two short, single-stranded DNA molecules that serve
as primers
6)Thin walled tubes Thermal cycler (a device that can
change temperatures dramatically in a very short
period of time)

15. PCR
The DNA, DNA
polymerase, buffer,
nucleoside
triphosphates, and
primers are placed in
a thin-walled tube
and then these tubes
are placed in the PCR
thermal cycler
PCR Thermocycler

16. Reaction Components
DNA
template
Primers
DNA
Polymerase
dNTPs
Mg
2+
Buffers

17. 1-DNA template
DNA containing
region to be
sequenced
Size of target DNA
to be amplified : up
to 3 Kb

18. PCR primers are short, single stranded DNA
molecules (15-40 bp)
They are manufactured commercially and can
be ordered to match any DNA sequence
Primers are sequence specific, they will bind to
a particular sequence in a genome
As you design primers with a longer length (15
→ 40 bp), the primers become more selective.
DNA polymerase requires primers to initiate
replication
2-Primers

19. Primers (ctnd)
2 sets of primers
Synthetically
produced
Complimentary to the
3’ ends of target DNA
Not complimentary to
each other

20. Primers (ctnd)
Not containing inverted repeat sequences to
avoid formation of internal structures
40-60% GC content preferred for better
annealing
Tm of primers can be calculated to
determine annealing T
0
Tm= 41(%G+C) + 16.6log(J
+
) + 81.5
(where J
+
is the concentration of
monovalent ions)

21. 3-Enzyme
Usually Taq Polymerase or anyone of the
natural or Recombinant thermostable
polymerases
Stable at Temperature up to 95
0
C
High processivity
Taq Polymerase lacks 3’-5’exonuclease
activity, no proofreading

22. DNA Polymerase
DNA Polymerase is the enzyme responsible for copying
the sequence starting at the primer from the single DNA
strand
Commonly use Taq, an enzyme from the
hyperthermophilic organisms Thermus aquaticus,
isolated first at a thermal spring in Yellowstone National
Park
This enzyme is heat-tolerant useful both because it is
thermally tolerant (survives the melting T of DNA
denaturation) which also means the process is more
specific, higher temps result in less mismatch –more
specific replication

23. The PCR Cycle
Comprised of 3 steps:
1.Denaturation of DNA
2.Primer hybridization (Annealing)
3.DNA synthesis (Primer Extension)

24. Three Steps
Denaturation: Double Stranded DNA is denatured by
heat into single strands.
Annealing: Short Primers for DNA replication are
added to the mixture.
Extension: DNA polymerase catalyzes the production
of complementary new strands.
Copying The process is repeated for each new strand
created
All three steps are carried out in the same vial but at
different temperatures

25. The three main steps of PCR
The basis of PCR is temperature changes and the effect that these temperature
changes have on the DNA.
In a PCR reaction, the following series of steps is repeated 20-40 times
(note: 25 cycles usually takes about 2 hours and amplifies the DNA fragment of
interest 100,000 fold)
Step 1: DenatureDNA
At 95C, the DNA is denatured (i.e. the two strands are separated)
Step 2: Primers Anneal
At 40C-65C, the primers anneal (or bind to) their complementary
sequences on the single strands of DNA
Step 3: DNA polymerase Extendsthe DNA chain
At 72C, DNA Polymerase extends the DNA chain by adding
nucleotides to the 3’ ends of the primers.

26. Step 1: Separation
Combine Target Sequence, DNA primers template,
dNTPs, TAQ Polymerase
Target Sequence: Usually fewer than 3000 bp
–Identified by a specific pair of DNA primers-usually
oligonucleotides that are about 20 nucleotides
Heat to 95
o
C to separate strands (for 0.5-2 minutes)
–Longer times increase denaturation but decrease enzyme
and template

27. Magnesium as a Cofactor
Stabilizes the reaction between:
–oligonucleotides and template DNA
–DNA Polymerase and template DNA

28. Heat Denatures DNA by uncoiling the
Double Helix strands.

29. Step 2: Priming
Decrease temperature by 15-25 degrees
Primers anneal to the end of the strand
0.5-2 minutes
Shorter time increases specificity but
decreases yield
Requires knowledge of the base sequences of
the 3’ -end

31. Selecting a Primer
Primer length
Melting Temperature (T
m
)
Specificity
Complementary Primer Sequences
G/C content and Polypyrimidine (T, C) or polypurine
(A, G) stretches
3’-end Sequence
Single-stranded DNA

32. Step 3: Polymerization
Since the Taq polymerase works best
at around 75
o
C (the temperature of
the hot springs where the bacterium
was discovered), the temperature of
the vial is raised to 72-75
o
C
The DNA polymerase recognizes the
primer and makes a complementary
copy of the template which is now
single stranded.
Approximately 150 nucleotides/sec

33. Potential Problems with Taq
Lack of proof-reading of newly synthesized DNA.
Potentially can include diNucleotriphosphates
(dNTPs) that are not complementary to the
original strand.
Errors in coding result
Recently discovered thermostable DNA
polymerases, Tli and Pfu, are less efficient, yet
highly accurate.

35. Amplification

36. Advantages
Automated, fast, reliable (reproducible)
results
Contained :(less chances of
contamination)
High output
Sensitive
Broad uses
Defined, easy to follow protocols

37. PCR Applications
Genome mapping and gene function determination
(Detection of variations and mutations in genes,
detection of diseases from the past, detection of
variations and mutations in genes)
Biodiversity studies ( e.g. evolution studies)
Diagnostics ( prenatal testing of genetic diseases, early
detection of infectious diseases, cancer, viral
infections...)
Detection of drug resistance genes
Forensic (DNA fingerprinting)

38. Detection of infectious
diseases
-AIDS Virus
-Otitis Media-middle ear infection
-Lyme Disease-joint inflammation from tick
bites
-Detect 3 sexually transmitted diseases in one
swab-herpes, papillomarvirus, chlamydia
-Test to see if mother and baby have compatible
blood group-saves lives of babies

39. Detection of Variations and
Mutations in Genes
Detects people with inherited disorders
Lets us know who carries deleterious
variations (mutations)
Direct way of distinguishing among the
confusion of different mutations in a single
gene. Ex: Duchenne muscular dystrophy
Track presence or absence of DNA
abnormalities characteristic to cancer

40. Future of PCR:
Copying larger pieces of DNA
Miniaturization of hardware (chip-sized
devices)
Computer automated test and analysis
Taking PCR on the road and getting on the
spot DNA analysis
Diagnose infection or genetic disorder right
in the doctors office

41. References
“Polymerase Chain Reaction-Xeroxing DNA”
http://www.accessexcellence.org/AB/IE/PCR_Xeroxing_DNA.html
“The Polymerase Chain Reaction”
http://avery.rutgers.edu/WSSP/StudentScholars/project/archives/onions/pcr.ht
ml
“Polymerase Chain reaction”
http://www.tulane.edu/~wiser/methods/handouts/pcr.PDF
Diagrams from : http://allserv.rug.ac.be/~avierstr/principles/pcrani.html
Purves, Sadava, Orians, Heller. “Life.” 6
th
ed. Sinauer Associates, 2001.
“Mechanism of PCR.” http://usitweb.shef.ac.uk/~mba97cmh/tutorial/pcr.htm
“The polymerase Chain Reaction
”www.faseb.org/opar/bloodsupply/pcr.html
Fundamentals of Biochemistry ( Voet, Voet, Pratt)
Molecular Cell Biology ( Lodish, Darnell.)

42. Mr. Rahul K. Lodha
Assistant Professor,
Department of Biotechnology,
PVP College of Arts, Science & Commerce,
Pravaranagar, Loni, Maharashtra, India .
Email: [email protected]
Contact: [phone]
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