This document provides an overview of polymerase chain reaction (PCR). It discusses: 1. The key discoveries and scientists that developed PCR, including Kary Mullis who won the 1993 Nobel Prize for his conception of PCR. 2. The basic steps and components of PCR including DNA template, primers, Taq polymerase enzyme, repeated temperature cycling of denaturation, annealing and extension to exponentially amplify the target DNA region. 3. Applications of PCR including DNA cloning, medical diagnostics, forensics, and research. PCR is used to amplify specific genes or DNA regions for various downstream analyses and applications.

1. 1

2. Naeem Ahmed Niazi
M.Shahbaz
M.Summar Sultan
M.Salman Tariq
Group Members
2

3. Polymerase Chain Reaction
3

4. Introduction
 1966, Thomas Brock discovers Thermus Aquaticus, a thermo-
stable 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 Thermo-stable Taq
Polymerase (from T.Aquaticus), which revolutionized PCR
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5. Key Points
 Polymerase chain reaction, or PCR, is a technique to make
many copies of a specific DNA region in vitro (in a test tube
rather than an organism).
 PCR relies on a thermo-stable DNA
polymerase, Taq polymerase, and requires
DNA primers designed specifically for the DNA region of
interest.
5

6.  In PCR, the reaction is repeatedly cycled through a series of
temperature changes, which allow many copies of the target
region to be produced.
 PCR has many research and practical applications. It is
routinely used in DNA cloning, medical diagnostics, and
forensic analysis of DNA.
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7. What is PCR?
 Polymerase chain reaction (PCR) is a common
laboratory technique used to make many copies (millions
or billions!) of a particular region of DNA. This DNA
region can be anything the experimenter is interested in.
For example, it might be a gene whose function a
researcher wants to understand, or a genetic marker used
by forensic scientists to match crime scene DNA with
suspects.
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8.  Typically, the goal of PCR is to make enough of the target
DNA region that it can be analyzed or used in some other
way. For instance, DNA amplified by PCR may be sent
for sequencing, visualized by gel electrophoresis,
or cloned into a plasmid for further experiments.
 PCR is used in many areas of biology and medicine,
including molecular biology research, medical diagnostics,
and even some branches of ecology.
8

9. Reaction Components
 DNA template
 Primers
 Enzyme
 dNTPs
 Mg2+
 Buffers
9

10. Taq polymerase
 Like DNA replication in an organism, PCR requires a DNA
polymerase enzyme that makes new strands of DNA, using
existing strands as templates. The DNA polymerase typically
used in PCR is called Taq polymerase, after the heat-
tolerant bacterium from which it was isolated
(Thermus aquaticus).
10

11.  T. aquaticus lives in hot springs and hydrothermal vents. Its
DNA polymerase is very heat-stable and is most active
around (a temperature at which a human or E. coli DNA
polymerase would be nonfunctional). This heat-stability
makes Taq polymerase ideal for PCR. As we'll see, high
temperature is used repeatedly in PCR to denature the
template DNA, or separate its strands.
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12. PCR primers
 Like other DNA polymerases, Taq polymerase can only
make DNA if it's given a primer, a short sequence of
nucleotides that provides a starting point for DNA synthesis.
In a PCR reaction, the experimenter determines the region of
DNA that will be copied, or amplified, by the primers she or
he chooses.
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13.  PCR primers are short pieces of single-stranded DNA, usually
around 20 nucleotides in length. Two primers are used in each
PCR reaction, and they are designed so that they flank the
target region (region that should be copied). That is, they are
given sequences that will make them bind to opposite strands
of the template DNA, just at the edges of the region to be
copied.The primers bind to the template by complementary
base pairing.
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14. 14

15.  When the primers are bound to the template, they can be
extended by the polymerase, and the region that lies between
them will get copied.
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16. 16

17. The steps of PCR
 The key ingredients of a PCR reaction are Taq polymerase,
primers, template DNA, and nucleotides (DNA building
blocks). The ingredients are assembled in a tube, along with
cofactors needed by the enzyme, and are put through repeated
cycles of heating and cooling that allow DNA to be
synthesized.
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18. The basic steps are:
 Denaturation (96°C): Heat the reaction strongly to separate,
or denature, the DNA strands. This provides single-stranded
template for the next step.
 Annealing (55 - 65°C): Cool the reaction so the primers can
bind to their complementary sequences on the single-stranded
template DNA.
 Extension (72°C): Raise the reaction temperatures
so Taq polymerase extends the primers, synthesizing new
strands of DNA.
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19. 19

20.  This cycle repeats 25–35 times in a typical PCR reaction,
which generally takes 2–4 hours, depending on the length
of the DNA region being copied. If the reaction is
efficient (works well), the target region can go from just
one or a few copies to billions.
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21.  That’s because it’s not just the original DNA that’s used as a
template each time. Instead, the new DNA that’s made in one
round can serve as a template in the next round of DNA
synthesis. There are many copies of the primers and many
molecules of Taq polymerase floating around in the reaction,
so the number of DNA molecules can roughly double in each
round of cycling.
21

22. This pattern of exponential growth
is shown in the image below.
22

23. Using gel electrophoresis to
visualize the results of PCR
 The results of a PCR reaction are usually visualized (made
visible) using gel electrophoresis. Gel electrophoresis is a
technique in which fragments of DNA are pulled through a gel
matrix by an electric current, and it separates DNA fragments
according to size. A standard, or DNA ladder, is typically
included so that the size of the fragments in the PCR sample
can be determined.
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24.  DNA fragments of the same length form a "band" on the gel,
which can be seen by eye if the gel is stained with a DNA-
binding dye. For example, a PCR reaction producing
a 400 base pair (bp) fragment would look like this on a gel:
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25. 25

26.  A DNA band contains many, many copies of the target DNA
region, not just one or a few copies. Because DNA is
microscopic, lots of copies of it must be present before we
can see it by eye. This is a big part of why PCR is an
important tool: it produces enough copies of a DNA sequence
that we can see or manipulate that region of DNA.
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27. Applications of PCR
 Using PCR, a DNA sequence can be amplified millions or
billions of times, producing enough DNA copies to be
analyzed using other techniques. For instance, the DNA may
be visualized by gel electrophoresis, sent for sequencing , or
digested with restriction enzymes and cloned into a plasmid.
27

28.  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). PCR can also be used
to test for a bacterium or DNA virus in a patient's body: if the
pathogen is present, it may be possible to amplify regions of
its DNA from a blood or tissue sample.
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29. PCR in forensics
 Suppose that you are working in a forensics lab. You have
just received a DNA sample from a hair left at a crime scene,
along with DNA samples from three possible suspects. Your
job is to examine a particular genetic marker and see whether
any of the three suspects matches the hair DNA for this
marker.
29

30.  The marker comes in two alleles, or versions. One contains a
single repeat (brown region below), while the other contains
two copies of the repeat. In a PCR reaction with primers that
flank the repeat region, the first allele produces a 200 bp DNA
fragment, while the second produces a 300 bp DNA fragment:
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31. 31

32. PCR on the four DNA samples and
visualize the results by gel electrophoresis,
as shown below:
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33. More about PCR and forensics
 In real forensic tests of DNA from a crime scene, technicians
would do an analysis conceptually similar to the one in the
example above. However, a number of different markers (not
just the single marker in the example) would be compared
between the crime scene DNA and the suspects' DNA.
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34.  Also, the markers used in a typical forensic analysis don't
come in just two different forms. Instead, they're
highly polymorphic (poly = many, morph = form). That is,
they come in many alleles that vary in tiny increments of
length.
 The most commonly used type of markers in forensics,
called short tandem repeats (STRs), consist of many
repeating copies of the same short nucleotide sequence
(typically, 2 to 5 nucleotides long). One allele of an STR
might have 20 repeats, while another might have 18, and
another just 101
.
34

35.  By examining multiple markers, each of which comes in
many allele forms, forensic scientists can build a unique
genetic "fingerprint" from a DNA sample. In a typical STR
analysis using 13 markers, the odds of a false positive (two
people having the same DNA "fingerprint") are less
than 1 in 10 billion start superscript, 1, end superscript!
 Although we may think of DNA evidence being used to
convict criminals, it has played a crucial role in exonerating
falsely accused people (including some who had been jailed
for many years). Forensic analysis is also used to establish
paternity and to identify human remains from disaster scenes.
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36. Gel electrophoresis
36

37. Key Points
 Gel electrophoresis is a technique used to separate DNA
fragments according to their size.
 DNA samples are loaded into wells (indentations) at one
end of a gel, and an electric current is applied to pull them
through the gel.
 DNA fragments are negatively charged, so they move
towards the positive electrode. Because all DNA fragments
have the same amount of charge per mass, small fragments
move through the gel faster than large ones.
37

38.  When a gel is stained with a DNA-binding dye, the DNA
fragments can be seen as bands, each representing a group of
same-sized DNA fragments.
38

39. Introduction
 Suppose you have just done a PCR reaction, making many
copies of a target DNA region. Or perhaps you’ve done
some DNA cloning, trying to "paste" a gene into a circular
DNA plasmid.
 Now, you want to check and see whether your PCR
worked, or whether your plasmid has the right gene in it.
What technique can you use to visualize (directly observe)
the fragments of DNA?
39

40. Gel electrophoresis
 Gel electrophoresis is a technique used to separate DNA
fragments (or other macromolecules, such as RNA and
proteins) based on their size and charge. Electrophoresis
involves running a current through a gel containing the
molecules of interest. Based on their size and charge, the
molecules will travel through the gel in different directions or
at different speeds, allowing them to be separated from one
another.
40

41.  All DNA molecules have the same amount of charge per mass.
Because of this, gel electrophoresis of DNA fragments separates
them based on size only. Using electrophoresis, we can see how
many different DNA fragments are present in a sample and how
large they are relative to one another. We can also determine the
absolute size of a piece of DNA by examining it next to a
standard "yardstick" made up of DNA fragments of known
sizes.
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42. What is a gel?
 As the name suggests, gel electrophoresis involves a gel: a slab
of Jello-like material. Gels for DNA separation are often made
out of a polysaccharide called agarose, which comes as dry,
powdered flakes. When the agarose is heated in a buffer (water
with some salts in it) and allowed to cool, it will form a solid,
slightly squishy gel. At the molecular level, the gel is a matrix
of agarose molecules that are held together by hydrogen bonds
and form tiny pores.
42

43. At one end, the gel has pocket-like
indentations called wells, which are where
the DNA samples will be placed:
43

44.  Before the DNA samples are added, the gel must be placed in
a gel box. One end of the box is hooked to a positive
electrode, while the other end is hooked to a negative
electrode. The main body of the box, where the gel is placed,
is filled with a salt-containing buffer solution that can conduct
current. Although you may not be able to see in the image
above , the buffer fills the gel box to a level where it just
barely covers the gel.
44

45.  The end of the gel with the wells is positioned towards the
negative electrode. The end without wells (towards which the
DNA fragments will migrate) is positioned towards the
positive electrode.
45

46. How do DNA fragments move
through the gel?
 Once the gel is in the box, each of the DNA samples we want to
examine (for instance, each PCR reaction or each restriction-
digested plasmid) is carefully transferred into one of the wells.
One well is reserved for a DNA ladder, a standard reference that
contains DNA fragments of known lengths. Commercial DNA
ladders come in different size ranges, so we would want to pick
one with good "coverage" of the size range of our expected
fragments.
46

47.  Next, the power to the gel box is turned on, and current
begins to flow through the gel. The DNA molecules have a
negative charge because of the phosphate groups in their
sugar-phosphate backbone, so they start moving through the
matrix of the gel towards the positive pole. When the power is
turned on and current is passing through the gel, the gel is
said to be running.
47

48. 48

49.  DNA samples are loaded into wells at negative electrode end
of gel. Power is turned on and DNA fragments migrate
through gel (towards the positive electrode). After the gel has
run, the fragments are separated by size. The largest
fragments are near the top of the gel (negative electrode,
where they began), and the smallest fragments are near the
bottom (positive electrode).
49

50.  As the gel runs, shorter pieces of DNA will travel through
the pores of the gel matrix faster than longer ones. After the
gel has run for awhile, the shortest pieces of DNA will be
close to the positive end of the gel, while the longest pieces
of DNA will remain near the wells. Very short pieces of
DNA may have run right off the end of the gel if we left it
on for too long (something I've most definitely been guilty
of!).
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51. Visualizing the DNA fragments
 Once the fragments have been separated, we can examine the
gel and see what sizes of bands are found on it. When a gel is
stained with a DNA-binding dye and placed under UV light,
the DNA fragments will glow, allowing us to see the DNA
present at different locations along the length of the gel.
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52. 52

53.  The bp next to each number in the ladder indicates how
many base pairs long the DNA fragment is.
 A well-defined “line” of DNA on a gel is called a band. Each
band contains a large number of DNA fragments of the same
size that have all traveled as a group to the same position. A
single DNA fragment (or even a small group of DNA
fragments) would not be visible by itself on a gel.
 By comparing the bands in a sample to the DNA ladder, we
can determine their approximate sizes. For instance, the bright
band on the gel above is roughly 700 base pairs (bp) in size.
53

54. Check your understanding
 Four lanes are numbered on the gel above. (A lane is a
corridor through which DNA passes as it leaves a well.)
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55. DNA sequencing
55

56. Key points:
 DNA sequencing is the process of determining the sequence
of nucleotides (As, Ts, Cs, and Gs) in a piece of DNA.
 In Sanger sequencing, the target DNA is copied many times,
making fragments of different lengths. Fluorescent “chain
terminator” nucleotides mark the ends of the fragments and
allow the sequence to be determined.
 Next-generation sequencing techniques are new, large-scale
approaches that increase the speed and reduce the cost of
DNA sequencing.
56

57. What is sequencing?
 DNA sequencing is the process of determining the sequence of
nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA. Today,
with the right equipment and materials, sequencing a short piece
of DNA is relatively straightforward.
 Sequencing an entire genome (all of an organism’s DNA)
remains a complex task. It requires breaking the DNA of the
genome into many smaller pieces, sequencing the pieces, and
assembling the sequences into a single long "consensus."
However, thanks to new methods that have been developed over
the past two decades, genome sequencing is now much faster
and less expensive than it was during the Human Genome
Project.
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58.  In this article, we’ll take a look at methods used for DNA
sequencing. We'll focus on one well-established method,
Sanger sequencing, but we'll also discuss new ("next-
generation") methods that have reduced the cost and
accelerated the speed of large-scale sequencing.
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59. Sanger sequencing: The chain
termination method
 Regions of DNA up to about 900 base pairs in length are
routinely sequenced using a method called Sanger
sequencing or the chain termination method. Sanger
sequencing was developed by the British biochemist Fred Sanger
and his colleagues in 1977.
59

60.  In the Human Genome Project, Sanger sequencing was used to
determine the sequences of many relatively small fragments of
human DNA. (These fragments weren't necessarily 900 bp or
less, but researchers were able to "walk" along each fragment
using multiple rounds of Sanger sequencing.) The fragments
were aligned based on overlapping portions to assemble the
sequences of larger regions of DNA and, eventually, entire
chromosomes.
60

61.  Although genomes are now typically sequenced using other
methods that are faster and less expensive, Sanger
sequencing is still in wide use for the sequencing of
individual pieces of DNA, such as fragments used in DNA
cloning or generated through polymerase chain
reaction (PCR).
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62. Ingredients for Sanger sequencing
 Sanger sequencing involves making many copies of a target
DNA region. Its ingredients are similar to those needed
for DNA replication in an organism, or for polymerase chain
reaction (PCR), which copies DNA in vitro. They include:
 A DNA polymerase enzyme
 A primer, which is a short piece of single-stranded DNA that
binds to the template DNA and acts as a "starter" for the
polymerase
 The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)
62

63.  The template DNA to be sequenced
 However, a Sanger sequencing reaction also contains a unique
ingredient:
 Dideoxy, or chain-terminating, versions of all four
nucleotides (ddATP, ddTTP, ddCTP, ddGTP), each labeled
with a different color of dye
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64. 64

65.  Dideoxy nucleotides are similar to regular, or deoxy,
nucleotides, but with one key difference: they lack a hydroxyl
group on the 3’ carbon of the sugar ring. In a regular
nucleotide, the 3’ hydroxyl group acts as a “hook," allowing a
new nucleotide to be added to an existing chain.
 Once a dideoxy nucleotide has been added to the chain, there
is no hydroxyl available and no further nucleotides can be
added. The chain ends with the dideoxy nucleotide, which is
marked with a particular color of dye depending on the base
(A, T, C or G) that it carries.
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66. Method of Sanger sequencing
 The DNA sample to be sequenced is combined in a tube with
primer, DNA polymerase, and DNA nucleotides (dATP,
dTTP, dGTP, and dCTP). The four dye-labeled, chain-
terminating dideoxy nucleotides are added as well, but in
much smaller amounts than the ordinary nucleotides.
66

67.  The mixture is first heated to denature the template DNA
(separate the strands), then cooled so that the primer can bind
to the single-stranded template. Once the primer has bound,
the temperature is raised again, allowing DNA polymerase to
synthesize new DNA starting from the primer. DNA
polymerase will continue adding nucleotides to the chain
until it happens to add a dideoxy nucleotide instead of a
normal one. At that point, no further nucleotides can be
added, so the strand will end with the dideoxy nucleotide.
67

68.  This process is repeated in a number of cycles. By the time
the cycling is complete, it’s virtually guaranteed that a
dideoxy nucleotide will have been incorporated at every
single position of the target DNA in at least one reaction.
That is, the tube will contain fragments of different lengths,
ending at each of the nucleotide positions in the original
DNA (see figure below). The ends of the fragments will be
labeled with dyes that indicate their final nucleotide.
68

69. 69

70.  After the reaction is done, the fragments are run through a
long, thin tube containing a gel matrix in a process
called capillary gel electrophoresis. Short fragments move
quickly through the pores of the gel, while long fragments
move more slowly. As each fragment crosses the “finish
line” at the end of the tube, it’s illuminated by a laser,
allowing the attached dye to be detected.
70

71.  The smallest fragment (ending just one nucleotide after the
primer) crosses the finish line first, followed by the next-
smallest fragment (ending two nucleotides after the primer),
and so forth. Thus, from the colors of dyes registered one
after another on the detector, the sequence of the original
piece of DNA can be built up one nucleotide at a time. The
data recorded by the detector consist of a series of peaks in
fluorescence intensity, as shown in
the chromatogram above. The DNA sequence is read from
the peaks in the chromatogram.
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72. Uses and limitations
 Sanger sequencing gives high-quality sequence for relatively
long stretches of DNA (up to about 900 base pairs). It's
typically used to sequence individual pieces of DNA, such
as bacterial plasmids or DNA copied in PCR.
 However, Sanger sequencing is expensive and inefficient for
larger-scale projects, such as the sequencing of an entire
genome or metagenome (the “collective genome” of a
microbial community). For tasks such as these, new, large-
scale sequencing techniques are faster and less expensive.
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73. Next-generation sequencing
 The name may sound like Star Trek, but that’s really what
it’s called! The most recent set of DNA sequencing
technologies are collectively referred to as next-generation
sequencing.
 There are a variety of next-generation sequencing techniques
that use different technologies. However, most share a
common set of features that distinguish them from Sanger
sequencing:
73

74.  Highly parallel: many sequencing reactions take place at the
same time
 Micro scale: reactions are tiny and many can be done at once
on a chip
 Fast: because reactions are done in parallel, results are ready
much faster
 Low-cost: sequencing a genome is cheaper than with Sanger
sequencing
 Shorter length: reads typically range from 50 -
700 nucleotides in length
74

75.  Conceptually, next-generation sequencing is kind of like
running a very large number of tiny Sanger sequencing
reactions in parallel. Thanks to this parallelization and small
scale, large quantities of DNA can be sequenced much more
quickly and cheaply with next-generation methods than with
Sanger sequencing. For example, in 2001, the cost of
sequencing a human genome was almost $100millionm. In
2015, it was just $1245dollar sign, 12452
!
75

76.  Why does fast and inexpensive sequencing matter?
The ability to routinely sequence genomes opens new
possibilities for biology research and biomedical
applications. For example, low-cost sequencing is a step
towards personalized medicine – that is, medical treatment
tailored to an individual's needs, based on the gene variants
in his or her genome.
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78. THE ENDTHE END
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