TERM PAPER

OF
BTY 312
ON THE TOPIC
PCR
Submitted to Submitted by
Ms. Kuljot Satveer kaur
Roll no-21
D 7701

INDEX
• Introduction
• History and discovery
• The technique explained
• Types of PCR
• Principle of PCR
• PCR stages
• PCR optimization
• The evolution of PCR
• Limitations and difficulties
• Overview of PCR
• Problems of PCR
• Applications of PCR
• The future of PCR
• Refernces

INTRODUCTION
PCR (polymerase chain reaction) is a technique in molecular genetics that
permits the analysis of any short sequence of DNA (or RNA ) even in
samples containing only minute quantities of DNA or RNA. PCR is used to
reproduce (amplify) selected sections of DNA or RNA for analysis.
Previously, amplification of DNA involved cloning the segments of interest
into vectors for expression in bacteria , and took weeks. But now, with PCR
done in test tubes, it takes only a few hours. PCR is highly efficient so that
untold numbers of copies can be made of the DNA. What is more, PCR uses
the same molecules that nature uses for copying DNA:

• Two "primers", short single-stranded DNA sequences that are

synthesized to correspond to the beginning and ending of the DNA
stretch to be copied;
• An enzyme called polymerase that moves along the segment of DNA,
reading its code and assembling a copy; and
• A pile of DNA building blocks that the polymerase needs to make that
copy.

OR

PCR (polymerase chain reaction) is a technique that is central to molecular

biology research. It used in applications such as cloning, gene expression
analysis, genotyping, sequencing, and mutagenesis to name a few.
Invitrogen offers an array of products to help you achieve PCR success. For
enzymes, our AccuPrime™ technology provides specific primer-template
hybridization in every cycle of PCR, while our Platinum® and AmpliTaq
Gold® 360 technologies provide efficient hot starts for robust PCR
performance.

Three major steps are involved in a PCR. These three steps are repeated for
30 or 40 cycles. The cycles are done on an automated cycler, a device which
rapidly heats and cools the test tubes containing the reaction mixture. Each
step -- denatauration (alteration of structure), annealing (joining), and
extension -- takes place at a different temperature:

1. Denaturation: At 94 C (201.2 F), the double-stranded DNA melts and

opens into two pieces of single-stranded DNA.
2. Annealing: At medium temperatures, around 54 C (129.2 F), the
primers pair up (anneal) with the single-stranded "template" (The
template is the sequence of DNA to be copied.) On the small length of
double-stranded DNA (the joined primer and template), the
polymerase attaches and starts copying the template.
3. Extension: At 72 C (161.6 F), the polymerase works best, and DNA
building blocks complementary to the template are coupled to the
primer, making a double stranded DNA molecule .

With one cycle, a single segment of double-stranded DNA template has

thus been amplified into two separate pieces of double-stranded DNA. These
two pieces are then available for amplification in the next cycle. As the
cycles are repeated, more and more copies are generated and the number of
copies of the template is increased exponentially.

HISTORY AND DISCOVERY

PCR was invented by Kary Mullis. At the time he thought up PCR in 1983,
Mullis was working in Emeryville, California for Cetus, one of the first
biotechnology companies. There, he was charged with making short chains
of DNA for other scientists. Mullis has written that he conceived of PCR
while cruising along the Pacific Coast Highway 128 one night on his
motorcycle. He was playing in his mind with a new way of analyzing
changes (mutations) in DNA when he realized that he had instead invented a
method of amplifying any DNA region. Mullis has said that before his
motorcycle trip was over, he was already savoring the prospects of a Nobel
Prize. He shared the Nobel Prize in chemistry with Michael Smith in 1993.
The Technique Explained
A mixture is created, with optimized concentrations of the DNA template,
polymerase enzyme, primers and dNTPs. The ability to heat the mixture
without denaturing the enzyme allows for denaturing of the double helix of
DNA sample at temperatures in the range of 94 degrees Celsius. Following
denaturation, the sample is cooled to a more moderate range, around 54
degrees, which facilitates the annealing (binding) of the primers to the
single-stranded DNA templates. In the third step of the cycle, the sample is
reheated to 72 degrees, the ideal temperature for Taq DNA Polymerase, for
elongation. During elongation, DNA polymerase uses the original single
strand of DNA as a template to add complimentary dNTPs to the 3’ ends of
each primer and generate a section of double-stranded DNA in the region of
the gene of interest. Primers that have annealed to DNA sequences that are
not an exact match do not remain annealed at 72 degrees, thus limiting
elongation to the gene of interest
This process of denaturing, annealing and elongation is repeated multiple
(30-40) times, thereby increasing exponentially the number of copies of the
desired gene in the mixture. Although this process would be quite tedious if
performed manually, samples can be prepared and incubated in a
programmable Thermocycler, now commonplace in most molecular
laboratories, and a complete PCR reaction can be performed in 3-4 hours.
Each denaturing step stops the elongation process of the previous cycle, thus
truncating the new strand of DNA and keeping it to approximately the size
of the desired gene. The duration of the elongation cycle can be made longer
or shorter depending on the size of the gene of interest, but eventually,
through repeated cycles of PCR, the majority of templates will be restricted
to the size of the gene of interest alone, as they will have been generated
from products of both of the primers.

A number of factors will affect whether or not a PCR is successful. Some of

these are the quality of the original DNA sample, quantity of template DNA
and suitability of the primers. The most widely used method to test for PCR
product is gel electrophoresis which is used to separate DNA fragments
based on size and charge. The fragments are then visualized using dyes or
radioisotopes.

Types of
PCR -
Polymerase
Chain
Reaction
- Types as follows:

I.Inverse PCR.
II.Anchored PCR.
III. PCR for site directed mutagenesis.
IV. Cloning and expression of the PCR product.
V. Asymmetric PCR for DNA sequencing.

Inverse PCR - Polymerase Chain Reaction –

In this technique, the amplification of those DNA sequences takes place,
which are away from the primers and not those which are flanked by the
primers.

For instance if the border sequences of a DNA segment are not known and
those of a vector are known, then the sequence to be amplified may be
cloned in the vector and border sequences of vector may be used as primers
in such a way that the polymerization proceeds in inverse direction i.e. away
from the vector sequence flanked by the primers and towards the DNA
sequence of inserted segment.

Anchored PCR - Polymerase Chain Reaction –

In the basic PCR technique and the inverse PCR, one has to use two primers
representing the sequences lying at the ends of sequences LO be amplified.
But sometimes, we may have knowledge about sequence at only one of the
two ends of the DNA sequence to be amplified.

In such cases, anchored PCR may be used, which will utilize only one
primer instead of two primers. In. this technique, due to the use of one
primer, only one strand will be copied first, after which a poly G tail will be
attached at the end of the newly synthesized strand.

This newly synthesized strand with poly G tail at its 3' end will then become
template for the daughter strand synthesis utilizing an anchor primer with
which a Poly C sequence is linked to complement with poly G of the
template. In the next cycle, both the original primer and anchored primer
will be used for gene amplification.

Principle of the PCR

The purpose of a PCR (Polymerase Chain Reaction) is to make a huge
number of copies of a gene. This is necessary to have enough starting
template for sequencing.

1. The cycling reactions : There are three major steps in a PCR, which
are repeated for 30 or 40 cycles. This is done on an automated cycler,
which can heat and cool the tubes with the reaction mixture in a very
short time.
1. Denaturation at 94°C :
During the denaturation, the double strand melts open to single
stranded DNA, all enzymatic reactions stop (for example : the
extension from a previous cycle).
2. Annealing at 54°C :
The primers are jiggling around, caused by the Brownian
motion. Ionic bonds are constantly formed and broken between
the single stranded primer and the single stranded template. The
more stable bonds last a little bit longer (primers that fit
exactly) and on that little piece of double stranded DNA
(template and primer), the polymerase can attach and starts
copying the template. Once there are a few bases built in, the
ionic bond is so strong between the template and the primer,
that it does not break anymore.
3. extension at 72°C :
This is the ideal working temperature for the polymerase. The
primers, where there are a few bases built in, already have a
stronger ionic attraction to the template than the forces breaking
these attractions. Primers that are on positions with no exact
match, get loose again (because of the higher temperature) and
don't give an extension of the fragment.
The bases (complementary to the template) are coupled to the
primer on the 3' side (the polymerase adds dNTP's from 5' to 3',
reading the template from 3' to 5' side, bases are added
complementary to the template)
 The different steps in PCR.

Because both strands are copied during PCR, there is an exponential

increase of the number of copies of the gene. Suppose there is only one copy
of the wanted gene before the cycling starts, after one cycle, there will be 2
copies, after two cycles, there will be 4 copies, three cycles will result in 8
copies and so on.

Is there a gene copied during PCR and is it the right

size ?
 Before the PCR product is used in further applications, it has to be checked
if :

1. There is a product formed.

Though biochemistry is an exact science, not every PCR is successful.
There is for example a possibility that the quality of the DNA is poor,
that one of the primers doesn't fit, or that there is too much starting
template
2. The product is of the right size
It is possible that there is a product, for example a band of 500 bases,
but the expected gene should be 1800 bases long. In that case, one of
the primers probably fits on a part of the gene closer to the other
primer. It is also possible that both primers fit on a totally different
gene.
3. Only one band is formed.
As in the description above, it is possible that the primers fit on the
desired locations, and also on other locations. In that case, you can
have different bands in one lane on a gel.

PCR stages
The PCR process can be divided into three stages:
Exponential amplification: At every cycle, the amount of product is doubled
(assuming 100% reaction efficiency). The reaction is very sensitive: only
minute quantities of DNA need to be present.[12]

Levelling off stage: The reaction slows as the DNA polymerase loses activity
and as consumption of reagents such as dNTPs and primers causes them to
become limiting.

Plateau: No more product accumulates due to exhaustion of reagents and

enzyme.

PCR optimization
In practice, PCR can fail for various reasons, in part due to its sensitivity to
contamination causing amplification of spurious DNA products. Because of
this, a number of techniques and procedures have been developed for
optimizing PCR conditions.[13][14] Contamination with extraneous DNA is
addressed with lab protocols and procedures that separate pre-PCR mixtures
from potential DNA contaminants.[6] This usually involves spatial separation
of PCR-setup areas from areas for analysis or purification of PCR products,
use of disposable plasticware, and thoroughly cleaning the work surface
between reaction setups. Primer-design techniques are important in
improving PCR product yield and in avoiding the formation of spurious
products, and the usage of alternate buffer components or polymerase
enzymes can help with amplification of long or otherwise problematic
regions of DNA.

The Evolution of PCR

Since the discovery of PCR, DNA polymerases other than the original Taq
have been discovered. Some of these have better “proof-reading” ability or
are more stable at higher temperatures, thus improving the specificity of
PCR and reducing errors from insertion of the incorrect dNTP.

Some variations of PCR have been designed for specific applications and are
now used regularly in molecular genetic laboratories. Some of these are
Real-Time PCR and Reverse-Transcriptase PCR. The discovery of PCR has
also lead to the development of DNA sequencing, DNA fingerprinting and
other molecular techniques.

Limitations/Difficulties
While a very powerful technique, PCR can also be very tricky. The
polymerase reaction is very sensitive to the levels of divalent cations
(especially Mg2+) and nucleotides, and the conditions for each particular
application must be worked out. Primer design is extremely important for
effective amplification. The primers for the reaction must be very specific
for the template to be amplified. Cross reactivity with non-target DNA
sequences results in non-specific amplification of DNA. Also, the primers
must not be capable of annealing to themselves or each other, as this will
result in the very efficient amplification of short nonsense DNAs. The
reaction is limited in the size of the DNAs to be amplified (i.e., the distance
apart that the primers can be placed). The most efficient amplification is in
the 300 - 1000 bp range, however amplification of products up to 4 Kb has
been reported. Also, Taq polymerase has been reported to make frequent
mismatch mistakes when incorperating new bases into a strand.

The most important consideration in PCR is contamination. If the sample

that is being tested has even the smallest contamination with DNA from the
target, the reaction could amplify this DNA and report a falsely positive
identification. For example, if a technician in a crime lab set up a test
reaction (with blood from the crime scene) after setting up a positive control
reaction (with blood from the suspect) cross contamination between the
samples could result in an erroneous incrimination, even if the technician
changed pipette tips between samples. A few blood cells could volitilize in
the pipette, stick to the plastic of the pipette, and then get ejected into the
test sample. The powerful amplification of PCR may be able to detect this
cross contamination of samples. Modern labs take account of this fact and
devote tremendous effort to avoiding this problem.

OVERVIEW OF PCR
An overview of the polymerase chain reaction (PCR) analysis
(a) DNA is obtained from a cell and placed in a test tube with appropriate
other materials.
(b) The enzyme DNA polymerase duplicates the target DNA millions of
times. (r With multiple copies of the DNA present, the DNA probe can
easily locate its complementary binding site

Problems with PCR

But the PCR is not without problems. Chief among these is contamination. If
any contaminating DNA is present, it is amplified along with the target
DNA. To eliminate this possibility, great care must be taken in sample
preparation. Such preparation tends to be labor intensive and costly.
Moreover, the PCR is not quantitative - it can determine that a particular
DNA segment is present but it cannot determine how much of it was
originally present. And, because the process is so new, reproducible results
may be difficult to obtain and a period of time for standardization will be
required. Despite these drawbacks, the PCR used in conjunction with DNA
probes holds great promise for the future
Applications of PCR

The Polymerase Chain Reaction (PCR) has found widespread application in

many areas of genetic analysis. This is a list of some of these applications

Medical applications
PCR has been applied to a large number of medical procedures:

• The first application of PCR[1] was for genetic testing, where a sample
of DNA is analyzed for the presence of genetic disease mutations.
Prospective parents can be tested for being genetic carriers, or their
children might be tested for actually being affected by a disease. DNA
samples for Prenatal testing can be obtained by amniocentesis,
chorionic villus sampling, or even by the analysis of rare fetal cells
 circulating in the mother's bloodstream. PCR analysis is also essential
to Preimplantation genetic diagnosis, where individual cells of a
developing embryo are tested for mutations.

• PCR can also be used as part of a sensitive test for tissue typing, vital
to organ transplantation. As of 2008, there is even a proposal to
replace the traditional antibody-based tests for blood type with PCR-
based tests[2].

• Many forms of cancer involve alterations to oncogenes. By using

PCR-based tests to study these mutations, therapy regimens can
sometimes be individually customized to a patient.

Infectious disease applications

• Some disease organisms, such as that for Tuberculosis, are difficult

to sample from patients and slow to be grown in the laboratory. PCR-
based tests have allowed detection of small numbers of disease
organisms (both live or dead), in convenient samples. Detailed genetic
analysis can also be used to detect antibiotic resistance, allowing
immediate and effective therapy. The effects of therapy can also be
immediately evaluated.

• The spread of a disease organism through populations of domestic or

wild animals can be monitored by PCR testing. In many cases, the
appearance of new virulent sub-types can be detected and monitored.
The sub-types of an organism that were responsible for earlier
epidemics can also be determined by PCR analysis.

Forensic applications
The development of PCR-based genetic (or DNA) fingerprinting protocols
has seen widespread application in forensics:

• In its most discriminating form, Genetic fingerprinting can uniquely

discriminate any one person from the entire population of the world.
Minute samples of DNA can be isolated from a crime scene, and
compared to that from suspects, or from a DNA database of earlier
evidence or convicts. Simpler versions of these tests are often used to
rapidly rule out suspects during a criminal investigation. Evidence
from decades-old crimes can be tested, confirming or exonerating the
people originally convicted.

• Less discriminating forms of DNA fingerprinting can help in

Parental testing, where an individual is matched with their close
relatives. DNA from unidentified human remains can be tested, and
compared with that from possible parents, siblings, or children.
Similar testing can be used to confirm the biological parents of an
adopted (or kidnapped) child. The actual biological father of a
newborn can also be confirmed (or ruled out).

Research applications
PCR has been applied to many areas of research in molecular genetics:

• PCR allows rapid production of short pieces of DNA, even when

nothing more than the sequence of the two primers is known. This
ability of PCR augments many methods, such as generating
hybridization probes for Southern or northern blot hybridization.
PCR supplies these techniques with large amounts of pure DNA,
sometimes as a single strand, enabling analysis even from very small
amounts of starting material.

• The task of DNA sequencing can also be assisted by PCR. Known

segments of DNA can easily be produced from a patient with a
genetic disease mutation. Modifications to the amplification technique
can extract segments from a completely unknown genome, or can
generate just a single strand of an area of interest.

THE FUTURE OF PCR

The present technology for doing PCR, about the size of a microwave oven
and costing several thousand dollars, seems destined for further radical
improvement. By tinkering with variables such as chemical reagents and pH,
researchers have already reported success at copying larger and larger pieces
of DNA, including the entire genome of HIV.

Extraordinary miniaturization of the hardware is also underway, as

experimenters squeeze PCR onto chip-sized devices. Crisscrossed with the
tiniest of troughs to hold the reagents and the DNA, the chips are heated
electrically and cool down much faster than the present generation of
machines, so amplification is even speedier than today's swift process.
Already researchers have reported using a handheld battery-powered gadget
to copy pieces of DNA that contained eight different cystic fibrosis mutation
sites.

While such experimental chip-based devices are not yet ready for prime
time, they are hastening the day when scientists can take them on the road,
and patients will be able to get on-the-spot readouts of their DNA. Before
long it may be quite routine to diagnose an infectious or genetic disorder, or
even detect an inherited predisposition to cancer or heart disease, right in the
doctor's office.

PCR is doing for genetic material what the invention of the printing press
did for written material-making copying easy, inexpensive, and accessible.
In principle, PCR can reproduce the genetic material of any organism in
essentially unlimited quantities, so it can be used to analyze any cells
containing that material. Whether they are germs, rare medicinal plants, or
human beings, eventually we can know whatever is recorded in their DNA.
With simple organisms, to know their DNA will be to know almost
everything about them. With complicated ones, like people, DNA is only
part of the story, but a very big part. Thanks to PCR, we will be probing the
genetic past, and peering into the genetic future, for many years to come.

Reference:
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genomic sequences and restriction site analysis for diagnosis of sickle cell
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 ^ a b Saiki, RK; Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT,
Mullis KB, Erlich HA (1988). "Primer-directed enzymatic amplification of
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 ^ a b Kary Mullis Nobel Lecture, December 8, 1993
 ^ Cheng S, Fockler C, Barnes WM, Higuchi R (1994). "Effective
amplification of long targets from cloned inserts and human genomic DNA".
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PMID 8202550.
 ^ a b Joseph Sambrook and David W. Russel (2001). Molecular Cloning:
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Harbor Laboratory Press. ISBN 0-87969-576-5. Chapter 8: In vitro
Amplification of DNA by the Polymerase Chain Reaction
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