Principle of the PCR

This technique is invented in 1984 by Kary Mulis. The purpose of a PCR

(Polymerase Chain Reaction) is to make a huge number of copies of a gene if the
flanking sequences of that gene are known.
PCR is a rapid, inexpensive and simple way of copying specific DNA fragments from
minute quantities of source DNA material, even when that source DNA is of
relatively poor quality.

PCR technique involves the preparation of the sample, the master mix and the
primers, followed by detection and analysis of the reaction products.

Applications of PCR:

1). Medical applications

A. Genetic testing, for the presence of genetic disease mutations.
B. Tissue typing, vital to organ transplantation.
C. In cancer detection, and in monitoring cancer therapy.
2). Infectious disease applications

A. The Human Immunodeficiency Virus (or HIV), Infections can be detected

earlier,
B. Diagnosis of Tuberculosis,
C. The spread of a disease organism through populations
of domestic or wild animals that were responsible for earlier epidemics
3). Forensic applications

A. Genetic fingerprinting can uniquely discriminate any one person from the
entire population of the world.
B. In Parental testing, where an individual is matched with their close relatives.
4). Research applications

A. Hybridization probes for Southern or northern blot hybridization.

B. DNA sequencing

C. DNA cloning.

D. Gene expression.

E. In genetic mapping
Typical components of a PCR include:
DNA: the template used to synthesize new DNA strands.

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 DNA polymerase: an enzyme that synthesizes new DNA
strands.
Two PCR primers: short DNA molecules (oligonucleotides) that
define the DNA sequence to be amplified.
Deoxynucleotide triphosphates (dNTPs): the building blocks
for the newly synthesized DNA strands.
Reaction buffer: a chemical solution that provides the optimal
environmental conditions (tris-EDTA).
Divalent metals: Mg or Mn. a necessary cofactor for DNA
polymerase activity.
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 thermal cycler, which can heat and cool the tubes
with the reaction mixture in a very short time.

1. Denaturation at 94C: During the denaturation, the double strand

melts open to single stranded DNA, all enzymatic reactions stop.

2. Annealing at 54C : 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.

3. Extension at 72C: This is the ideal working temperature for the

polymerase. 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)

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Figure 1 : 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, and three cycles will result in 8 copies and so on.

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 Figure 2 : The exponential amplification of the gene in PCR.

Types of PCR
Multiplex PCR: It employs different primer pairs in the same reaction for
simultaneous amplification of multiple targets.

RT-PCR, reverse transcriptase (RT): is used to copy all of the

mRNAs in an RNA sample into single stranded DNA complementary to mRNA
(cDNA). This cDNA can then be amplified by PCR using primers that anneal to
a specific cDNA.

Factors Affecting the PCR:

1. Denaturing Temperature and time

2.Annealing Temperature and Primer Design

3.Primer Length

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4.Elongation Temperature and Time

5. Cycle Number: The number of amplification cycles necessary to

produce a band visible on a gel

PCR controls

1. No-template control: A NTC reaction contains all PCR components except

the template. Detection of a positive signal in an NTC reaction indicates the
presence of contaminating nucleic acids.
2. Positive control :A positive control can be an absolute standard, which is
a nucleic acid from an established cell line, a plasmid containing cloned
sequences, or in vitro transcribed RNA, .A positive control can also be a
known positive sample, which is usually a substitute for an absolute standard
and used only to test for the presence or absence of a target.

Annealing Temperature and Primer

Design
Primers: A primer is a short segment of nucleotides which is
complementary to a section of the DNA which is to be amplified in the PCR reaction.
Primers are annealed to the denatured DNA template to provide an initiation site for
the elongation of the new DNA molecule. Primers can either be specific to a
particular DNA nucleotide sequence or they can be "universal." Universal primers
are complementary to nucleotide sequences which are very common in a particular
set of DNA molecules. Thus, they are able to bind to a wide variety of DNA
templates.
Bacterial ribosomal DNA genes contain nucleotide sequences that are common to
all bacteria. Thus, bacterial universal primers can be made by creating primers
which are complementary to these sequences. Examples of bacteria universal
primer sequences are:

Forward 5' GAT CCT GGC TCA GGA TGA AC 3' (20 mer)
Reverse 5' GGA CTA CCA GGG TAT CTA ATC 3' (21 mer)

Animal cell lines contain a particular sequence known as the " Alu gene". There are
approximately 900,000 copies of the Alu gene distributed throughout the human
genome, and multiple copies distributed through the genome of other animal cells,
as well. Thus, the Alu gene provides the sequence for a universal primer for animal
cell lines. The Alu primer is especially useful in that it binds in both forward and
reverse directions.The Alu universal primer seqeunce is as follows:

5' GTG GAT CAC CTG AGG TCA GGA GTT TC 3' (26mer)

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PCR primer design
Optimal primer sequences and appropriate primer concentrations are essential for
maximal specificity and efficiency in PCR. The table, Guidelines for the design and
use of primers provides an overview of primer design and use for standard and
multiplex PCR, as well as one-step RT-PCR. Molar conversions can be found in the
table Molar conversions for PCR primers.
The following points should be considered when designing PCR primers and are
common to all types of PCR:
1. primers should be 17-28 bases in length;
2. base composition should be 50-60% (G+C)
3. primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of
ends and increases efficiency of priming
4. Tms between 55-80oC are preferred
5. Tm calculation: 2C x (A+T) + 4C x (G+C)
6. Avoid complementarity in the 23 bases at the 3' end of the primer pairs
7. Avoid runs of 3 or more Cs or Gs at the 3' end of the primer ,this may
promote mispriming at G or C-rich sequences (because of stability of
annealing), and should be avoided;
8. Avoid complementarity within primers and between the primer pair
9. Avoid a T as ultimate base at the 3' end
10.Ensure primer sequence is unique for the template sequence
11.Use a concentration of 0.11.0 M of each primer. For many applications, a
primer concentration of 0.2 M will be sufficient
12.3'-ends of primers should not be complementary (ie. base pair), as otherwise
primer dimers will be synthesised preferentially to any other product

Lyophilized primers should be dissolved in a small volume of distilled water or TE to

make a concentrated stock solution. Prepare small aliquots of working solutions
containing 10 pmol/l to avoid repeated thawing and freezing. Store all primer
solutions at 20C. Primer quality can be checked on a denaturing polyacrylamide
gel; a single band should be seen.

Annealing Temperature and Primer Design

Melting temperature of NA duplex increases both with its length, and with
increasing (G+C) content:
a simple formula for calculation of the Tm is

Tm = 4(G + C) + 2(A + T)oC.

Thus, the annealing temperature chosen for a PCR depends directly on length and
composition of the primer(s). One should aim at using an annealing
temperature (Ta) about 5oC below the lowest Tm of ther pair of primers to be
used

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 1- Preparation of Master Mix
The Master Mix contains all of the components necessary to make new strands of DNA in the
PCR process. The Master Mix reagents include (table 1):

If want to make 10 reactions, Multiply all components (except DNA template)

by 10 total number of the reactions

Solve for the volume of the sterile water needed per reaction by subtracting
the volumes of all other reaction components from 50 ul:

50 ul (final reaction volume) 10 ul buffer 5 ul (forward primer ) 5

( Reverse primer) 1 ul (Taq Polymerase) - 4 ul ( dTNP) - 5 ul (DNA template)
= ul sterile water

Protocol:
Table 1: To make the master mix for one reaction add:

Master mix components For one Volume Final

reaction for 10 conc
reactions
1. 10 ul 100 ul 1X
5X PCR buffer
2. 4ul 40 ul 200 uM
Each nucleotide mixture,25 mM
(dATP, dCTP, dGTP, dTTP))
3. 5ul 50 ul 0.2 uM
forward primer
4. 5ul 50 ul 0.2 uM
Reverse primer
5. 1ul 10 ul 5 units
1 ul Taq DNA polymerase ( 5U/ul)

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 6. 20 ul 200 ul
water
7. 45 ul 450 ul
Total volume

volume
Master mix 45ul
DNA template 5 ul

Notes on the Master Mix

The Master Mix buffer is often stored as a 5X stock solution (100 mM Tris-
HCL, pH 8.3, 500 mM KCL, 75 mM MgCl2) which is diluted to 1X for use. Both
the Master Mix buffer and the purified water can be stored at room
temperature. Store the deoxynucleotides, primers and Taq DNA polymerase
enzyme at -20oC.
ALWAYS run a no-DNA control to check for contamination!!!!!!
Final Component Purpose
Conc.

1X Buffer keeps the master mix at the proper pH so the PCR reaction
will take place.

200uM Deoxynu cleotides provide both the energy and nucleosides for
the synthesis of DNA. It is important to add equal amounts of each
nucleotide (dATP, dTTP, dCTP,GTP) to the master mix to prevent
mismatches of bases.

0.2-1.0uM Primers Short pieces of DNA (20-30 bases) that bind

to the DNA template allowing Taq DNA polymerase
enzyme to initiate incorporation of the
deoxynucleotides.Both specific and universal primers
can be used.

2.5U/100ul Taq Polymerase A heat stable enzyme that adds the

deoxynucleotides to the DNA template.

0.05-1.0ug Template DNA The DNA which will be amplified by the PCR reaction.

2- Programming of PCR machine:

Thermal cycler
The thermal cycler (also known as a thermocycler, PCR
machine or DNA amplifier) is a laboratory apparatus most commonly used
to amplify segments of DNA via the polymerase chain reaction (PCR).

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Thermal cyclers may also be used in laboratories to facilitate other
temperature sensitive reactions, including but not limited to restriction
enzyme digestion or rapid diagnostics. The device has a thermal block with
holes where tubes holding the reaction mixtures can be inserted. The cycler
then raises and lowers the temperature of the block in discrete, pre-
programmed steps

Programming of PCR machine, Each PCR cycle include:-

Denaturation

Denaturation for 30 seconds at 94-95oC is routinely used to amplify linear

DNA molecules whose GC content is <55% and higher temperature for
template and/or target DNAs whose GC content is >55%.

Annealing of primers to template DNA

Annealing is usually carried out 3-5o C lower than the calculated melting
temperature at which the oligonucleotide primers dissociate from their
templates. It is best to optimize the annealing conditions by performing a
series of trial PCRs at temperatures ranging from 2C to 10 C below the lower
of melting temperatures calculated for the two oligonucleotide primers.
optimization is achieved by exposing a single PCR to a sequential series of
annealing temperatures in successive cycles of the reaction. The
polymerization rate of Taq polymerase is ~2000 nucleotides /minute at the
optimal temperature (72oC) .For the last cycle of PCR, many investigators use
an extension time that is 3 times longer than the previous cycles, to allow
completion of all amplified products.

Extension of oligonucleotide primers

Is carried out at or near the optimal temperature for DNA synthesis

catalyzed by the thermostable polymerase, which in the case of Taq
polymerase is 72-78oC.

Number of cycles

The number of cycles required for amplification depends on the number of

copies of template DNA present at the beginning of the reaction and the
efficiency of primer extension and amplification. At least 25 cycles are
required to achieve acceptable levels of amplification of single copy target
sequences in mammalian DNA templates

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 Stages Temp time cycles

Initial denaturation 94 2 minutes 1

denaturation 94 30 s

annealing 65 10 s 30 cycles

extension 72 1 minute

Final extension 72 5 minutes

hold 4 0 ( infinity)

Detection and analysis of the reaction product

Take 1/10th - 1/3rd of the reaction mix this with some gel loading buffer (1:1 - 1:5
mix: loading buffer): this is TBE containing 10 - 20% glycerol or sucrose and a dash
of bromophenol blue (BPB) tracking dye. Load 5 - 30ul of sample into wells of 0.8 -
3.0% submarine agarose gel made up in TBE, preferably containing 50ng/ml
ethidium bromide. Run at 80 -100 volts (not too slow or small products diffuse;
not too fast or bands smear) until BPB reaches end of gel (large products) or 2/3
down gel (small products). Use DNA markers going from 2kb down to 100 bp or less
(recommend BM PCR markers). View on UV light box at 254 - 300 nm, photo 1 - 5
sec.

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Figure 3 : Verification of the PCR product on gel. Lane 1 : PCR fragment is
approximately 1850 bases long. Lane 2 and 4 : the fragments are approximately
800 bases long. Lane 3 : no product is formed, so the PCR failed. Lane 5 : multiple
bands are formed because one of the primers fits on different places.

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