Dr.

Shikha Yashveer
Deptt. of MBB&B,
CCS Haryana Agricultural University, Hisar
 
Lecture Title: PCR and Gene Amplification
• The technique of gene cloning permits to obtain identical copies of a DNA fragment.
• This technique discovered around 1975 became an essential tool in all molecular biology laboratories.
• In 1985, another remarkable tool in molecular biology was discovered, Polymerase chain reaction (PCR).
• It is a relatively simple technique that amplifies a DNA template to produce specific DNA fragments in
vitro.
• Traditional methods of cloning a DNA sequence into a vector and replicating it in a living cell often require
days or weeks of work, but amplification of DNA sequences by PCR requires only hours.
• Most biochemical analyses, including nucleic acid detection with radioisotopes, require the input of
significant amounts of biological material, the PCR process requires very little quantity of DNA in
nanograms.
• Thus, PCR can achieve more sensitive detection and higher levels of amplification of specific sequences in
less time than previously used methods.
• These features make the technique extremely useful, not only in basic research, but also in commercial
uses, including genetic identity testing, forensics, industrial quality control and in vitro diagnostics.
• This chapter provides an overview of basic PCR technique, different types of PCR methods, and
applications. 
Basic PCR
• All of you must be familiar with the process of DNA replication in the cells.
• DNA replication involves addition of nucleotides to the 3’ OH end of an RNA primer using
the information present in the template DNA strand. DNA polymerase enzyme is required
for the process.
• In PCR, a similar reaction is carried out in vitro conditions in an Eppendorf tube in which a
template strand having the DNA fragment to be amplified along with all the four
deoxynucleotides, polymerase enzyme and primer in the form of a deoxy oligonucleotide is
added.
• There are three basic steps in the reaction (Fig 1).
• In the first step, the reaction tube is heated around 90 -98 ℃ which helps in denaturation of
the template strand.
• The temperature is then lowered to 40 -60 ℃. At this step, primers bind to their
complementary sequences in the template strand. This step is called annealing and the
temperature at which the primers bind to their complementary sequence is called annealing
temperature.
• The temperature is again raised to 72 ℃ at which DNA polymerase
adds deoxynucleotides to the 3’ OH end of the primers. This step is
called primer extension.
• All these three steps denaturation, annealing and extension constitute
one cycle and are repeated again and again.
• Initially E. coli DNA polymerase enzyme was used for the process.
but this enzyme become denatured at high temperatures used in the
process so after each cycle fresh aliquot of the enzyme needs to be
added.
• Discovery of Taq DNA polymerase isolated from Thermus aquaticus
growing in hot springs resulted in the automation of PCR reaction in a
thermocycler.
• This enzyme acts best at 72 ℃ which is the temperature of the
extension step of the reaction and denaturation temperature of 94℃
does not affect its enzymatic activity.
• Other thermostable enzymes like Pflu DNA polymerase isolated from
Pyrococcus furiosus and Vent polymerase isolated from
Thermococcus litoralis have been discovered and more efficient than
Taq polymerase
Different types of PCR
There are several variations to the basic PCR technique.

Inverse PCR
• In the basic PCR reaction, amplification of those sequences takes place Fig 2: Inverse
PCR
which are flanked by the primers.
• Thus, the sequencing of the ends of the DNA fragment to be amplified, has
to be done to design the primers.
• As the polymerization always proceeds in 5’ to 3’ direction, primers bind
(are complementary) to the 3’ end of the fragment to be amplified.
• In inverse PCR, amplification of those DNA sequences takes place which
are away from the primers and not those which are flanked by the primers.

• E.g., if the border sequences of a fragment are not known but those of a vector are known, then the sequence
to be amplified is cloned in the vector and border sequences of the 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 inserted segment.
• Similarly, if the gene sequence is known, its border sequences can be used in an inverse PCR reaction to
amplify the sequences flanking this gene e.g., its promoter or other regulatory sequences.
PCR for site directed mutagenesis
• Usually, the primers are completely complementary to the 3’end of the DNA fragment.
• PCR for site directed mutagenesis is used for introducing mutations at desired place in a DNA
fragment by altering the sequences of primers.
• Since mutations are introduced only through primers, they are limited to the ends of the gene
sequence.
• A variation of this technique known as overlap extension method allows the
introduction of mutation at any place of interest in the gene.
• In this method, a particular gene fragment is amplified in two separate gene
fragments in two separate PCR reactions.
• In each of these reactions there is one primer at the end of the gene and the
other with the desired modification for mutation, internal to the fragment, so
that the amplified products have their ends internal to the gene sequence and
overlap each other. The internal modified primers used in two reactions are
complementary to one another.
• The internal ends of two PCR products are mixed where they function as primers and are extended by Taq
Polymerase to form a complete gene with mutations incorporated at the internal sites.
Anchored PCR
• In the basic PCR reaction two primers are required lying at the
ends of the sequences to be amplified.
• Sometimes we may have knowledge about only one of the two
ends of the sequence so that only one primer can be designed.
• In such situation, anchored PCR is used in which only one strand
is copied first due to the use of one primer.
• A poly G tail is then attached at the end of the newly synthesized
strand.
• This newly synthesized strand with poly G tail at its 3’ end then
acts as a template for synthesis of the daughter strand using an
anchor primer in which a poly C sequence is attached to
complement with poly G of the template.
• Further amplification is carried out using both original and
anchored primers.
PCR for DNA sequencing
• PCR can be used to produce single stranded DNA for sequencing.
• This is called asymmetric PCR in which two primers are used in 100:1 ratio, so that after 20 -25 cycles
of amplification, one primer is exhausted and single stranded DNA is produced for the next 5-10 cycles.
 Applications of PCR
 Study of DNA polymorphism
• A variety of molecular markers such as RAPD, SSR, AFLP and SNP
have been developed using PCR.
• These markers can be used to study DNA polymorphism.

 PCR for confirming the presence of transferred gene

• PCR can be used to confirm the transfer and maintenance of transferred
gene by designing primers based on the transferred gene sequence.

 PCR in sex determination of embryos

• Sex of human and livestock embryos fertilized in vitro, can be
determined by PCR, by using primers and DNA probes specific for sex
chromosomes.
• Further, this technique is also useful to detect sex -linked disorders in
fertilized embryos.
 Prenatal diagnosis of inherited diseases
• PCR can be employed in the prenatal diagnosis of inherited diseases by using chorionic villus samples or
cells from amniocentesis.
• Thus, diseases like sickle-cell anaemia, β-thalassemia and phenylketonuria can be detected.

 DNA fingerprinting
• DNA fingerprinting technique was originally developed by
a British scientist Alec Jeffreys in 1984.
• Some well characterized sequences of microsatellites are
being used for designing primers to identify individual
cultivars or for characterization of germplasm.