Biotechnology Engineering

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Lecture- 29

2. Applications of PCR
 The discovery of PCR is a breakthrough in increasing the limits
of knowledge in molecular biology. It is a basic research tool in
a molecular biology lab.
 Molecular biologists use PCR for identifying specific genes in
many different species and to make many copies of the
identified piece of DNA .
 Forensic technicians use it to help in identification of suspects
and victims based on the amplification patterns of the DNA. It
is also used in disease diagnosis, evolutionary genetics, and
genome sequencing.
 The advent of PCR has dramatically impacted many areas of
our lives and promises to yield many more exciting
discoveries. PCR can also be exploited in both site-directed and
localized mutagenesis and the generation of specific gene (and
hence protein) fusions.
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3. Important Applications of PCR
 Isolation of Gene
 Cloning of Gene
 cDNA Synthesis and Rapid Amplification of cDNA Ends
(RACE)
 PCR based Mutagenesis
 PCR-based Molecular Markers
 Study of Fossil DNA Using PCR
 Analysis of Environment by PCR
 Medical Diagnosis by PCR
 PCR Use in Forensic Science
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4. Isolation of gene
 Using standard PCR method, a particular gene can be
isolated and amplified within the known boundaries.
 Several methods have been developed for the
amplification of DNA sequences that flank regions of
known sequences, e.g., inverse PCR and gene walking.
 By this method, one is able to obtain valuable
amounts of DNA without cloning.
 Even regions from relatively uncharacterized
organisms can also be amplified on the basis of
sufficient sequence homology by using degenerate
primers.
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5. Cloning of a gene
 The technique of PCR has its application in the cloning of
gene of interest. Various cloning procedures are-
1. Cloning of Amplicon by Adding Restriction Sites
2. TA Cloning
3. TOPO TA Cloning
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6. Cloning of An Amplicon by Adding
Restriction Sites
 PCR product can be easily cloned by adding sequences of
restriction sites at its ends
 Primers are designed with restriction sites in their 5'ends.
 Generally, two different sites are added in a pair of primers that
will facilitate the cloning experiment.
 The amplified fragment having restriction sites at its ends can
be used for directional cloning. The purified insert and the
vector DNA are digested with the appropriate restriction
enzymes, ligated, and used for transformation of E. coli.
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7. Limitations
 (i) After the PCR reaction, the presence of unused DNA
polymerase and dNTPs can generate blunt ended DNA on
digestion of amplicon by restriction enzymes.
 (ii) The efficiency of restriction digestion of amplicon
may also decrease because of the presence of residual
primers and primer dimers as these compete for restriction
reaction.
 (iii) Various restriction enzymes fail to digest recognition
sequences located close to the ends of amplicon, e.g.,
Hind III, Not I, Sal I, Xba I, and Xho I.
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8. TA Cloning
 TA cloning is one of the most popular methods of cloning
the amplified PCR product by exploiting the properties of
Taq and other DNA polymerases.
 As these polymerases have a non-template dependent
terminal transferase activity that adds a single dATP
residue to the 3'ends of the double stranded amplicon, this
type of amplified product can be directly cloned in a
linearized vector having complementary 3' T-overhangs.
Such vectors are called T-vectors.
 T-vectors may be generated in the following ways:
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9. T-vector generation
 The vector DNA is digested with Xcm I, Hph I or Mbo I which
generate 3'-terminal deoxythymidine.
 T residue is also added at the 3'-end with the help of terminal
transferase enzyme in the presence of dideoxy TTP.
 T-vectors are also created by utilizing the template independent
terminal transferase activity of Taq DNA polymerase that
catalyzes the addition of T residues at the 3'-end of linearized
vector.
 T vectors are provided by many suppliers as a component of T-
vector cloning kit, for example, pCR Script (SK+)(Stratagene),
pCR II (Invitrogen), and pGEM-T (Promega)
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10. TOPO TA Cloning
 TOPO TA cloning is designed for cloning PCR products
directly from a PCR reaction in just 5 min. This method of
cloning uses a pCR®-TOPO® vector (Invitrogen) having
covalently bound topoisomerase I for fast cloning and
obtaining >95% recombinants.
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11. TOPO vector
 This vector has 3' T overhangs for direct ligation of Taq
DNA polymerase amplified PCR products.
 Ampicillin and kanamycin resistance genes for selection
of X-gal and IPTG for screening in E. coli.
 Eco RI sites flanking the PCR product insertion site for
easy excision of insert.
 Topoisomerase I covalently bound to the vector (referred
to as 'activated' vector).
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