2. Introduction to PCR
• Method widely used in molecular
biology to make many copies of a
specific DNA segment.
• Using PCR, copies of DNA
sequences are exponentially
amplified to generate thousands
to millions of more copies of that
particular DNA segment.
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6. Multiplex PCR
• Used for multiple pathogens
• Using multiple primer sets, each one
targeting a particular pathogen
• It permits analysis of different target
pathogens within a single sample
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7. • targets areas of the genome that exhibit
length variation
• Analysis of smaller VNTR segments known
as short tandem repeats (or STRs) is the
basis for DNA fingerprinting databases
• VNTRs are amplified and run on agarose
to produce unique DNA fragments
Variable Number of Tandem Repeats
(VNTR) PCR
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8. • Used to increase the specificity of
DNA amplification
• Two sets of primers are used in two
successive reactions
• Nested PCR is often more successful
in specifically amplifying long DNA
products than conventional PCR, but
it requires more detailed knowledge
of the sequence of the target
Nested PCR
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9. • Used to measure the specific amount of
target DNA (or RNA) in a sample
• The amount of measured product more
accurately reflects the initial amount of target
• Quantitative Real-Time PCR (QRT-PCR)
methods use fluorescent dyes or fluorophore-
containing DNA probes, to measure the
amount of amplified product
Quantitative PCR
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10. • The annealing temperature in the early cycles
is usually 3–5 °C above the standard Tm of the
primers used
• The annealing temperature is gradually
decreased in later cycles
• The initial higher annealing temperature leads
to greater specificity for primer binding, while
the lower temperatures permit more efficient
amplification at the end of the reaction
Touchdown PCR
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11. • synthesis of long DNA structures by
performing PCR on a pool of long
oligonucleotides with short overlapping
segments, to assemble two or more
pieces of DNA into one piece
• It involves an initial PCR with primers that
have an overlap and a second PCR using
the products as the template that
generates the final full-length product
Assembly PCR
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13. • Uses primers selected from
segments repeated throughout a
genome to produce a unique
fingerprint of amplified product
lengths
InterSequence-Specific PCR
(or ISSR-PCR)
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14. • Target DNA fragments (or cDNA) are
first inserted into a cloning vector
• A single set of primers are designed
for the areas of the vector flanking
the insertion site
• Amplification occurs for whatever
DNA has been inserted.
Extended Colony PCR
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( Vec tor Primers)
15. • Reduced primer-dimer formation
• Increases the number of assays in
multiplex PCR
• The method utilizes primers with a
cleavable block on the 3’ end that is
removed by the action of a
thermostable RNase HII enzyme
RNase H-dependent PCR
(rhPCR)
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