Download lecture 4 DNA

Variants of PCR
Lecture 4
• Colony PCR
• Multiplex PCR
• Nested PCR
• Reverse transcriptase PCR
• Quantative PCR
• Hot Start PCR
• Touch down PCR
Colony PCR
• PCR screening of bacterial (E Coli) and
yeast transformants containing cloned
inserts
• to determine insert size and/or orientation in
the vector.
• The presence of an insert and its size
can be determined by growing each
colony in liquid
• Plasmid purified by a boiling or
alkaline preparation protocol,
• Digestion of the plasmid with
restriction enzyme(s) that excises the
insert
• separation by agarose gel
electrophoresis
Multiplex PCR
• several pairs of primers annealing to
different target sequences.
• This permits the simultaneous
analysis of multiple targets in a single
sample.
• testing for genetic mutations, six or
more amplifications might be
combined
• analysis of microsatellites and SNPs.
Nested PCR
• Two pairs of primers instead of one pair are
used to amplify a fragment.
• In the first PCR, one pair of primers is used
to generate DNA products, which may
contain products amplified from non-target
areas.
• Template (product of first PCR) in a second
PCR, using one ('hemi-nesting') or two
different primers whose binding sites are
located (nested) within the first set, thus
increasing specificity.
Reverse transcriptase
• detect RNA expression levels. qualitative
study of gene expression.
• detect gene expression through creation
of complementary DNA (cDNA) transcripts
from RNA.
• quantification of RNA, by incorporating qPCR
into the technique
• qRT-PCR, RT-qPCR
• most powerful, sensitive, and quantitative
assay for the detection of RNA levels as
compared to northern blot.
• expression analysis of single or multiple
genes
• expression patterns for identifying
infections and diseases.
• May be either one step or two step RTPCR
Real time or Quantative PCR
• amplify and quantify targeted DNA molecule.
• amplified DNA is detected as the reaction progresses
in real time.
• 1) non-specific fluorescent dyes that interclate with
any double-stranded DNA
• 2) sequence-specific DNA probes oligonucleotides
labeled with a fluorescent reporter which permits
detection only after hybridization of the probe with its
complementary DNA target
Applications
 rapidly detect nucleic acids that are
diagnostic of, infectious diseases,cancer
and genetic abnormalities.
 detect newly emerging diseases, such as
new strains of flu
 in the fields of food safety, food spoilage
and fermentation and for the microbial risk
assessment of water quality.
 It is also used for the determination of
zygosity of transgenic animals used in
research
Hot start PCR
• Hot Start PCR avoids non-specific
amplification of DNA by inactivating the taq
polymerase at lower temperature.
• dsDNA is denatured by heating the sample at
its denaturing temperature and then the
temperature is suddenly reduced to 55 degree
C at which primer and Taq-polymerase is
added
• specific antibodies are used to
block this Taq-polymerase at
annealing temperature.
• When the temperature raises for
amplification to 72 degrees, the
specific antibody detaches from
Taq-polymerase and the
amplification with greater specificity
starts.
Touchdown PCR
• Primers will avoid amplifying nonspecific
sequences.
• The annealing temperature during a
polymerase chain reaction determines the
specificity of primer annealing.
• The melting point of the primer sets the
upper limit on annealing temperature. At
temperatures just below this point, only
very specific base pairing between the
primer and the template will occur.
• Touchdown PCR uses a cycling program
annealing temperature is gradually
reduced (e.g. 1-2°C /every second cycle).
• The initial annealing temperature should
be several degrees above the estimated
Tm of the primers.
• The annealing temperature is then
gradually decreased until it reaches the
calculated annealing temperature of the
primers or some degrees below.
Amplification is then continued.