6. PCR
• The PCR technique is basically a primer extension reaction for
amplifying specific nucleic acids in vitro.
• PCR will allow a stretch of DNA (usually fewer than 3000bp) to
be amplified to about a million fold.
• The particular stretch of DNA to be amplified, called the target
sequence
7. Composition of the reaction mixture
Template DNA:
• Usually the template DNA amount is in the range of
0.01-1ng for plasmid or phage DNA and 0.1-1µg for genomic
DNA, for a total reaction mixture of 50µl.
• Higher template DNA amounts usually increase the yield of
nonspecific PCR products.
8. PRIMERS
• A primer is a short segment of nucleotides that is
complementary to a section of the DNA that 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
that are very common in a particular set of DNA molecules.
Thus, they are able to bind to a wide variety of DNA templates.
Primers length should be from 15-30
not less than 15 because it might be non specific, where multiple
regions have the same sequence
not more than 30 because it might anneal with each other
9. PRIMERS cont…
• 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)
• When using universal primers the annealing temperature on
the thermal cycler is lowered to 40-55 degrees C.
10. Other components
• dNTPs
• Taq DNA polymerase – heat resistant polymerase
• The Master mix buffer (Tris-HCL, pH 8.3, KCL,mM MgCl2)
11. Principle of the PCR
• The cycling reactions:
– Denaturation
– Annealing
– Extension
where you take the primers ? it depends,, if you know the DNA that
you are going to amplify you will construct the primer based on the
DNA sequence that you know" the primer's sequence that flank 5'
and 3' region
if you don’t know the sequence we have
to add the DNA of interest which is unknown to a plasmid that you
know the sequence of the flanking region to use these flanking
regions to form primers
