Nucleic Acid Manipulation

The Polymerase Chain Reaction

 DNA polymerase
• Enzyme that carries the replication of genetic material.
• These initiate the synthesis of DNA starting from a primer bound
to a template.

DNA replication. In the first step, the double helix shown above in blue is
unwound by a helicase. Next, a molecule of DNA polymerase shown in green
binds to one strand of the DNA. It moves along the strand, using it as a template
for assembling aleading strand shown above in red of nucleotides and reforming
a double helix. A second DNA polymerase molecule (also green) is used to bind
to the other template strand as the double helix opens. This molecule must
synthesize discontinuous segments of polynucleotides (called Okazaki fragment
s). Another enzyme, DNA ligase shown in violet, then stitches these together into
the lagging strand.
 Polymerase Chain Reaction
• With this, any particular stretch of genetic material can be
pinpointed and replicated numerous times simply by selecting a
pair of primers that flank the desired stretch of DNA.
 Cycle
• One repetition of three steps of PCR
The PCR cycle.  Step A, denaturation of the DNA template;
Set B, annealing of primers to the single-stranded DNA
template; Step C, extension of the prmer to make a
complementary copy of the DNA template.  These three
steps make up one cycle of PCR.  Generally, 25 to 30 cycles
of PCR are performed.  Theoretically, each cycle amplifies
the amount of DNA exponentially (doubles it).  Thus, 25
cycles should yield a 225 increase in DNA.  Practically, a
106-fold increase in DNA is achieved because the efficiency
of amplification is not perfect.  But this increase is enough to
allow visualization of the DNA. 
 Three Temperature Incubations/ Steps

• Denaturation
• Annealing
• Extension or Elongation
 Denaturation
• The two strands of target DNA molecule are separated by
heating the DNA to 94° C to break the hydrogen bonds between
bases, yielding two separate strands.
 Annealing
• Two primers hybridize to complementary sequences in the
single strands.

• The primers are short (20-30 bases in length) , synthetic

stretches of single stranded DNA.
• They are selected so that one primer is complementary to one
end of the gene interest on one strand, while the second primer
is complementary to the opposite end on the other strand.
• The primers form hydrogen bonds with (anneal to) their
complementary sequences, forming stable, double stranded
molecules.

• Annealing temperatures range between 37° and 60° C.

 Extension/ Elongation
• The primers are extended by the thermostable DNA polymerase
at 72° C.

The ribosome then moves 1 codon down the mRNA in a 5' to 3' direction. This is
achieved by a translocase enzyme. As the process of ribosome translocation
continues, the "old" tRNA is released to bind another amino acid and go in search of a
new codon. The binding of a new aminoacid is mediated by an enzyme called amino-
acyl-tRNA synthase
• First, the double-stranded DNA has to be heated to 96°C in
order to separate the strands. This step is called melting; it
breaks apart the hydrogen bonds that connect the two DNA
strands. Prior to the first cycle, the DNA is often melted for an
extended time to ensure that both the template DNA and the
primers have completely separated and are now single-strand
only.
• After separating the DNA strands, the temperature is lowered so
the primers can attach themselves to the single DNA strands.
This step is called annealing. The temperature of this stage
depends on the primers and is usually 5°C below their melting
temperature. A wrong temperature during the annealing step
can result in primers not binding to the template DNA at all, or
binding at random.
• Finally, the DNA-Polymerase has to fill in the missing strands. It
starts at the annealed primer and works its way along the DNA
strand. This step is called elongation. The elongation
temperature depends on the DNA-Polymerase. The time for this
step depends both on the DNA-Polymerase itself and on the
length of the DNA fragment to be amplified.
Figure 2 : Schematic drawing of the PCR cycle.
(1) Melting at 96°C. (2) Annealing at 68°C. (3) Elongation at 72°C (P=Polymerase). (4)
The first cycle is complete. The two resulting DNA strands make up the template DNA
for the next cycle, thus doubling the amount of DNA duplicated for each new cycle.
 Site-directed mutagenesis
• Technique use in studying the effects of mutations.

• It introduces point mutations at specific sites.

Site-directed mutagenesis reprograms DNA
Using site-directed mutagenesis the information in the genetic material can
be changed. A synthetic DNA fragment is used as a tool for changing one
particular code word in the DNA molecule. This reprogrammed DNA molecule
can direct the synthesis of a protein with an exchanged amino acid. Michael
Smith's method has become one of biotechnology's most important
• One of the primers is designed with a sequence
complementary to the region in the target DNA, but with the
desired substitution, insertion or deletion.

• The mutagenic sequence within the primer must be either at the

5´ end of the primer or internal to the primer, but never at the 3´
end of the mutagenic primer.
• The primer 3´ end of the mutagenic primer (at least 6-10 bp
long) must be totally complementary to the target DNA to permit
full annealing of the primer to its target and allow the
polymerase to extend the primer.

• The PCR is carried out initially (first 5-10 cycles) under low
stringency conditions, to allow the mismatch to occur.
• Once a few mutagenized templates are produced during the
PCR, these will serve as targets and will be fully complementary
to the primer.

• The end products will contain the mutation at the desired end.
 References:
• http://www.google.com.ph/imglanding?q=denaturation&um=1&hl=tl&safe=vss&sa=G&biw=1024&bih=611&tbs=isch:1&tbnid=PfLfiDoy71s2
EM:&imgrefurl=http://qwickstep.com/search/denaturation-of-proteins.html&imgurl=http://www.elmhurst.edu/~chm/vchembook/images/568
denaturation.gif&zoom=1&w=340&h=357&iact=hc&oei=VfKjTIWxKIOWcaXBwJkI&page=1&tbnh=153&tbnw=146&start=0&ndsp=12&ved=
1t:429,r:0,s:0
• http://www.uq.edu.au/vdu/DNATranslation.htm
• http://www.google.com.ph/imglanding?q=cycle+of+pcr&um=1&hl=tl&safe=vss&sa=G&biw=1024&bih=611&tbs=isch:1&tbnid=I90mk5lpqB
w1OM:&imgrefurl=http://ag.arizona.edu/swes/maier_lab/kartchner/pcr_graphic.html&imgurl=http://ag.arizona.edu/swes/maier_lab/kartchn
er/images/research/nonculture/pcrdiagram.jpg&zoom=1&w=700&h=858&iact=hc&oei=Q_KjTMvnBIP4cNf6rKYI&page=1&tbnh=166&tbnw
=135&start=0&ndsp=15&ved=1t:429,r:0,s:0
The end