nucleic acid manipulation

8/8/2019 Nucleic Acid Manipulation

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Nucleic Acid Manipulation

The Polymerase Chain Reaction


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DNA polymeraseEnzyme that carries the replication of genetic material.

These initiate the synthesis of DNAstarting from a primer bound to atemplate.

DNA replication. In the first step, the double helix shown above in blue isunwound by a helicase . Next, a molecule of DNA polymerase shown in greenbinds to one strand of the DNA. It moves along the strand, using it as a templatefor assembling a leading strand shown above in red of nucleotide s and reforming adouble helix. A second DNA polymerase molecule (also green) is used to bind tothe other template strand as the double helix opens. This molecule mustsynthes ize discontinuous segments of polynucleot ides (called Okazakifragment s). Another enzyme, DNA ligase shown in violet, then st itches thesetogether into the lagging strand.


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Polymerase Chain ReactionWith this, any particular stretch of genetic material can be pinpointedand replicated numerous timessimply by selecting a pair of primersthat flank the desired stretch of DNA.


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CycleOne repetition of three steps of PCR


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The PCR cycle. Step A, denaturation of the DNA template;Set B, annealing of primers to the single-stranded DNAtemplate; Step C, extension of the prmer to make acomplementary copy of the DNA template. These threesteps make up one cycle of PCR. Generally, 25 to 30 cyclesof PCR are performed. Theoretically, each cycle amplifies

the amount of DNA exponentially (doubles it). Thus, 25cycles should yield a 225 increase in DNA. Practically, a106-fold increase in DNA is achieved because the efficiencyof amplification is not perfect. But this increase is enough toallow visualization of the DNA.


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Denaturation

The two strands of target DNAmolecule are separated by heatingthe DNA to 94 C to break thehydrogen bonds between bases,yielding two separate strands.


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AnnealingTwo primers hybridize tocomplementary sequences in thesingle strands.

The primers are short (20-30 bases inlength) , synthetic stretches of singlestranded DNA.


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They are selected so that one primeris complementary to one end of thegene interest on one strand, whilethe second primer is complementaryto the opposite end on the otherstrand.


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The primers form hydrogen bondswith (anneal to) their complementarysequences, forming stable, doublestranded molecules.

Annealing temperatures rangebetween 37 and 60 C.


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Extension/ElongationThe primers are extended by thethermostable DNA polymerase at 72C.

The ribosome then moves 1 codon down the mRNA in a 5' to 3' direction. This isachieved by a translocase enzyme. As the process of ribosome translocationcontinues, the "old" tRNA is released to bind another amino acid and go in search of anew codon. The binding of a new aminoacid is mediated by an enzyme called amino-acyl-tRNA synthase


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First, the double-stranded DNA hasto be heated to 96C in order toseparate the strands. This step iscalled melting; it breaks apart thehydrogen bonds that connect the twoDNA strands. Prior to the first cycle,

the DNA is often melted for anextended time to ensure that boththe template DNA and the primershave completely separated and arenow sin le-strand onl .


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After separating the DNA strands, thetemperature is lowered so theprimers can attach themselves to thesingle DNA strands. This step iscalled a nne a ling. The temperature of this stage depends on the primers

and is usually 5C below theirmelting temperature. A wrongtemperature during the annealingstep can result in primers not bindingto the tem late DNA at all or bindin


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Finally, the DNA-Polymerase has tofill in the missing strands. It starts atthe annealed primer and works itsway along the DNA strand. This stepis called elong a tion. The elongationtemperature depends on the DNA-

Polymerase. The time for this stepdepends both on the DNA-Polymerase itself and on the lengthof the DNA fragment to be amplified.


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F igure 2 : Schematic drawing of the PCR cycle.(1) Melting at 96C. (2) Annealing at 68C. (3) Elongation at 72C (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.


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Site-directed mutagenesisTechnique use in studying the effectsof mutations.

It introduces point mutations atspecific sites.


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Site-directed mutagenesis reprograms DNAUsing site-directed mutagenesis the information in the genetic material canbe changed. A synthetic DNA fragment is used as a tool for changing oneparticular code word in the DNA molecule. This reprogrammed DNA

molecule can direct the synthesis of a protein with an exchanged aminoacid. M ichael Smith's method has become one of biotechnology's most


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One of the primers is designed witha sequence complementary to theregion in the target DNA, but withthe desired substitution, insertion ordeletion.

The mutagenic sequence within theprimer must be either at the 5 endof the primer or internal to the

primer, but never at the 3 end of


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The primer 3 end of the mutagenicprimer (at least 6-10 bp long) mustbe totally complementary to thetarget DNA to permit full annealing of the primer to its target and allow thepolymerase to extend the primer.

The PCR is carried out initially (first5-10 cycles) under low stringency

conditions, to allow the mismatch to


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Once a few mutagenized templatesare produced during the PCR, thesewill serve as targets and will be fullycomplementary to the primer.

The end products will contain themutation at the desired end.


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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=PfLfiDoy71s2EM:&imgrefurl=http://qwickstep.com/search/denaturation-of-proteins.html&imgurl=http://www.elmhurst.edu/~chm/vchembook/images/568denaturation.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:0http://www.uq.edu.au/vdu/DNATranslation.htmhttp://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=I90mk5lpqBw1OM:&imgrefurl=http://ag.arizona.edu/swes/maier_lab/kartchner/pcr_graphic.html&imgurl=http://ag.arizona.edu/swes/maier_lab/kartchner/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


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nucleic acid manipulation

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