Polymerase chain reaction

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Polymerase chain reaction

• The ultimate purpose of this reaction is to
  amplify a defined section of DNA
• Make millions of copies of a specific region

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• Thermostable DNA polymerase is used for the PCR.
  These enzymes do not denature at high temperature
  and remain active through the entire PCR
  reaction. Therefore, in order to perform PCR we
  just need to mix in the reaction tube
• 1. DNA containing the gene to be amplified
• 2. Primers complementary to the flanking regions
  of the gene.

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• 3. Thermostable DNA polymerase
• 4. Nucleoside triphosphates (dATP, dGTP, dCTP,
  dTTP)
• and then place the reaction mixture in the PCR
  machine that can rapidly change the temperature
  of the reaction mixture.

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Design of PCR Primers

• The amplification product of a PCR reaction is
  defined by the sequence of the PCR primers. The
  primers anneal to complementary sequences on the
  DNA template and thereby determine the boundaries
  of the amplified product.
• Once a DNA target has been chosen, there are
  several rules of thumb for primer design that are
  important to consider. These general rules
  arePrimer length Choose primers that will
  anneal to complementary sequences that are 18-24
  nucleotides long.Duplex stability Both primers
  in a PCR reaction should have similar melting
  temperatures (Tm) to ensure that they will have
  the same hybridization kinetics during the
  template annealing phase.Non-complementary
  primer pairs The two primers cannot share
  complementarity at the 3 ends or else they will
  give rise to primer dimer products.No hairpin
  loops Each primer needs to be devoid of
  palindromic sequences which can give rise to
  stable intrastrand structures that limit primer
  annealing to the template DNA.

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• Agarose gel electrophoresis is used to
  characterize PCR products from a temperature
  optimization experiment. This photograph of an
  ethidium bromide stained gel below illustrates
  the effect of temperature on product yield and
  reaction specificity.

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• Tm 2(NA NT) 4(NG NC)

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gagtccccag gagcttacat

• 1 caggaaggcg gcaacgaagc gagtccccag gagcttacat
  cagtaagtga
• 51 ctggggtgaa cgacggcagc caacgcacat gcaactcgaa
  gtatgacggg
• 101 taaatgggtg agcttatgct cggaacaacg cattattcag
  ggggttaatt
• 151 gatagattga ccagccactg ccggacgtca tttgaggatt
  gtctgaatct
• 201 cttgccactc tagtttaatc atgtgaggta acaatatatg
  ctgatgaaga
• 251 gcaaatgggc tttaagcaat aattaacacg ataacaagga
  tgtactatgg
• 301 atacagttga agagctgggc gggacgtact tttatgcagg
  caagccgaat
• 351 ttaaaagcca gtgagctact atttatgatt ttctgtgaga
  atactgccag
• 401 ccagtttggt atgcaggatt tcggggctgt ggttgcgatt
  atttcgggta
• 451 gaagcaatct cttaacaaga ggaaagccca taggtgcaac
  aaaaggcaca

gccttgttgc gtaataagtc
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• gagtccccag gagcttacat cagtaagtga ctggggtgaa
• ctcaggggtc ctcgaatgta gtcattcact gaccccactt
• cgacggcagc caacgcacat gcaactcgaa gtatgacggg
• gctgccgtcg gttgcgtgta cgttgagctt catactgccc
• taaatgggtg agcttatgct cggaacaacg cattattcag
• atttacccac tcgaatacga gccttgttgc gtaataagtc

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Applications of PCR
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Cloning
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Diagnostics
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Forensics
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PCR mutagenesis

• The most common method is restriction site
  addition which utilizes PCR primer pairs that
  incorporate restriction enzyme recognition sites
  into the 5 end of the oligonucleotide.

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cDNA Production

• Reverse transcriptase can be used to convert mRNA
  into complementary DNA and thus was born the term
  cDNA.
• cDNA is a more convenient way to work with the
  coding sequence than mRNA because RNA is very
  easily degraded by omnipresent Rnases, and mRNAs
  cannot be directly cloned.
• By definition, cDNA is double-stranded DNA that
  was derived from mRNA which can be obtained from
  prokaryotes or eukaryotes.
• The Four basic steps in constructing a cDNA
  library
• 1. Purification of mRNA using chemical
  extraction and oligo-dT purification.2. First
  strand cDNA synthesis using oligo-dT, random
  pdN6, or specific primers.3. Second strand cDNA
  synthesis requires a priming event done with
  RNaseH.4. Repair of cDNA termini and ligation
  of adaptor oligos clone into vector.

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Why is RNA so unstable

• 1. Endoribonucleases (RNases) are very stable
  enzymes that cannot be easily inactivated . In
  fact, human hands are a rich source of RNase and
  it is therefore necessary to wear clean latex
  gloves during RNA isolation procedures and to use
  RNase-free labware.
• 2. RNA is thermodynamically less stable than DNA
  because of the 2 hydroxyl group on the ribose
  ring that promotes hydrophilic attack on the
  5-3 phosphodiester bond to form a 2-3 cyclic
  phosphate. This cyclic phosphate intermediate is
  stabilized by Mg, a component of many
  biochemical reactions. It is critical that the
  RNA be isolated intact and pure so that it can
  function as a faithful template for first strand
  synthesis. One method is to purify mRNA from
  tissue culture cells using guanidinium
  thiocyanate and oligo dT cellulose. Total RNA
  remains in the aqueous phase under acidic pH
  conditions following phenolchloroform
  extraction. After precipitation with isopropanol,
  the RNA solution is adjusted to high salt (0.5M
  NaCl) and loaded onto an oligo-dT cellulose
  column.

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Step 1a. Harvest total RNA
Inactivates endogenous RNases
P. Chomczynski, N. Sacchi, "Single-step method of
RNA isolation by guanidinum    
thiocyanate-phenolchloroform extraction,"
Analytical Biochemistry, 162 156-9, 1987.
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Step 1b. Selectively purify mRNA
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ISOLATION OF mRNA
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• 2. First strand cDNA synthesis
• Three different types of primers can be used.

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• Insertion of cDNA into plasmid.
• To complete our construction of a useful cDNA
  library we need a way to maintain and propagate
  our cDNA.
• We can accomplish this by inserting the cDNA into
  an appropriate plasmid.
•  Linker addition

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• Linkers
• An alternate method to insert cDNA fragments into
  a library vector is through the addition of
  "linkers".
• Linkers are short oligonucleotides (18 to 24
  mers) which are typically palindromic and contain
  a single or repeated restriction endonuclease
  recognition sequence.
• The palindromic nature allows the linker
  oligonucleotide to self-hybridize to form a blunt
  ended duplex.
• If the ends of the cDNA fragments are blunt, then
  the linker can be ligated to both ends to
  introduce useful terminal restriction sites.

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• Identifying and Analyzing Cloned DNA
• Suppose youve isolated a protein using a
  biological assay, you want to know the gene
  sequence that codes for this protein. Suppose
  that you also have constructed a cDNA library
  that should contain the mRNA for your protein,
  the question is how you fish that one mRNA out of
  thousands of other cDNA clones.
• A. One very laborious method
• 1.     pool numerous cDNA-containing plasmids and
  bind to nitrocellulose
• 2.     cDNA-containing plasmids are denature,
  hybridize to total cellular mRNA
• 3.     Bound mRNA are eluted and analyzed by in
  vitro translation
• 4.     subfraction of the previous pool of
  cDNA-containing plasmids are used to repeat the
  experiment until a single conlony is identified.
• This method proved infeasible for detecting mRNAs
  of rarer proteins.

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• B. Using antibodies to screen a cDNA expression
  library
• 1.     requires cDNAs are cloned into inducible
  bacteria vectors containing selectable markers
  (antibiotics).
• 2.     also requires that mRNA are properly
  transcribed and translated in E coli

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• C. Screening a cDNA library with an
  oligonucleotide probe
• A six-amino-acid sequence can be used to derive
  an 18-nt oligo, take into account the degeneracy
  (one could use inosine at wobble positions (3rd
  base of the codon), for instance).

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RT-PCR
Primer 1

Primer 2
Primer 1 2 or nested primers