PCR and Forensics YouTube - CSI Intro

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PCR and ForensicsYouTube - CSI Intro
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What well be doing

• Lesson 1 DNA chemistry
• Lesson 2 Theory of PCR
• Lesson 3 Prac 330 PCR using supplied template
  (set up and run PCRs)
• Lesson 4 Run gel of PCRs from Lesson 3, Prac 225
  do restriction digests.
• Lesson 5 Run restriction digests on gels and
  produce DNA fingerprints

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Lesson 1 The chemistry of DNA
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In a nutshell, PCR

• Produces enough target DNA that it can be
  analysed, either on a gel or in a machine which
  measures fluorescence.
• DNA (deoxyribonucleic acid) is the chemical which
  carries the genetic code of life on this planet.
• YouTube - Polymerase Chain Reaction

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DNA bases
Pratt Ch. 3. page 54, 55
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The bases are linked to a five-carbon sugar to
form nucleosides. In RNA, the sugar is ribose.
In DNA, the sugar is 2 deoxyribose.
page 55 Nucleosides.
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The structure of DNA The linkage between the
nucleotides is called phosphodiester
bond. Linked nucleotides form a polymer in which
the the backbones are the phosphate-sugar groups
and the bases project out.
One strand
page 56 The structure of DNA.
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The opposing strands of DNA pair anti-parallel
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5
5
3
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Exercise

• Draw a 3 base section of DNA indicating the 5
  and 3 regions
• Look at the next slide and explain, using your
  knowledge of electronegativity and molecular
  geometry, explain how/why the hydrogen bonds
  shown exist
• Heres a really tough one hydrogen bonding
  doesnt have an effect on double strand
  stability, why not?
• The pKA of the phosphoryl groups is about 3.
  Explain why DNA is negatively charged at pH 7.0

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DNA contains two strands
DNA contains 2 strands (double helix) as their
bases pair through hydrogen bonding.
Which is stronger??
page 57 DNA base pairs.
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How is DNA measured?

• Size of DNA is measured in base-pairs (bp) or,
  for example, Kilobase pairs (1000bp1kb).
• Most DNA in cells is thousands to millions of bp

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• Genome Sizes of Model SystemsModel system
  Size (Million Bases) Genes
• Escherichia coli (Bacterium)  4.6
  3,000
• S. cerevisiae (Yeast) 15
• Neurospora crassa (Fungus) 39.9
  10,000

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What is the mass of one human genome?

• Assume
• Genome is 109bp
• The average mass of a nucleotide residue is
  300g/mole

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DNA replication issues

• DNA polymerase must have a primer (a bit of DNA
  or RNA on which to elongate the DNA strand)
• The raw material used to construct DNA is dNTPs
• DNA polymerase can only synthesise 5? 3

Draw a diagram illustrating how DNA is
synthesised.
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Lesson 2 The theory of PCR
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POLYMERASE CHAIN REACTION
Polymerase chain reaction (PCR) uses
thermostable DNA polymerase and specific
oligonucleotide primers to amplify genes. The
first thermostable DNA polymerase was
Taq polymerase from the bacterium Thermus
aquaticus which was isolated from hot springs.
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Figure 3.08 A DNA melting curve.
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The rate of renaturation depends on the length
of the DNA fragment Short fragment anneal faster
than long fragments.
Figure 3.09 Renaturation of DNA.
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POLYMERASE CHAIN REACTION
Denature DNA sample to separate DNA
strands (94oC, 5 min)
Hybridise primers to DNA strands (30-65oC, 30 s)
20-30 cycles
Denature to separate new DNA strands (94oC, 30 s)
Taq polymerase synthesises new DNA
strands (65-75oC, 2-5 min)
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POLYMERASE CHAIN REACTION
Target DNA
3 5
5 3
Denature DNA
3
5
5
3
Anneal specific primers
3
5
5
3
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POLYMERASE CHAIN REACTION
3
5
5
3
Extend primers with Taq polymerase
5 3
3 5
5 3
3 5
Denature DNA and anneal specific primers
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3
5
5
3
5
3
5
3
Extend primers with Taq polymerase
3
5
5
3
5
3
5
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Size of PCR fragment
Repeat cycles
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POLYMERASE CHAIN REACTION
Cycle number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Target DNA 0 1 2 4 8 16 32 64 128 256 512 1024 204
8 4096 8192
Cycle number 16 17 18 19 20 21 22 23 24 25 26 27 2
8 29 30
Target DNA 16384 32768 65536 131072 262144 524288
1048576 2097152 4194304 8388608 16777216 33544432
67108864 134217728 268435456
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The PCR Song and a myth

• YouTube - PCR
• YouTube - The PCR Song
• YouTube - CSI PCR in 60 seconds BS
• There was a time when to amplify DNA,
• You had to grow tons and tons of tiny cells.
• Then along came a guy named Dr. Kary Mullis,
• Said you can amplify in vitro just as well.
• Just mix your template with a buffer and some
  primers,
• Nucleotides and polymerases, too.
• Denaturing, annealing, and extending.
• Well its amazing what heating and cooling and
  heating will do.
• PCR, when you need to detect mutations.
• PCR, when you need to recombine.
• PCR, when you need to find out who the daddy is.
• PCR, when you need to solve a crime.

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POLYMERASE CHAIN REACTION
PCR is now used routinely for

• cloning genes
• DNA sequence analysis
• site directed mutagenesis
• generating DNA probes
• random PCR to differentiate individuals
• cloning flanking DNA
• Producing DNA from unculturable microorganisms

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Cancer detection
Valeries normal breast
Valeries breast tumour
Valeries peripheral blood
control
std
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Typical PCR reaction mixture
Calculate the final concentrations of the
components in the table below.
COMPONENT VOLUME FINAL CONCENTRATION
1. autoclaved ultra-filtered water (pH 7.0) 20.7µL -
2. 10x PCR Buffer 2.5µL
3. dNTPs mix (25 mM each nucleotide) 0.2µL
4. primer mix (25 pmoles/µL each primer) 0.4µL
5. Taq DNA polymerase (native enzyme) 0.2µL 1 Unit/25 µL
6. genomic DNA template (100 ng/µL) 1.0µL
The PCR buffer used was made after the
recommendations of the manufacturer/vendor
(Perkin Elmer Cetus). The 10x PCR buffer
contains 500 mM KCl 100 mM Tris-HCl (pH 8.3)
15 mM MgCl2 (the final concentrations of these
ingredients in the PCR mix are 50 mM KCl 10 mM
Tris-HCl 1.5 mM MgCl2).It is useful to prepare
a larger volume of this buffer (10-15ml), aliquot
it and store the vials at -20 C for years.
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Things Are a Little Different Now...
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Now try the PCR/bioinformatics exercise