6.3 Advanced Molecular Biological Techniques

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6.3 Advanced Molecular Biological Techniques

• 1. Polymerase chain reaction (PCR)
• 2. Restriction fragment length polymorphism
  (RFLP)
• 3. DNA sequencing

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Polymerase Chain Reaction (PCR)

• Until the late 1980s, many copies of a desired
  DNA fragment could only be made by inserting the
  DNA sequence into plasmids
• Problem The plasmids had to be extracted from
  bacteria, and then the desired DNA fragment had
  to be excised
• Solution Direct method of making copies of a
  desired DNA sequence, called polymerase chain
  reaction (PCR) Kary Mullins, 1985

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Polymerase Chain Reaction (PCR)

• PCR Amplification of DNA sequence by repeated
  cycles of strand separation and replication
• Small sample of DNA can be amplified to make
  multiple copies of a desired DNA fragment
• Each PCR cycle doubles the copies of a desired
  DNA fragment, resulting in exponential growth
• ie. after 30 cycles, gt 1 000 000 000 copies (230)
  are made

http//users.ugent.be/avierstr/principles/pcrcopi
es.gif
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Polymerase Chain Reaction (PCR)

• One cycle
• Double-stranded DNA is denatured using heat
  (94oC96oC) to separate strands by breaking
  hydrogen bonds
• No DNA helicase or DNA gyrase
• DNA primers (5-3) anneal to complementary
  template DNA that bracket the desired DNA
  sequence (50oC65oC)
• No RNA primer
• Taq polymerase add complementary nucleotides to
  synthesize the new DNA strand (72oC)
• No DNA polymerase III
• Repeat cycle (steps 1-3)

http//croptechnology.unl.edu/animationThumbnails/
1020458324.gif
http//www.cbs.dtu.dk/staff/dave/roanoke/genetics9
80211.html
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PCR Length of DNA strands

• Targeted DNA sequence is not completely isolated
  in the first few cycles of PCR
• Variable-length strands Mixture of replicated
  DNA strands of unequal length
• After first cycle, variable-length strands start
  at target region on one end and extends beyond
  the target region on the other end
• Constant-length strands Mixture of replicated
  DNA strands of equal length
• After second cycle, two of the replicated strands
  start at target region on one end and terminates
  at target region on the other end
• By third cycle, number of copies of targeted DNA
  strands increases exponentially

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www.maxanim.com/genetics/PCR/PCR.htm


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Restriction Fragment Length Polymorphism (RFLP)

• Polymorphism
• any difference in DNA sequence (coding or
  non-coding) that can be detected between
  individuals
• Restriction Fragment Length Polymorphism Analysis
  
• technique that compares different lengths of DNA
  fragments produced by restriction endonucleases
  to determine genetic differences between
  individuals by using complementary radioactive
  probes

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Restriction Fragment Length Polymorphism Analysis

• Digest DNA using restriction enzyme(s)
• Run digested DNA on gel using gel electrophoresis
• Smear - Many DNA fragments with slight
  differences in length
• Expose gel to a chemical to denature
  double-stranded DNA to become single-stranded
• Southern blotting

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RFLP Analysis

• 4. Southern blotting
• Transfer DNA from gel to nylon membrane
• Expose nylon membrane to solution with
  radioactive complementary nucleotide probes that
  hybridize to specifically chosen DNA sequences on
  nylon membrane
• Place nylon membrane against X-ray film, where
  hybridized radioactive probes cause exposure of
  X-ray film, producing an autoradiogram

http//www.cbs.dtu.dk/staff/dave/roanoke/genetics9
80211.html
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RFLP analysis

• Differences in pattern to detect polymorphisms

Animation
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DNA Sequencing

• Determine sequence of base pairs for genes
• Sanger dideoxy method DNA sequencing technique
  based on DNA replication using dideoxynucleoside
  triphosphate

http//www.sanger.ac.uk/Info/Intro/gfx/fred_bw.jpg
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Sanger dideoxy method

• Into 4 reaction tubes, add
• Double-stranded DNA to be sequenced is denatured
  to become single-stranded
• Radioactively labelled primer to end of the DNA
  template
• DNA polymerase
• Free nucleotides (dATP, dTTP, dGTP, dCTP)
• Into each of the 4 reaction tubes, add a
  different radioactively labelled dideoxy analogue
  (nucleoside triphosphate that has no hydroxyl
  group on the 2 and 3 carbon of ribose sugar)

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Sanger dideoxy method

• If dideoxy analogue is missing 3-OH on the
  deoxyribose sugar, DNA polymerase cannot add the
  next complementary base?synthesis stops

• Chain termination resulting in different DNA
  fragment lengths
• Separate different DNA lengths by gel
  electrophoresis, loading each reaction tube in a
  separate well/lane
• Sequence can be read from the gel in ascending
  order

http//www.cbs.dtu.dk/staff/dave/roanoke/genetics9
80211.html
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Sanger Method Animation

• http//www.mefeedia.com/watch/21777157

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Human Genome Project

• To determine the genetic sequence of the 46 human
  chromosomes
• Used similar sequencing technique, but used
  fluorescently tagged ddNTPs that could be read by
  a computer