DNA Technology

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DNA Technology

• VA SOL 12.e 3.b-c 6.f-i

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What is DNA?

• Deoxyribonucleic acid (DNA) is the molecule that
  contains the biological instructions that makes
  each species unique.
• Basically, DNA provides the instructions for
  building and maintaining an organism.
• These instructions are stored in the order or
  sequence of the four chemical bases adenine (A),
  guanine (G), cytosine (C), and thymine (T).

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• Understanding the structure and function of DNA
  has allowed scientists to make huge advancements
  in a variety of different areas like medicine,
  agriculture, bioinformatics and forensics.

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• DNA technology enabled scientists to
• Identify species
• Map entire genomes
• Identify specific genes and traits
• Diagnose diseases
• Determine paternity
• Recombine specific genes
• Solve crimes
• Clone organisms

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• DNA is used to identify organisms.

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• The Bush to Base scientists are currently
  studying chimpanzees in Tanzania. Understanding
  the food they eat can provide these scientists
  with a wealth of information.
• Plant samples were collected and returned to
  Virginia Tech to be identified.

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• With a small sample of DNA, scientists can follow
  a simple set of steps to identify the sequence of
  the DNA and learn so much.
• DNA Extraction
• Polymerase Chain Reaction (PCR)
• DNA Purification
• Electrogelphoresis
• DNA Sequencing/BLAST
• Additional Tools

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• DNA Extraction

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• DNA can be extracted from most cells using a
  simple procedure. Once extracted, the DNA can be
  analyzed.

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• There are three basic steps in DNA extraction
• Breaking the cells open by grinding them up to
  expose the DNA.
• Removing the cell membrane by using a detergent.
• Precipitating the DNA using alcohol.

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• Polymerase Chain Reaction
• (PCR)

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• Polymerase chain reaction (PCR) is a technique
  used to make multiple copies of any piece of DNA.

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• The idea behind PCR is very simple.
• Millions of copies of DNA can be produced in a
  few hours by incubating the DNA under specific
  conditions with the enzyme DNA polymerase,
  nucleotides, and short pieces of complementary
  DNA called primers.

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• The process of PCR is composed of cycles composed
  of each of the following steps
• Denaturing
• Annealing
• Extending

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Denaturing

• The DNA is heated to break the hydrogen bonds
  between the complementary strands allowing the
  two strands to separate.

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Annealing

• Once separated, the DNA is cooled to allow the
  primers, forward and reverse, to bond to the ends
  of the targeted sequence.

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Extending

• Once the primers have bonded, the DNA polymerase
  begins to extend the primers by adding
  nucleotides using the longer strands of DNA as
  templates.

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• Since the copies at as templates themselves,
  millions of copies are produced within a few
  dozen cycles.
• The number of copies grows exponentially!

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• PCR can be performed using minute amounts of DNA,
  so it makes it possible for scientists to amplify
  DNA from a wide variety of sources.

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History Behind PCR

• PCR makes it possible to replicate DNA quickly
  whereas it can take weeks to clone a piece of DNA
  using a plasmid.
• Developed in 1983, Kary B. Mullins was awarded
  the Nobel Prize for Chemistry in 1993 for
  revolutionizing the study of DNA.

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• Taq polymerase, an enzyme isolated from the
  bacterium Thermus aquaticus found in the hot
  springs at Yellowstone, makes PCR possible
  because it is a heat stable DNA polymerase.

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• PCR involves thermal cycling which means the
  solution is continually heated and cooled, the
  Taq polymerase is able to withstand the high
  temperatures that breaks down or denatures most
  enzymes.

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• DNA Purification/
• PCR Clean Up

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• DNA purification is basically a way to clean up a
  sample especially PCR product.
• Even though a single strand of DNA can produce
  millions of copies using PCR, the sample has much
  more than the targeted DNA sequence, so it needs
  to be cleaned or purified.

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• The purification process removes any contaminants
  from the PCR product like the primers and
  nucleotides that were initially added.

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• Gel Electrophoresis

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• Gel electrophoresis is a technique used to
  separate macromolecules including DNA, RNA, or
  proteins.

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• DNA fragments are placed at the negative end of a
  porous gel. Since the phosphate groups found in
  DNA have a negative charge, when an electrical
  current is applied to the gel the fragments
  migrate toward the positive end of the chamber.

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• DNA molecules are too large to analyze, so
  scientists can cut DNA into smaller pieces using
  restriction enzymes.
• PCR can also be used to generate multiple copies
  of specific, shorter pieces of DNA using forward
  and reverse primers.

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• The different sized pieces of DNA will separate
  as they move through the gel - the smaller
  fragments moving faster and farther than the
  larger fragments.

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• It is important to have a ladder or mixture of
  DNA fragments for comparison.

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• Bands of DNA can be easily seen using a UV light
  on a gel that has had ethidium bromide (EtBr)
  added.
• Unfortunately, EtBr is a carcinogen, so schools
  will need to other dyes to visualize the bands.

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• Gel electrophoresis can be used to
• Confirm the presence of DNA.
• Ensure that a PCR has been successful
• Compare genomes of different organisms or
  different individuals
• Locate and identify one particular gene out of
  thousands of genes within a genome

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• DNA Sequencing

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• Sequencing is the process of determining the
  order of the nucleotide bases, adenine (A),
  guanine (G), cytosine (C), and thymine (T) in a
  sample of DNA.
• Sequences provide scientists with the basis they
  need to study specific genes with the genome of
  an organism.

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• Sequencing is done at facilities like Virginia
  Bioinformatics Institute (VBI).
• The fragments are analyzed on a capillary
  electrophoresis machine and detected by a laser
  to generate a string of nucleotides representing
  the DNA sequence of the starting template.

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• Once the sequence is generated, scientists can
  perform BLAST (Basic Local Alignment Search Tool)
  search.

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• Information from a BLAST can be used to infer
  functional and evolutionary relationships between
  sequences as well as help identify members of
  gene families.

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• The research done this summer helped to identify
  10 plant samples found in the Mahale Mountains
  National park in Tanzania.
• These plants are food sources for the chimpanzees
  being studied in this national park.

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Additional Tools

• There additional tools that need to be used
  during the process of sequencing DNA.
• Centrifuge
• Nanospectrophotometer
• Finch TV

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Centrifuge

• The centrifuge spins a solution separating the
  solid particles from the liquid portion of a
  solution.
• One important rule to remember is to
  counterbalance the centrifuge!

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Nanospectrophotometer

• The nanospectrophotometer is used to determine
  the concentration of DNA is a sample.
• DNA needs to be at specific concentrations in
  order to run a PCR or have a sample sequenced.

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Useful Programs

• Finch TV is a DNA sequence viewer allows you to
  view chromatograms and conduct BLAST searches.

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• ClustalX is a multiple sequence alignment program
  allows you to compare sequences from BLAST
  results.

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Final Results

• Six weeks is a short amount of time to correctly
  identify an organism using DNA, but it is plenty
  of time to provide the experience.
• Upon completing this process, I came up with a
  list of possibilities for each of the samples.

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Sample Identifications

• Sample 1 Ficus sp. Moore (fig tree)
• Sample 2 No DNA isolated from two samples
• Sample 3 Polysphaeria macrophylla
• Sample 4 Ficus sp. Moore (fig tree)

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• Sample 5 Vangueria infausta, Tricalysia
  elliotii, Sarcocephalus latifolius, Pseudosabicea
  floribunda or Stipularia elliptica
• Sample 6 Ficus sp. Moore (fig tree)
• Sample 7 - Vangueria infausta, Tricalysia
  elliotii, Sarcocephalus latifolius, Pseudosabicea
  floribunda, Kraussia floribunda or Stipularia
  elliptica

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• Sample 8 Vangueria infausta, Tricalysia
  elliotii, Pseudosabicea floribunda, Kraussia
  floribunda, Rutidea schlechteri or Stipularia
  elliptica
• Sample 9 Ficus sp. Moore (fig tree)
• Sample 10 - Ensete ventricosum or Sarcocephalus
  latifolius

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• Narrowing down the search to one specific species
  takes more analysis and a lot more time!

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References

• Campbell, Neil. Biology. 3rd ed. Redwood City
  Benjamin/Cummings, 1993.
• Miller, Kenneth, and Joseph Levine. Biology.
  Upper Saddle River Pearson Prentice Hall, 2006.
• Life Science Learning Center. 2007. Media
  Animations. 12 Aug. 2008 http//lifesciences.envme
  d.rochester.edu/animation.html
• PCR Animation. 13 Aug. 2008 http//www.ncvs.org/nc
  vs/groups/cmb/graphics/pcranimated.gif
• Thermas aquaticus. 13 Aug. 2008
  http//home.planet.nl/evanhove/Microbiologie/JQui
  z/thermus.jpg
• Electrophoresis. 12 Aug. 2008 http//fig.cox.miami
  .edu/cmallery/150/gene/c7.20.8.electrophoresis.jp
  g
• Kary Mullis. 13 Aug. 2008 http//www.foruminvest.r
  o/images/forum/kary_mullis.JPG
• Yellowstone hot springs. 13 Aug. 2008
  http//whyfiles.org/022critters/hot_bact.html