Working with DNA: Isolation and Fingerprinting

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Working with DNA Isolation and Fingerprinting
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Funding and support received from
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Todays Agenda

1. Introduction
2. Safety
3. Basic Practice Using a Pipetteman
4. DNA Isolation Procedures
5. Restriction Enzymes and Gels
6. Yellowstone National Park and Bacterial Mats
7. Practice DNA Fingerprinting Problems
8. Analysis of our Fingerprinting Gels
9. Bacteria and DNA Basics
10. Closing

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Our Research Project - What We Are Cloning and Why

• We hope to identify new hot spring bacteria that
  cannot be grown on lab media
• To study these organisms, we extract DNA from hot
  springs that contain unknown bacteria

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Our Research Project - What We Are Cloning and Why

• We clone a specific identification gene (the 16S
  gene) from the hot spring DNA
• We place each hot spring gene into E. coli, our
  cloning factory
• And then we fingerprint and DNA sequence each hot
  spring clone

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Words to the cautious

• Neither the E. coli we use nor the hot spring
  bacteria we study have ever been shown to be
  pathogenic. Although you will be working with E.
  coli, you will never come in contact with hot
  spring bacteria just their DNA after it has been
  extracted from the once-living cells.

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Introduction

• All living things contain cells
• Eukaryotes more than one cell
• Prokaryotes one cell organisms

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The Boring (Yawn!!) Eukaryotic Plant and Animal
Cells
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The Exciting Bacterial Cell
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Bacteria come in many different shapes and
sizestake a quick look
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Bacteria can replicate easily

• To grow, bacteria divide and divide and divide
  again.
• Problem If you started with only 1 bacterial
  cell, and it divided 10 times, how many bacteria
  would you then have??

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Bacteria are everywhere

• Dont panic!!
• This is a good thing.
• We have bacteria growing on our bodies which are
  supposed to be there.

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What are Bacteria?

• Bacteria are prokaryotes, meaning they are only
  one celled organisms. They are very small and
  can be harmful or beneficial.

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Bacteria can cause diseases, like we all know
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Bacteria can also have beneficial uses
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Bacterial Cell Components
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Plasmids can also be found in bacterial cells

• Plasmids are Mini-chromosomes found only in
  some bacteria
• (1,000-10,000 base pairs)
• Free-floating in the cytoplasm - not
  membrane-bound like chromosome
• Naturally carry many antibiotic resistance genes
• Replicate on their own

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Plasmids and Cloning

• Bacteria are used in genetic engineering and
  cloning because they serve as the factories for
  expressing foreign genes like insulin. Without
  plasmids, there would be no way to clone and
  express foreign genes.

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Now we are going to do some work!!!

• DNA Precipitation

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What are we using now?

• 3M Sodium acetate contributes ions to bind with
  positive phosphates open on DNA
• Isopropanol polar solution which attaches to
  DNA for precipitation
• - 80C Freezer Speeds the precipitation reaction
  with low amounts of DNA

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DNAthe code of life
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What do we know about DNA?

• Structure
• Composed of nucleotides (monomer) consisting of
• 1) phosphate group
• 2) deoxyribose sugar
• 3) one of four nitrogen bases

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What do we know about DNA?

• Structure
• Nitrogen bases are named
• - adenine (A)
• - guanine (G)
• - thymine (T)
• - cytosine (C)

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What do we know about DNA?

• Structure
• The structure of these nucleotides determines how
  they fit together.
• Adenine fits with Thymine
• Guanine fits with Cytosine

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What do we know about DNA?

• Structure
• DNA is double-stranded
• The nucleotides are linked together covalently
• Phosphate Sugar Phosphate Sugar etc.
• This is the backbone

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What do we know about DNA?

• Structure
• The two strands are oriented in opposite
  directions
• The two strands are wound around each other
  forming the helix structure

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What do we know about DNA?

• Function
• Codes for 80,000 genes, which form proteinsthe
  building blocks of life.

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Eukaryotic Deoxyribonucleic Acid

• DNA for Short
• Double helix - two strands made up of A, T, G,
  and C bases
• Complex organisms - many linear chromosomes
  (10,000,000,000 or more base pairs)

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Plant or Animal DNA Strand
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Prokaryotic Deoxyribonucleic Acid

• Bacteria - one circular chromosome (1,000,000
  base pairs)
• Chromosomes, in both cases, are held by proteins
  to the cell or nuclear membrane
• Most RNA is translated into proteins that have
  structural or functional jobs in cells

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Bacterial DNA Strand
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Lets get our samples now and continue on with
our isolation

• Centrifuge Spins solution at high speed to
  concentrate DNA at the bottom
• TE buffer at pH 8.0
• RNAse enzyme which removes RNA present in
  sample through digestion

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Restriction Enzymes and Gels
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Restriction Enzymes

• Cut specific sequences of DNA
• Many different kinds
• Named after organism they came from, enzyme
  number
• E.g. EcoR1

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Bacteria Produce Restriction Enzymes

• Uniquely bacterial protection mechanismwhy?
• Restriction enzymes are short nucleotide
  sequences isolated from bacteria cells that
  protect them from virus.

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Bacteria Produce Restriction Enzymes

• When a viral DNA enters the bacterial cell, the
  restriction enzyme is able to recognize a
  specific sequence (restriction site) on the DNA
  molecule, which is usually 4-8 nucleotides long.
  The restriction enzyme will cut the viral DNA at
  these sites and hence restrict the growth of the
  virus.

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Bacteria Produce Restriction Enzymes

• Several hundreds of these enzymes have been
  isolated from various organisms and most are
  available commercially. These enzymes are used to
  cut a segment of gene from a human DNA molecule.

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DNA Fingerprinting
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DNA Fingerprinting

• DNA fragments are separated using gel
  electrophoresis
• Each band represents the DNA which has been cut
  into smaller pieces using restriction enzymes

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Gel Electrophoresis

• From your studies of DNA, can you tell me what
  charge DNA has?

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Gel Electrophoresis

• Gel is made of water and agarose
• Wells on one end are where gels will be loaded
  with our samples

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Gel Electrophoresis

• The gel box contains water and buffer to keep the
  pH constant
• Gel box has platinum wire that conducts protons
  and electrons
• Gel box will be wired to the power source
  following the load

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Gel Electrophoresis

• To the strand of DNA moving through the agarose,
  the gel looks like a big mesh-like maze
• The DNA travels through the maze as fast as its
  size will allow

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Gel Electrophoresis

• DNA moves from the negative towards the positive
• Smaller faster
• Larger slower
• Where will these three end?

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Gel Electrophoresis

• Review
• DNA travels to
• When the power supply turns off, we can
  see where the bands are and infer which are
  bigger and smaller
• Small goes far
• Large goes not far

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DNA Fingerprinting Questions and Answers

• Do you know the answers to these questions?

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DNA Fingerprinting

• How good (accurate) is it at identification. For
  example, is it as good as classical fingerprints?

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Question 1

• How good (accurate) is it at identification. For
  example, is it as good as classical fingerprints?
  
• Answer In theory, with the exception of
  identical twins, EVERYONE on this planet has a
  different DNA fingerprint. That is, DNA
  fingerprinting IS as good (distinctive/unique/spec
  ific) as classical fingerprinting for
  identification.

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Question 2

• What are its advantages?
• Answer In theory DNA fingerprinting will work
  with much smaller amounts of material than a
  classical fingerprint DNA lasts much longer
  than classical fingerprints. DNA-containing
  samples that are many years old (up to 25 million
  yr.) are still usable. Only very tiny quantities
  of DNA are required in order to carry out a
  highly accurate test. For example, dried blood,
  semen, spit, skin etc. on samples stored in dusty
  files for years are still usable. Samples of
  mixed DNA's can also be used. DNA containing
  evidence is much harder to clean up at a crime
  scene than other evidence, like classical
  fingerprints.

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Question 3

• What are its limitations?
• Answer There currently are no accepted Federal
  standards for controlling the quality of DNA
  testing nationwide. Poor quality poorly
  controlled testing can lead to QUESTIONABLE and
  SHODDY RESULTS.
• Even if there is a perfect match between DNA, you
  can not say HOW the DNA containing sample got
  there or WHEN. In the O.J. trial a VALID question
  was raised about the possibility of evidence
  being planted. What makes this charge so powerful
  is the EXTREME SENSITIVITY of the procedure.

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Now, how do we come up with those different bands?

• Answer Restriction Enzymes

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Lets do an example of DNA Fingerprinting
together
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DNA Fingerprinting Example

• Two men fitting the description of a robber were
  caught in the vicinity of the crime. Both had
  cuts on their arms which they explained away.
  DNA samples were taken from each suspect and from
  the broken window at the scene of the crime.

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DNA Fingerprinting Example

• Using DNA Fingerprinting and Restriction Enzymes,
  we can determine which of the men was the
  robber!!
• We can cut each sample (one from each suspect and
  one from the crime scene) with two different
  enzymes, run them on a gel and compare the results

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So, how do we organize what we know? We organize
the gel lanes

•   Lane      Description 
• 1 DNA sample from crime scene cut w/ Enzyme 1
• 2 DNA sample from crime scene cut w/ Enzyme 2
• 3 DNA sample from Suspect 1 cut with Enzyme 1
• 4 DNA sample from Suspect 1 cut with Enzyme 2
• 5 DNA sample from Suspect 2 cut with Enzyme 1
• 6 DNA sample from Suspect 2 cut with Enzyme 2

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Fingerprinting Gel Projected Results
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Practical applications of DNA technology
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Practical applications of DNA technology

• Diagnosis of diseases includes
• Huntingtons
• PKU
• cystic fibrosis
• Duchennes muscular dystrophy

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Practical applications of DNA technology

• Human gene therapy
• Somatic cell therapy versus germ cell therapy

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Practical applications of DNA technology

• Pharmaceutical products
• Insulin
• human growth hormone
• Protection from viral infection

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Practical applications of DNA technology

• Forensic uses
• DNA fingerprinting
• RFLPs and simple tandem repeats (microsatellite
  DNA repeats of different lengths)

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Practical applications of DNA technology

• Environmental uses
• Genetically engineered microbes for mining,
  cleaning up toxic wastes, etc.

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Practical applications of DNA technology

• Agricultural uses
• Animal husbandry
• Transgenic animals
• Gene knock-in or knock-out animals (requires
  homologous recombination)
• Cloned animals
• Genetic engineering in plants
• Can grow many plants from a single cell