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Working with DNA: Isolation and Fingerprinting

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We hope to identify new hot spring bacteria that cannot be grown on lab media ... Bacteria are prokaryotes, meaning they are only one celled organisms. ... – PowerPoint PPT presentation

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Title: Working with DNA: Isolation and Fingerprinting


1
Working with DNA Isolation and Fingerprinting
2
Funding and support received from…
3
Todays Agenda
  • Introduction
  • Safety
  • Basic Practice Using a Pipetteman
  • DNA Isolation Procedures
  • Restriction Enzymes and Gels
  • Yellowstone National Park and Bacterial Mats
  • Practice DNA Fingerprinting Problems
  • Analysis of our Fingerprinting Gels
  • Bacteria and DNA Basics
  • Closing

4
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

5
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

6
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.

7
Introduction
  • All living things contain cells
  • Eukaryotes more than one cell
  • Prokaryotes one cell organisms

8
The Boring (Yawn!!) Eukaryotic Plant and Animal
Cells…
9
The Exciting Bacterial Cell…
10
Bacteria come in many different shapes and
sizes…take a quick look…
11
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??

12
Bacteria are everywhere…
  • Dont panic!!
  • This is a good thing.
  • We have bacteria growing on our bodies which are
    supposed to be there.

13
What are Bacteria?
  • Bacteria are prokaryotes, meaning they are only
    one celled organisms. They are very small and
    can be harmful or beneficial.

14
Bacteria can cause diseases, like we all know…
15
Bacteria can also have beneficial uses…
16
Bacterial Cell Components…
17
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

18
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.

19
Now we are going to do some work!!!
  • DNA Precipitation

20
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

21
DNA… the code of life
22
(No Transcript)
23
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

24
What do we know about DNA?
  • Structure
  • Nitrogen bases are named
  • - adenine (A)
  • - guanine (G)
  • - thymine (T)
  • - cytosine (C)

25
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

26
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

27
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

28
What do we know about DNA?
  • Function
  • Codes for 80,000 genes, which form proteins…the
    building blocks of life.

29
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)

30
Plant or Animal DNA Strand
31
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

32
Bacterial DNA Strand
33
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

34
Restriction Enzymes and Gels
35
Restriction Enzymes
  • Cut specific sequences of DNA
  • Many different kinds
  • Named after organism they came from, enzyme
    number
  • E.g. EcoR1

36
Bacteria Produce Restriction Enzymes
  • Uniquely bacterial protection mechanism…why?
  • Restriction enzymes are short nucleotide
    sequences isolated from bacteria cells that
    protect them from virus.

37
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.

38
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.

39
DNA Fingerprinting
40
DNA Fingerprinting
  • DNA fragments are separated using gel
    electrophoresis
  • Each band represents the DNA which has been cut
    into smaller pieces using restriction enzymes

41
Gel Electrophoresis
  • From your studies of DNA, can you tell me what
    charge DNA has?

42
Gel Electrophoresis
  • Gel is made of water and agarose
  • Wells on one end are where gels will be loaded
    with our samples

43
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

44
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

45
Gel Electrophoresis
  • DNA moves from the negative towards the positive
  • Smaller faster
  • Larger slower
  • Where will these three end?

46
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

47
DNA Fingerprinting Questions and Answers
  • Do you know the answers to these questions?

48
DNA Fingerprinting
  • How good (accurate) is it at identification. For
    example, is it as good as classical fingerprints?

49
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.

50
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.

51
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.

52
Now, how do we come up with those different bands?
  • Answer Restriction Enzymes

53
Lets do an example of DNA Fingerprinting
together…
54
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.

55
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

56
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

57
Fingerprinting Gel Projected Results
58
Practical applications of DNA technology
59
Practical applications of DNA technology
  • Diagnosis of diseases includes
  • Huntingtons
  • PKU
  • cystic fibrosis
  • Duchennes muscular dystrophy

60
Practical applications of DNA technology
  • Human gene therapy
  • Somatic cell therapy versus germ cell therapy

61
Practical applications of DNA technology
  • Pharmaceutical products
  • Insulin
  • human growth hormone
  • Protection from viral infection

62
Practical applications of DNA technology
  • Forensic uses
  • DNA fingerprinting
  • RFLPs and simple tandem repeats (microsatellite
    DNA repeats of different lengths)

63
Practical applications of DNA technology
  • Environmental uses
  • Genetically engineered microbes for mining,
    cleaning up toxic wastes, etc.

64
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
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