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Recombinant DNA Technology and Genomics

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Title: Recombinant DNA Technology and Genomics


1
Chapter 3
  • Recombinant DNA Technology and Genomics

2
Agarose Gel Electrophoresis
  • Electrophoresis is a molecular technique that
    separates nucleic acids and proteins based on
  • Size
  • and
  • -Charge-

Shape
3
Agarose Gel Electrophoresis
  • DNA is a negatively charged molecule and
    therefore is attracted to positive charges.

4
Agarose Gel Electrophoresis
  • Agarose provides a matrix through which DNA
    molecules migrate.
  • Larger molecules move through the matrix slower
    than small molecules
  • The higher the concentration of agarose, the
    better the separation of smaller molecules

5
Agarose Gel Electrophoresis
  • How to make an agarose gel
  • Weigh out a specified amount of agarose powder.
  • Add the correct amount of buffer.
  • Dissolve the agarose by boiling the solution.
  • Pour the gel in a casting tray.
  • Wait for the gel to polymerize.

6
Agarose Gel Electrophoresis
  • How to make an agarose gel
  • Place gel in chamber and cover with buffer
  • Add loading dye to the sample
  • Load sample on to the gel.

7
Agarose Gel Electrophoresis
  • How to make an agarose gel
  • Stain the gel
  • Take a picture of the gel
  • Analyze results

8
Agarose Gel Electrophoresis
Electrophoresis Animation
9
Recombinant DNA
  • Recombinant DNA technology
  • Allows DNA to be combined from different sources
  • Also called genetic engineering or transgenics

10
Recombinant DNA
  • Vector DNA source which can replicate and is
    used to carry foreign genes or DNA fragments.
  • Recombinant DNA A vector that has taken up a
    foreign piece of DNA.

11
Restriction Enzymes
  • Restriction enzyme an enzyme which binds to DNA
    at a specific base sequence and then cuts the DNA
  • Restriction enzymes are named after the bacteria
    from which they were isolated.
  • Bacteria use restriction enzymes to chop up
    foreign viral DNA

12
Restriction Enzymes
  • Recognition site specific base sequence on DNA
    where a restriction enzyme binds.
  • All recognition sites are palindromes, which
    means they read the same way forward and
    backward.
  • example RACECAR or GAATTC
  • CTTAAG
  • Each restriction enzyme has its own unique
    recognition site.

13
Restriction Enzymes
14
Restriction Enzymes
  • After cutting DNA with restriction enzymes, the
    fragments can be separated on an agarose gel.
  • The smaller fragments will migrate further than
    the longer fragments in an electric field.
  • The bands are compared to standard DNA of known
    sizes. This is often called a DNA marker, or a
    DNA ladder.

15
Restriction Enzymes
  • Running a Restriction digest on an agarose gel

16
Restriction Enzymes
  • After analyzing your results, you draw a
    restriction map of the cut sites.
  • A restriction map is a diagram of DNA showing
    the cut sites of a series of restriction enzymes.

17
Restriction Enzymes
18
Restriction Enzymes
19
Restriction Enzymes
  • Most restriction enzymes cut within the
    recognition site.
  • When restriction enzymes cut in a zig zag
    pattern, sticky ends are generated.

20
Restriction Enzymes
  • Overhanging sticky ends will complementarily base
    pair, creating a recombinant DNA molecule.
  • DNA ligase will seal the nick in the
    phosphodiester backbone.

21
Restriction Enzymes
Restriction Enzyme Animation
22
Transformation
  • Transformation the process by which organisms
    take up and express foreign DNA

Griffiths experiment
23
Transformation
  • Bacterial Transformation
  • Bacteria, such as E.coli, can take up and express
    foreign DNA, usually in the form of a plasmid.

24
Transformation
  • Gene cloning using bacterial transformation to
    make lots of copies of a desired gene.

Gene Cloning Animation
25
Transformation
  • Steps of Bacterial Transformation
  • Choose a bacterial host
  • E. coli is a model organism
  • Well studied
  • No nuclear membranes
  • Has enzymes necessary for replication
  • Grows rapidly (20 min. generation time)
  • Inexpensive
  • Normally not pathogenic
  • Easy to work with and transform

26
Transformation
  • Steps of Bacterial Transformation
  • Choose a plasmid to transform
  • Characteristics of a useful plasmid
  • Single recognition site
  • Plasmid only cuts in one place, so this ensures
    that the plasmid is reformed in the correct
    order.
  • Origin of replication
  • Allows plasmid to replicate and make copies for
    new cells.
  • Marker genes
  • Identifies cells that have been transformed.
  • ? gene for antibiotic resistance bacteria is
    plated on media
  • with an antibiotic, and only bacteria
    that have taken up a
  • plasmid will grow
  • ? gene that expresses color bacteria that
    have taken up a
  • recombinant plasmid are a different
    color than bacteria
  • that have taken up a NONrecombinant
    vector.

27
Transformation
  • Steps of Bacterial Transformation
  • Prepare bacterial cells for transformation
  • Treat with calcium chloride softens the
    phospholipid bilayer of the cell membrane, which
    allows the plasmid to pass through
  • Electroporation brief electric pulse
  • Directly inject plasmid into bacterial host

28
Transformation
  • Steps of Bacterial Transformation
  • Plate transformation on appropriate media
  • Contains nutrients for bacteria and antibiotic to
    distinguish transformed bacteria from
    NONtransformed bacteria
  • Incubate plates overnight
  • E.coli grows at body temp. (37 C)
  • Analyze plates

Gene Cloning Animation
29
Gene Cloning
  • What makes a good vector?

30
Gene Cloning
  • What makes a good vector?

31
Gene Cloning
  • How do you identify and clone a gene of interest?
  • BUILD A LIBRARY!!
  • DNA library a collection of cloned DNA
    fragments from a particular organism
  • Can be saved for a relatively long period of time
    and screened to pick out different genes of
    interest
  • Two types of libraries
  • Genomic library contains DNA sequences from
    entire genome
  • cDNA library contains DNA copies of mRNA
    molecules expressed

Construction of a DNA library Animation
32
Gene Cloning
33
Gene Cloning
  • Steps to screen a library
  • Plate cells and transfer to nylon membrane
  • Lyse bacterial cells
  • Denature DNA
  • Add radioactively labeled probe that is
    complementary to gene of interest

34
Gene Cloning
  • Steps to screen a library
  • Wash off non-specifically bound probe
  • Expose membrane to x-ray film
  • Align exposed film with original plate
  • Grow cells containing gene of interest in culture.

35
Gene Cloning
  • Rarely is an entire gene cloned in one piece,
    even in a cDNA library, therefore must walk the
    chromosome until a start and stop codon are
    found.

36
Sequencing
  • Sequencing determining the order and
    arrangement of Gs, As, Ts and Cs in a segment
    of DNA.

37
Sequencing
  • Lets review replication..

38
Sequencing
  • The Sanger sequencing method uses
    dideoxy-nucleotides to generate all possible
    fragments of the DNA molecule to be sequenced.

deoxynucleotide
dideoxynucleotide
39
Sequencing
  • Set up four different reactions

40
Sequencing
  • Load the four reactions in different wells of a
    polyacrylamide gel to separate the fragments

41
Sequencing
Sequencing Animation
42
Human Genome Project
  • Initiated in 1990 with plan to identify all human
    genes
  • Analyze genetic variation among humans
  • Map and sequence genomes of model organisms
  • Develop new lab technology
  • Disseminate genome information
  • Consider ethical, legal, and social issues that
    accompany genetic research

43
Human Genome Project
  • Francis Collins
  • Craig Venter

44
Human Genome Project
  • Consider ethical, legal and social issues
  • Who owns your DNA?

45
Human Genome Project
  • Develop new lab technology
  • Automated Sequencing

46
Human Genome Project
  • Disseminate genome information
  • GenBank database

47
Human Genome Project
  • Analyze genetic variation among humans
  • The genome is approximately 99.9 identical
    between individuals of all nationalities and
    backgrounds.

48
Human Genome Project
  • Map and sequence genomes of model organisms
  • E.coli
  • Arabidopsis thaliana
  • Saccharomyces cerevisiae
  • Drosophila melangaster
  • Caenorhabditis elegans
  • mus musculus

49
PCR
  • Polymerase chain reaction (PCR)
  • A lab technique used to amplify segments of DNA

"PCR has transformed molecular biology through
vastly extending the capacity to identify,
manipulate and reproduce DNA. It makes abundant
what was once scarce -- the genetic material
required for experimentations."
50
PCR
  • Reaction requirements
  • Template DNA total genomic DNA isolated from an
    organism that contains a target region to be
    amplified
  • DNA primers - Short pieces of single stranded DNA
    that flank the target
  • Taq DNA polymerase - Attaches nucleotides on the
    growing strand of DNA
  • Nucleotides (GATC) Polymerase adds
    complementary nucleotides to the template

51
PCR
  • Reactions are placed in a machine called a
    thermal cycler. The machine cycles through three
    temperatures.

52
PCR
  • Heat samples to 94C for a minute or so to
    denature the double stranded template DNA.

53
PCR
  1. Drop temperature to around 50 or 60C to allow
    primers to anneal.

54
PCR
  1. Maintain temperature at 72C for a minute or two
    to allow the polymerase to elongate the new DNA
    strands.

55
PCR
  • The thermal cycler repeats the denaturing,
    annealing, and elongating temperatures
    approximately 30 times.

PCR Animation
56
PCR
  • PCR amplification is logarithmic, meaning the
    number of copies of the target is doubled every
    cycle. (2n)

57
PCR
PCR animation
58
PCR
  • Cloning by PCR
  • Design primer specific for gene of interest (must
    know some of the sequence)
  • Can use a T-vector because Taq polymerase adds an
    A to the 3 end of sequence

59
Applications of Recombinant DNA Technology
60
Chromosomal Location and Gene Copy Number
  • Fluorescence in situ hybridization (FISH)

61
Chromosomal Location and Gene Copy Number
  • Southern Blot - molecular technique where DNA is
    transferred onto a membrane from an agarose gel
    and a probe is hybridized.

62
Southern Blot
  • The first step in preparing a Southern Blot is to
    cut genomic DNA and run on an agarose gel.

63
Southern Blot
  • The next step is to blot or transfer single
    stranded DNA fragments on to a nylon membrane.

64
Southern Blot
  • The next step is to hybridize a radioactively
    labeled DNA probe to specific sequences on the
    membrane.

65
Southern Blot
  • The last step is to expose the radioactively
    labeled membrane to a large sheet of film.
  • You will only visualize bands where the probe
    hybridized to the DNA..

66
Southern Blot
Southern Blot Animation
67
Studying Gene Expression
  • Northern Blot
  • Isolate RNA from tissue of interest
  • Separate on agarose gel
  • Blot onto nylon membrane
  • Hybridize probe specific for desired transcript
  • Expose on film
  • Reverse Transcription PCR (RT-PCR)
  • Used if RNA produced is below detection level for
    Northern blot
  • Isolate RNA from tissue of interest
  • Convert into double stranded cDNA
  • Amplify by PCR
  • Run on agarose gel

68
Studying Gene Expression
  • Real Time PCR (qPCR)
  • Eliminates the need for running agarose gels
  • Is quantitative

69
Studying Gene Expression
  • Gene microarray

Microarray animation
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