Recombinant DNA Technology and Genomics

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Chapter 3

• Recombinant DNA Technology and Genomics

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Agarose Gel Electrophoresis

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

Shape
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Agarose Gel Electrophoresis

• DNA is a negatively charged molecule and
  therefore is attracted to positive charges.

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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

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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.

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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.

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Agarose Gel Electrophoresis

• How to make an agarose gel
• Stain the gel
• Take a picture of the gel
• Analyze results

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Agarose Gel Electrophoresis
Electrophoresis Animation
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Recombinant DNA

• Recombinant DNA technology
• Allows DNA to be combined from different sources
• Also called genetic engineering or transgenics

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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.

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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

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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.

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Restriction Enzymes
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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.

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Restriction Enzymes

• Running a Restriction digest on an agarose gel

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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.

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Restriction Enzymes
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Restriction Enzymes
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Restriction Enzymes

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

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Restriction Enzymes

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

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Restriction Enzymes
Restriction Enzyme Animation
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Transformation

• Transformation the process by which organisms
  take up and express foreign DNA

Griffiths experiment
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Transformation

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

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Transformation

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

Gene Cloning Animation
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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

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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.

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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

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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
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Gene Cloning

• What makes a good vector?

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Gene Cloning

• What makes a good vector?

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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
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Gene Cloning
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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

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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.

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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.

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Sequencing

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

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Sequencing

• Lets review replication..

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Sequencing

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

deoxynucleotide
dideoxynucleotide
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Sequencing

• Set up four different reactions

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Sequencing

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

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Sequencing
Sequencing Animation
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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

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Human Genome Project

• Francis Collins

• Craig Venter

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Human Genome Project

• Consider ethical, legal and social issues
• Who owns your DNA?

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Human Genome Project

• Develop new lab technology
• Automated Sequencing

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Human Genome Project

• Disseminate genome information
• GenBank database

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Human Genome Project

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

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Human Genome Project

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

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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."
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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

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PCR

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

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PCR

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

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PCR

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

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PCR

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

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PCR

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

PCR Animation
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PCR

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

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PCR
PCR animation
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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

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Applications of Recombinant DNA Technology
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Chromosomal Location and Gene Copy Number

• Fluorescence in situ hybridization (FISH)

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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.

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Southern Blot

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

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Southern Blot

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

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Southern Blot

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

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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..

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Southern Blot
Southern Blot Animation
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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

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Studying Gene Expression

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

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Studying Gene Expression

• Gene microarray

Microarray animation