Applications of Bacterial Enzymes

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Applications of Bacterial Enzymes

• Probe usage
• Isolation of DNA polymerases allowed for the
  synthesis of DNA in vitro
• e.g. Purified DNA sequences could be specifically
  labeled by nick translation with radioactive or
  fluorescent-tagged dNTPs and Pol I...

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Use of Labeled DNA Probes Hybridization analysis

• The self-complementary nature of DNA can be used
  to identify its presence (and the presence of
  gene products) in cells and tissues
• DNA can be denatured to single- stranded form
• Labeled probes can then be made to reanneal
  (hybridize) to the DNA in situ...

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• Heat treatment (as shown here) or treatment with
  bases (e.g. NaOH) will cause DNA to denature.

• Under the appropriate salt and temperature
  conditions, it can be made to reanneal (base
  pair) with a complementary strand

If one DNA strand (probe) is labeled, genes can
be detected...
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Probe DNA
Label and denature probe
Denature chromosomal DNA
Hybridize probe to chromosomes
Detect via auto-radiography
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Probe DNA
Label and denature probe
Denature chromosomal DNA
Hybridize probe to chromosomes
Detect via auto-radiography
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Mapping of the chromosomal location of the human
insulin gene by in situ hybridization
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Fluorescent In Situ Hybridization (FISH) p. 330

• Multiple probes tagged with different
  fluorescent markers can be used in the same
  hybridization experiment

• chromosome painting see pp. 455 626

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Sure its DNA, but whose?????
To paraphrase Gertrude Stein. DNA is DNA is
DNA...
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Applications of Bacterial Enzymes Synthesis and
Propagation (Cloning) of Recombinant DNA (ch. 9)

• Purified enzymes from microorganisms can be used
  as molecular scissors to break up DNA molecules
  and piece them together
• Autonomously replicating genetic elements in
  microorganisms (e.g. plasmids) can be used to
  propagate that DNA within individual cells...

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• Restriction Endonucleases
• have a specific recognition sequence (e.g 4-6
  bp)
• many enzymes have palindromic recognition sites
• DNA cut with these enzymes is broken within the
  strand
• some of the enzymes leave a single strand
  overhang (sticky end)

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Bacterial Plasmids have been engineered to serve
as vehicles (vectors) for the replication of
foreign DNA

• Properties
• origin of replication
• restriction sites to introduce DNA
• means of selection for plasmid in bacterial cell

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Cutting of a circular plasmid with restriction
enzyme yields a linear molecule with sticky
ends...
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• Digest foreign DNA with same enzyme
• if 6 base recognition site is random in genome
  cuts every 4096 bp (46)
• human genome 3 x 109 bpgt7 x 105 fragments

Linearize plasmids with restriction enzyme
Entire population of transformants represents
genomic library

• single fragments are ligated to individual
  plasmid DNAs
• transformation introduces individual plasmids
  (clones) into bacteria

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Viruses can also be used to clone foreign DNA
molecules via their ability to propagate in
bacteria...
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• digest lambda DNA
• remove middle section (contains genes for
  lysogeny)
• ligate foreign DNA to lamba arms
• mix with viral coat proteins to package
  recombinant DNA

• infect E. coli

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Creating a genomic library using phage vectors...
(CLONE)
All phage plaques taken together represent
genomic library...
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Cosmids

• Vectors that are hybrids between plasmids and
  lambda phage
• can replicate in a cell like a plasmid or be
  packaged like a virus
• they can carry larger DNA inserts than plasmids

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Classical genetic studies show gene a has an
interesting phenotypehow do we clone it?
Summary of Genomic library construction...
The genomic library may contain gt106 individual
clones...
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Information on protein sequence can lead to the
synthesis of short oligonucleotide probes.
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Screening using related sequences

• If a tissue is enriched in a particular mRNA, the
  mRNA could be used as a probe
• e.g. globin gene sequences
• Sometimes it is easier to isolate a sequence from
  one organism than another, and the related gene
  can be used to fish out the same gene from other
  genomes
• e.g. actin gene sequences

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• replica plate plaques or colonies onto filter
• denature DNA on filter
• incubate with radiolabeled probe
• detect cells that contain correct gene by
  autoradiography
• grow up appropriate clone

Genomic Library of bacteriophage lambda plaques
plated on petri dishes...
Each clone occupies a specific place on the
plate...
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• replica plate plaques or colonies onto filter
• denature DNA on filter
• incubate with radiolabeled probe
• detect cells that contain correct gene by
  autoradiography
• grow up appropriate clone

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• Construction of a cDNA library (pp. 316-17)
• provides a snapshot of the genes expressed in
  a particular tissue
• isolate mRNA from tissue of interest
• convert to d.s. DNA in vitro with reverse
  transcriptase and Pol I

• prepare ends for cloning (remove hairpins, add
  ligate on restriction sites)

• clone into vector and propagate in microorganism

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The Polymerase Chain Reaction (pp. 297-302)

• Allows for amplification (essentially cloning)
  of small amounts of DNA from biological samples
• provide specific primers flanking region of
  interest

• denature and anneal primers
• synthesize new DNA in vitro.
• Denature and repeat annealing and synthesis

1gt2gt4gt8gt16gt etc...
Procedure can be automated using DNA polymerases
from thermophilic organisms...
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DNA Sequencing the Chain Termination Method (pp.
302-07) dideoxynucleotides used to stop synthesis
at a specific base...
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• 4 different DNA synthesis reactions are run
• begin synthesis from specific priming point
• add components for DNA synthesis specific
  ddNTP for each of the four reactionse.g. ddATP

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Run the 4 different reactions on electrophoretic
gelsmallest fragments (closest to primer)
migrate farthest...
AATCTGGGCTATTCGGGCGT
For synthesized strand...
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• Automated DNA sequencing
• each reaction can be specifically labeled with a
  fluorescently tagged molecule

• since each terminated fragment is tagged with a
  different color, all reactions can set up
  together and run on the same lane.

• a laser built into the gel apparatus can now
  illuminate the bands and record the intensity of
  the fluorescent signal as the DNA band passes the
  detector...

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The output of the detector can be sent directly
to a computer and the DNA sequence recorded (in
real time) as the gel is being run
Example of output
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Pyrosequencing
DNA Bead
Polymerase
A A T C G G C A T G
C T A A A A G T C A
T
Annealed Primer
Sulfurylase Luciferase
Enzyme Bead
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Emulsion PCR

• Micro-reactors
• Water-in-oil emulsion generates millions of
  micelles.
• Each micelle contains all reagents/templates for
  a PCR reaction.
• 10 Million individual PCR reactions in a single
  tube.

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Load Beads into 454 Plate
Load Enzyme Beads
Load beads into PicoTiterPlate
Centrifugation
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Pyrosequencing Output
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Output Assembly

• Raw data is series of images

T
C

• Each wells data is extracted, quantized and
  normalized

G
A
T
dNTP Base Addition

• Read data converted into flowgrams

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Basecalling via Flowgram
TTCTGCGAA