Housekeeping

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Housekeeping

• Homework 3 due May 5
• Exam III-May 9
• Homework 4 due May 14th
• Lab
• Quiz 3 4/30-next week
• Quiz 4 5/7 (cumulative)

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Saga 2Help the sick cows!!
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What is a fast genetic test to detect a
bacteria from a poop sample?
How can we differentiate between different
bacterial isolates?
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Restriction Maps

• 1. Restriction digest of isolate
• Mutations/alterations changes of digest sites
• 2. Gel electrophoresis

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Restriction map analysis
Isolate
A
B
C
Why differences??
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Restriction maps

• Function?
• Map locations of restriction sites
• Detect variances between organisms
• Detect variances between isolates
• How do we create maps?
• Use one/multiple enzymes
• Use Whole genome/Genome piece

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One or more isolate affecting cows?
Restriction Digest of M. paratuberculosis isolates
Frequent or infrequent cutter?
Try multiple enzymes!
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Lets map 3 isolates from 3 different farms
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Digest results between several bacterial isolates
(20kb piece)
Farm 1
Farm 2
Farm 3
EcoRI
EcoRI
EcoRI
12 kb
8 kb
12 kb
6 kb
6 kb
6 kb
2 kb
4 kb
2 kb
CeuI
2 kb
CeuI
CeuI
10 kb
10 kb
8 kb
18 kb
2 kb
EcoRI and CeuI
2 kb
EcoRI and CeuI
EcoRI and CeuI
10 kb
8 kb
6 kb
6 kb
6 kb
4 kb
2 kb
2 kb
2 kb
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How similar is M. paratuberculosis from each
farm?
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Epidemiology of our bacteria in cows?

• gt 1 bacterial isolate infecting one herd?
• gt 1 bacterial isolate infecting multiple herds?
• Identify the potential source of the outbreak!!

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Genetic tools

• Restriction digest
• Gel electrophoresis
• Southern hybridization
• PCR

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The perils of tuberculosis
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Sequencing the M. tuberculosis genome

• Create a genomic library
• Sequence the genomic library
• Alignment of these sequences (what sequence goes
  where?)
• Identification of GENES

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Two approaches

• Directed sequencing
• Specific library (1 chromosome)

Fig. 8.23a
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Two approaches

• Random sequencing
• Random library (represents entire genome)

Fig. 8.23b
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Create a genomic library

• 1. Isolate genomic DNA
• Specific piece (directed)
• All genomic (random)
• 2. Shear DNA
• 3. Clone into a vector

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Shear DNA

• Restriction Digest
• What problems might result??
• Physical shearing
• Nebulization (air pressure)
• Sonication (extreme mixing)

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Create a genomic library

• 1. Isolate genomic DNA
• Specific piece (directed)
• All genomic (random)
• 2. Shear DNA
• 3. Clone into a vector

What do we need to do with our pieces?
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What do we want to do with these DNA pieces?

• Isolate them (purify them)
• Amplify them
• Sequence them

Clone them!