Housekeeping

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Housekeeping

• Homework 3 due May 5
• Question 4 P. multocida EcoRI BamHI 1.0, 2.0,
  5.0, 6.0 kb (no 3.0 kb)
• SKIP QUESTION 9
• Exam III-May 9
• Homework 4 due May 14th
• Lab
• Quiz 3 THIS WEEK
• Quiz 4 next week (cumulative)

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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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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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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!
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Whats a vector? Why do we need one?
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Reasons for a Vector

• Allow for transport of individual fragments
• Can get into its host
• Allow for replication of individual fragments
• Can replicate in its host
• Allow for purification of individual fragments
• Can extract it from its host

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Types of vectors
Vector
Vector Host
Insert size
Plasmids
Bacteria
Up to 15 kb
Bacteria virus (lambda)
Bacteria
Up to 25 kb
Bacteria
BAC (bacterial artificial chromosome)
100-500 kb
YAC (yeast artificial chromosome)
Yeast
250-1000 kb
30-45 kb
Bacteria
Cosmid
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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
• a. Ligate fragments into vector
• b. Insert vector( insert) into host
• c. Amplify vector (amplify host)
• d. Isolate vector

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Ligate sheared DNA into vector
Sheared DNA
Vector

• Have cut ends

• Cut so ends similar to sheared DNA

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Ligation What do we need?
Vector
DNA fragment
DNA ligase
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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
• a. Ligate fragments into vector
• b. Insert vector( insert) into host
• c. Amplify vector (amplify host)
• d. Isolate vector

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What host??
DEPENDS ON VECTOR AND HOST!!
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Vector
Vector Host
Insert size
Plasmids
Bacteria
Up to 15 kb
Bacteria virus (lambda)
Bacteria
Up to 25 kb
Bacteria
BAC (bacterial artificial chromosome)
100-500 kb
YAC (yeast artificial chromosome)
Yeast
250-1000 kb
30-45 kb
Bacteria
Cosmid
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What host??
DEPENDS ON VECTOR AND HOST!!
VECTOR PLASMID HOST BACTERIA (E. coli)
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Transformation
E. coli
E. coli

E. coli
E. coli
Competent!
E. coli
E. coli
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How do we know which E. coli cell has a vector?
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Vector Needs a marker
Ampicillin resistance gene
E. coli
E. coli
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How do we know the vector has an insert?
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lacZ gene Beta-galactosidase
Plasmid
Cut plasmid for ligation in lacZ region
Ligate genomic DNA
No Beta-galactosidase produced!
Amplify in X-gal and IPTG
Select white colonies
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What would a blue colony indicate?
Why are we growing in ampicillin?
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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
• a. Ligate fragments into vector
• b. Insert vector( insert) into host
• c. Amplify vector (amplify host)
• d. Isolate vector

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Amplify host
How do we grow up E. coli?
Anything important about the media?
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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
• a. Ligate fragments into vector
• b. Insert vector( insert) into host
• c. Amplify vector (amplify host)
• d. Isolate vector

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Isolate vector

• Separate plasmid from E. coli
• Separate from cell, proteins, carbs, etc..
• Separate from E. coli genomic DNA!

Genomic DNA (insert)
Ampicillin resistance gene
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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
• a. Ligate fragments into vector
• b. Insert vector( insert) into host
• c. Amplify vector (amplify host)
• d. Isolate vector

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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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How do we sequence?
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How do we sequence?

• Amplify the piece we want to sequence
• Identify the nucleotide order

What do we need??
Genomic DNA (insert)
Ampicillin resistance gene
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Sequencing ingredients
Ingredient
Function
Isolated Vector insert
Amplification and sequencing template
Need a way to differentiate between nucleotides
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Alter nucleotides

• Add some that cause termination of amplification
• No 3 hydroxyl end

• Also labelled!
• Radioactive
• Fluorscence

A
G
T
C
A
T
C
G
C