Lecture 2 catch up

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Lecture 3

• Lecture 2 catch up
• Vector structure
• Copy number control
• Selectable marker
• Plasmid DNA isolation
• ESTs
• DNA sequencing
• Entering the Honeybee database

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How are different sizes of DNA strands separated
on agarose gel?
Mixture of DNA molecules
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DNA separation is based on fragment size
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Size markers and their use to determine size
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Size markers and their use to determine size
Mobility of a DNA fragment is proportional to
the log of its size in bases.
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Plot of relative mobility of DNA vs log of size
on semi-log graph paper
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Varying concentrations of agarose
How does one make a 0.7, 1.0 1.5 agarose
gel? Why are there varying concentrations of
agarose gels?
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Varying concentrations of agarose
Why are there varying concentrations of agarose
gels? Higher concentrations provide better
resolution for smaller DNA fragments Lower
concentrations provide better resolution for
larger DNA fragments
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Experiment 1What is an EST?
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pT7T3-pac
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3 important parts of a vector

• Origin of replication
• Selectable marker
• Cloning sites

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Origin of replication considerations

• What is an origin of replication?
• Why do we need one?
• What properties should it have for easy plasmid
  DNA isolation?

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Replicon Contains all info necessary to begin
and end DNA replication Origin of replication
(Ori) is a defined location within the replicon
where DNA synthesis begins
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The ColE1 origin of replication
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ColE1 origin

• RNAII forms the primer for initiation of DNA
  synthesis
• RNAI and Rop protein are negative regulatory
  functions RNAI binds to RNAII stopping primer
  formation Rop protein stabilizes the complex
  formed by RNAI and RNAII.

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The ColE1 origin of replication
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RNAIRNAII interaction
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Rop protein negatively regulates the origin of
replication by stabilizing the RNAIRNAII
interaction.
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Copy number
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Why does pT7T3.pac have a high copy number?

• Point mutation in the origin that alters the
  initiation of RNAI transcription, such that the
  RNAIRNAII complex does not form as well as
  wild-type.
• The region of the origin that encodes Rop protein
  is deleted.

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Selectable Marker

• Ampicillin resistance
• Ampicillin is a penicillin derivative.
• Blocks cross linking of the bacterial cell wall
  causing the cells to burst/lyse.
• Ampicillin resistance gene encodes a secreted
  enzyme that cleaves the beta-lactam ring.

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Benzyl penicillin
b-lactam ring
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Mechanism of resistance
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Plasmid DNA isolation

• Separate chromosomal DNA from plasmid DNA
• Remove protein
• Remove RNA
• Easy

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Starting material
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Lysis and denaturation
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Neutralization and centrifugation
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Column

• Silica gel
• DNA binds at high salt.
• DNA is eluted at low salt.
• Removes glycogen and other cellular components.

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ESTs

• Expressed sequence tags?
• What are they?
• What do they represent?
• What do we use them for?
• What are their advantages?

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How is cDNA made?
First strand synthesis
GCGGCCGCGTTGCTTTTTTTTTTTTTT 3
3 AAAAAAAAAAAAAAAAAAANNNNNNNNNNNN
Reverse transcriptase
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Second strand synthesis
Remove mRNA with OH or enzymatically
DNA polymerase I
Cut hairpin
Add EcoRI adaptors GAATTCGGCACGAGG
Cut with NotI and insert into EcoRI NotI of vector
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Normalized Library
mRNAs accumulate to different levels in a mRNA
sample. Normalization is a method of evening out
the abundance levels using hybridization.
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T7 sequencing primer?
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DNA SEQUENCING
Dideoxy or Chain Termination Method
1974 Maxam/Gilbert (USA) (Chemical
Cleavage protocol) Sanger (England) (natural
process of DNA replication)

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Sanger Sequencing in 1989
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Example output
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Why is the size of the insert important?

• A sequencing run may only go 500-700 bases.
• What might you see and what might you not see?

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EST gel
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Clipping an EST sequence

• What are we clipping out?
• Why do we look for GAATTCGGCACGAGG and
  GCAACGCGGCCGC?
• Why do we look for AAAAAAn?

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Example

• GAATTCGGCACGAGG
• CGCGTTCTTGAAAAGACAGGTAAAATGCGAGTTCCAGAATGGGTAGAATT
  GTAAAGTCTGCACGATTCAAGGAACTTGCTCCATATGATCCAGATTGGTA
  TTATATTAGATGTGCTGCTTTAGTTCGTCATATTTATATTCGAAGTCCAA
  TTGGTGTTGGAGCAGTAACAAAAATTTTTGGAGGACGCAAACGTAATGGT
  ACTCATCCTAGCCATTTCTGTCGATCGGCAGGTGGTGTTGCTCGCAAAGC
  TCTTCAGAGCTTGGAACAACTTAAACTCATTGAAAAATCTCCAGTTGGTG
  GACGTAAACTTACCAGTCAAGGCCGTAGAGATTTAGATCGCATTGCTGCC
  CAAGTCAAAGCAAAAAGCAAAAAACAACTTAAGTTACAAGAAACTCTTGT
  TCTCTAATTTCTTTATTTCTATAATAAAAATTAAAAAATCGTACATTTAT
  ATTCAAATTTTATTTATTCTACTATTATAAGTTAATTTTAGAAGTCTTAA
  ATAATTTAATGGTAACAAAATATCACAAGACAATGTGCTATTATTTTTTT
  AATTGTTTATGAATTATCATAATTGAGGAAATTTTTAACTATAAATAGAT
  AAAGTAAGAAATATAAATTTATAATTTATGATTATGTATTTTATATCTAA
  ATGTATTTATTAATCTAAATATAAACAAATATACAT
• AAAAAAAAAAAAAAAAAAAAAAAA
• GCAACGCGGCCGC
• AAGCTTATTCCCTTTAGTGAGGGTTAATTTTAGCTTGGCACTGGCCGTCG
  TTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGC
  CTTGCAGCACATCCCCCTTTC

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Example output
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What could go wrong

• 18 of ESTs are in the reverse orientation.
• Potential of 2 inserts in the clone.
• What would this look like?

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Step into liquid
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Clipping a sequence I
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Clipping Sequence II
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Clipping Sequence III
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Clipping Sequence IV
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Step into liquid