Clone Selection

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Clone Selection

• Dr. Jason Linville
• University of Alabama at Birmingham
• jglinvil_at_uab.edu

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Clone Selection

• We have already determined ways to remove
  non-transformed cells and those containing a
  self-ligating vector.

How can we identify which clone contains the DNA
segment we want?
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Clone Selection

• Two Basic Strategies

• Direct Selection Only desired clones are grown.

• Identification from gene library Many colonies
  produced. Identify correct colony after growth.

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Clone Selection

• Direct Selection

Select medium on which only desired colonies will
grow

• Obvious example selection for antibiotic gene
• Weeding out self ligated vectors not an issue

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Clone Selection

• Direct Selection

Select medium on which only desired colonies will
grow

• Marker Rescue using mutant bacteria (auxotroph)
  that need the selected gene to survive.
• If looking for trpA gene, use trpA mutant
  bacteria that cant survive without the gene
  (vector).

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Clone Selection
Limits Mutant strain must be available for gene
in question
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Clone Selection

• Limitations of marker rescue

Often, auxotrophs do not exist for a gene.
Plus, genes from higher organisms often wont
function in bacteria or yeast.
The solution is to identify the desired colony in
a gene library.
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Clone Selection

• ID from gene library

Genomic Library colonies in sufficient number
to likely contain every gene present in a given
organism.
Total DNA purified, partially digested, and
cloned into vector
This is fine for small organisms, but what about
animals?
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Clone Selection

• ID from gene library

• Solution is to make a specialized library

• Not every gene is produced in specialized cells
  (liver, blood, brain cells)

• By cloning the mRNA (only expressed genes), the
  library size becomes reasonable.

Wait a minute, we cant clone RNA. Can we?
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Clone Selection gt Gene Library

• Cloning mRNA

• Single stranded mRNA cannot be cloned.
• Complementary DNA (of mRNA) is made.

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Clone Selection gt Gene Library

• Cloning mRNA

Complement DNA formed by reverse transcriptase.
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Clone Selection gt Gene Library

• Cloning mRNA

RNA partially degraded by RNase H.
DNA Polymerase I forms DNA compliment
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Clone Selection gt Gene Library

• Cloning mRNA

• DNA can then be added to vector.
• Clone library consists of all mRNAs (expressed
  genes)

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Clone Selection
Once total genomic library or specialized library
is formed, desired colony must be identified.
Two Methods

• Hybridization probing (detect DNA)
• Immunological screening (detect protein)

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Clone Selection

• Hybridization Probing

• Complimentary DNA or RNA will anneal to each
  other.

• Small differences wont prevent binding

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Clone Selection
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Clone Selection

• Hybridization Probing

• Radiolabeled probe
• Biotinylated probe
• Horseradish peroxidase probe
• Others.

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Clone Selection gt Hybridization

• Hybridization Probing Process

All colonies transferred to membrane
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Clone Selection gt Hybridization
Treatment with base and protease breaks open
cell, degrades protein, and denatures DNA.
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Clone Selection gt Hybridization
DNA fixed to membrane with heat or UV light
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Clone Selection gt Hybridization

• Labeled probe denatured
• Applied to membrane in solution favorable to
  hybridization

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Clone Selection gt Hybridization
For radiolabeled probe, x-ray film is used to
detect probe.
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Clone Selection gt Hybridization
For biotinylated probe, avidin and fluorescent
marker used.
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Clone Selection gt Hybridization
For horseradish peroxidase, luminol is used.
(oxidized at probe)
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Clone Selection gt Hybridization

• Hybridization Probing

• Clearly, the DNA sequence (or partial sequence)
  must be known in order to choose a proper probe.
• Clearly the sequence could be unknown if we want
  to clone it.

What gives?
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Clone Selection gt Hybridization

• Choosing the probe

• Gene expressed at high level
• AA sequence of protein product is known
• Gene is a member of a family of related genes

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Clone Selection gt Hybridization

• Gene expressed at high level

• Possible to use a clone from one colony to probe
  all the other colonies.
• If only a few light up, the gene is rare.
• If many light up it is likely the abundant gene
  that is being looked for.

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Clone Selection gt Hybridization

• AA sequence known

• Can use the genetic code to predict the likely
  sequence.
• Choose section with AAs that only have one or two
  codons associated with it.
• Make pool of all possible probes and use them to
  probe colonies.

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Clone Selection gt Hybridization
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Clone Selection gt Hybridization

• Heterologous probes

• If sequence is sufficiently similar, probe will
  stick (doesnt have to be exact).
• The gene sequence in one organism may be similar
  to the sequence for the same gene in a different
  organism.
• May be related genes in the same multigene family.

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Clone Selection gt Immunological Screen

• Detecting the Translated Product (Immunological
  Screening)

• Hybridization is the preferred method
• Still some circumstances where sequence of
  desired DNA clone is completely unknown.

Solution Search for the protein product
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Clone Selection gt Immunological Screen

• Immunological Screening

• Antibody specific to clone protein is used to
  detect presence of protein in colony

• Need gene to be expressed in colony
• Need antibody to protein

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Clone Selection gt Immunological Screen

• Antibody Production

• If foreign protein is injected into rabbit,
  rabbit will form antibodies to bind and degrade
  foreign molecule.

• Rabbit can be injected with protein days later
  antibody can be isolated from blood.

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Clone Selection gt Immunological Screen

• Gene expression

• Often, foreign genes wont be expressed in the
  vector.

• An expression vector can be used. Contain
  sequences to promote transcription of gene

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Clone Selection gt Immunological Screen

• Colonies transferred to membrane and lysed
• Antibodies bind to specific protein in colony

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Clone Selection gt Immunological Screen

• Labeled protein A can bind to antibody
• Antibodies themselves may be labeled or another
  labeled antibody may bind antibody