MCB 720: Molecular Biology

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MCB 720 Molecular Biology

• Gene libraries
• cDNA libraries
• Library screening

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Eukaryotic gene organization
enhancers silencers
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Genomic library construction
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Screening a genomic library using DNA
hybridization to a (radio-)labeled DNA probe
Note a cDNA is commonly (radio-)labeled and used
as a DNA probe to screen a genomic library
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Production of a (radio-)labeled DNA probe by the
random primer method uses the Klenow fragment of
DNA polymerase
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How many genomic clones must be screened to find
your gene?

• Theoretically, you will need to screen N clones
  where Nln(1-P)/ln(1-f) where Pthe probability
  of finding your gene and fthe average size of
  the cloned genomic sequence in your vector
  divided by the total genome size.
• How many clones must you screen to find your gene
  in a human gene library packaged in EMBL 3 with
  99 certainty?
• Nln(1-0.99)/ln(1-20kb/2.8 x 106kb) 6.4 x 105
  clones

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The first step in making a cDNA library
Purification of polyadenylated mRNA using
oligo(dT)-cellulose Note selection of the
proper source (organ, tissue) of the RNA is
critical here!
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Complementary DNA or cDNA cloningcDNA library
constructionNote ds cDNAs are typically
placed in a cloning vector such as bacteriophage
lambda (l) or a plasmid
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Bacteriophage lcloning system
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Bacteriophage l cloning system
Cos sites at the left and right ends
Cloning site
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There are several possible ways to screen a cDNA
library

• Using a DNA probe with a homologous sequence
  (e.g., a homologous cDNA or gene clone from a
  related species)
• Using an oligonucleotide probe based on a known
  amino acid sequence (requires purification of the
  protein and some peptide sequencing)
• Using an antibody against the protein of interest
  (note this requires use of an expression vector)
• Plus/minus or differential screening (the least
  specific way)

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Screening a cDNA library using DNA hybridization
to a (radio-)labeled DNA probe
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Screening a cDNA library with a labeled
oligonucleotide probe based on a known peptide
sequence
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Using polynucleotide kinase andg-32P-labeled ATP
to radiolabel oligonucleotide probes
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Immunological screening of an expression cDNA
library with a primary antibody and labeled
secondary antibody note the label is often an
enzyme label like alkaline phosphatase or
horseradish peroxidase, but it can also be
125INote see also MCB Chapter 9 for a related
animation http//bcs.whfreeman.com/lodish5e/pages/
bcs-main.asp?vcategorys00010n09000i09010.04
o005100061000520005300054000560005700059
00060000700007100001000020000300004000050
010000200003000040000500006000070000800009
000100001100012000130001400015000160001700
018000190002000021000220002300099000ns58
9
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Animations for two related uses of expression
vectors

• Expression cloning of receptor proteins-see MCB
  Chapter 9
• http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
  sp?vcategorys00010n09000i09010.04o00510
  006100052000530005400056000570005900060000
  700007100001000020000300004000050010000200
  00300004000050000600007000080000900010000
  110001200013000140001500016000170001800019
  0002000021000220002300099000ns589
• Looking for protein-protein interactions with the
  yeast two hybrid system-see MCB Chapter 11
• http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
  sp?s00010n11000i11010.01vcategoryo00510
  006100052000530005400056000570005900060000
  700007100001000020000300004000050010000200
  00300004000050000600007000080000900010000
  110001200013000140001500016000170001800019
  0002000021000220002300099000ns798tuid0
  rau0

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Plus/min (/-) or differential screening
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A cosmid cloning systemanother possible cloning
vector which can be used for genomic library but
not for cDNA libraries
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In summary, you have seen

• How to make and screen gene libraries
• How to make and screen cDNA libraries
• Several different cloning vectors including
  plasmids, bacteriophage lambda (l), and cosmids