Transcript PPT - Bruce Blumberg

Bio Sci 203 Lecture 20 - cDNA library screening
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Bruce Blumberg ([email protected])
– office - 4203 Bio Sci II
– 824-8573
– lab 5427 (x46873), 5305 (x43116)
– office hours Wednesday 1-2.
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http://blumberg-serv.bio.uci.edu/bio203-w2002/index.htm
http://blumberg.bio.uci.edu/bio203-w2002/index.htm
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Link is also main class web site
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Today
– wrap up cDNA library screening
– characterization of clones obtained from screening
– Protein protein binding assays
BioSci 203 lecture 20
page 1
©copyright
Bruce Blumberg 2001. All rights reserved
mRNA frequency and cloning
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mRNA frequency classes
– classic references
• Bishop et al., 1974 Nature 250, 199-204
• Davidson and Britten, 1979 Science 204, 10521059
– abundant
• 10-15 mRNAs that together represent 10-20% of
the total RNA mass
• > 0.2%
– intermediate
• 1,000-2,000 mRNAs together comprising 4045% of the total
• 0.05-0.2% abundance
– rare
• 15,000-20,000 mRNAs comprising 40-45% of
the total
• abundance of each is less than 0.05% of the total
• some of these might only occur at a few copies
per cell
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How does one go about identifying genes that might only
occur at a few copies per cell?
BioSci 203 lecture 20
page 2
©copyright
Bruce Blumberg 2001. All rights reserved
Normalization and subtraction
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How does one go about identifying genes that might only
occur at a few copies per cell?
– Improve your chances by altering the representation
of the cDNAs in a library or probe
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Normalization - process of reducing the frequency of
abundant and increasing the frequency of rare mRNAs
– Bonaldo et al., 1996 Genome Research 6, 791-806
– normalization is claimed to bring all cDNAs into the
same order of magnitude abundance, i.e., within 10
fold of each other
• rarely works this well.
• More commonly, abundant genes are reduced 10
fold and rare ones increased 3-10 fold.
• Intermediate class genes do not change much at
all
– Approach
• make a population of cDNAs single stranded
– tester
• hybridize with a large excess of cDNA or mRNA
to Cot =5.5
– driver
• Cot value is critical for success of normalization
– 5-10 is optimal
– higher values are not better
BioSci 203 lecture 20
page 3
©copyright
Bruce Blumberg 2001. All rights reserved
Normalization and subtraction (contd)
– Approach (contd)
• various approaches to make driver
– use mRNA - may not be easy to get
– make ssRNA by transcribing library
– make ssDNA by gene II/ExoIII treating
inserts digested from plasmid library
– PCR amplification of library
• experience has demonstrated that the best
approach is to use driver derived from the same
library by PCR
– rapid, simple and effective
– other approaches each have various
technical difficulties
– see the Bonaldo review for details.
– What are normalized libraries good for?
• EST sequencing
• gene identification
– biggest use is to reduce the number of
cDNAs that must be screened
– good general purpose target to screen
» subtracted libraries are useful but
limited in utility
– Drawbacks
• Not trivial to make
• Size distribution of library changes
– Longer cDNAs lost
BioSci 203 lecture 20
page 4
©copyright
Bruce Blumberg 2001. All rights reserved
Normalization and subtraction (contd)
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Subtraction - removing cDNAs (mRNAs) expressed in
two populations leaving only differentially expressed
– Sagerström et al. (1997) Ann Rev. Biochem 66, 751783
+/- screening St. John and Davis (1979) Cell 16, 443-452.
– Hybridize the same library with probes prepared
from two different sources and compare the results
• example - hybridize normal liver cDNA library
with probes from normal and cancerous liver
– Colonies or plaques that are expressed in
target tissue (tumor) compared with control
are picked
– Why aren’t all colonies labeled in normal
tissue?
BioSci 203 lecture 20
page 5
©copyright
Bruce Blumberg 2001. All rights reserved
Normalization and subtraction (contd)
• What about rare mRNAs? These might be
differential but not abundant enough to detect
– Do reverse experiment -> select for absence
of a signal
– example - hybridize a tumor cDNA library
with probe prepared from normal liver
– select for genes absent in tumor
» Get genes lost from normal tissue and
gained in tumor by this approach
BioSci 203 lecture 20
page 6
©copyright
Bruce Blumberg 2001. All rights reserved
Normalization and subtraction (contd)
– Advantages
• Relatively simple approach
• Doesn’t require difficult manipulations on
probes
– Disadvantages
• Housekeeping genes often appear to be
differential
• Sensitivity less than subtracted screening
– +/- screening typically requires >10 fold difference
in expression levels using standard methods
• not widely used any longer BUT
– microarray analysis is really just a refined version of
+/- screening
• fluorescence ratios give good internal standards
– more precise quantitation
– increased sensitivity
BioSci 203 lecture 20
page 7
©copyright
Bruce Blumberg 2001. All rights reserved
Normalization and subtraction (contd)
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Subtractive screening - Sargent and Dawid (1983)
Science 222, 135-139.
– Make 1st strand cDNA from a tissue and then
hybridize it to excess mRNA from another
• larger Cot is best
– remove double stranded materials -> common seqs
– make a probe or library from the remaining single
stranded cDNA
– 10-100 fold more sensitive than +/- screening
BioSci 203 lecture 20
page 8
©copyright
Bruce Blumberg 2001. All rights reserved
Normalization and subtraction (contd)
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Subtractive screening (contd)
– benefits
• sensitive
• can simultaneously identify all cDNAs that are
differentially present in a population
• good choice for identifying unknown, tissue
specific genes
– drawbacks
• easy to have abundant housekeeping genes slip
through
– multistage subtraction is best
– in effect normalize first, then subtract
• libraries have limited applications
– may not be useful for multiple purposes
– rule of thumb
• make a high quality representative library from a
tissue of interest
• save subtraction and other fancy manipulations
for making probes to screen such libraries with
– unlimited screening
– easy to use libraries for different purposes,
e.g. the liver library
» hepatocarcinoma
» cirrhosis
» regeneration specific genes
BioSci 203 lecture 20
page 9
©copyright
Bruce Blumberg 2001. All rights reserved
How to identify your gene of interest
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Screening methods depend on what type of information
you have in hand.
– Related gene from another species?
• Low stringency hybridization
– A piece of genomic DNA?
• Hybridization
– A mutant
• Complementation
• Positional cloning
– A functional assay?
• Expression screening
– An antibody?
• Expression library screening
– A partial amino acid sequence?
• Oligonucleotide screening
– A DNA element required for expression of an
interesting gene?
• Various binding protein strategies
– An interacting protein?
• Interaction screening
– A specific tissue or embryonic stage?
• Subtracted or +/- screening
BioSci 203 lecture 20
page 10
©copyright
Bruce Blumberg 2001. All rights reserved
How to identify your gene of interest (contd)
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If you wish to identify a cDNA, what is the most
important piece of information you need?
– Information on where the mRNA is expressed
• either what tissue or
• what time during development
– such information is indispensable!!
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First step in any hybridization based method (high or low
stringency) is to get information on expression
– straightforward with high stringency homologous
screening - Northern analysis
– cross species screening requires more care
• perform a genomic Southern to identify
hybridization and washing conditions that
identify a small number of hybridizing
fragments
– standard hybridization conditions are 1 M
Na+, 43% formamide, 37° C
– begin washing at RT in 2 x SSC and expose
– increase stringency until reasonable
signal/noise ratio is obtained
– use these conditions for Northern.
• If Northern is unsuccessful - obtain a genomic
clone and repeat the screening at high stringency
– this approach will never fail to identify a
homologous gene
BioSci 203 lecture 20
page 11
©copyright
Bruce Blumberg 2001. All rights reserved
How to identify your gene of interest (contd)
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Cloning by complementation
– generally only useful with manipulable genetic
systems
• yeast
• Drosophila
• C. elegans
• zebrafish
– presumes that complemented mutant is readily
observable
– Approach
• transfer pooled cDNA libraries in expression
vectors into the mutant
– or mRNA pools derived from libraries
• assay for rescue
• subdivide positive pools and repeat
– advantages
• direct functional test
• rapid compared with chromosome walking
– disadvantages
• fairly tedious
• dependent on library quality
• requires easily observable rescue
BioSci 203 lecture 20
page 12
©copyright
Bruce Blumberg 2001. All rights reserved
How to identify your gene of interest (contd)
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Positional cloning
– If your mutant results from a transposon insertion
then this can be recovered
– If insertion is a P-element or such
• Make genomic library from mutant
– What type of library will you make? Why?
• Screen with transposon
– Recover positives, sequence flanking region
• Use flanking sequence to screen normal genomic
library
– What type of library will you screen?
– If insertion is a gene trap or related
• You can digest mutant DNA with an enzyme that
linearizes the vector
• Ligate and transform
• Colonies that form should have flanking region
– sequence
• Use this to screen normal library
• OR
• Use inverse PCR to get flanking sequence from
plasmid and use this to probe library
BioSci 203 lecture 20
page 13
©copyright
Bruce Blumberg 2001. All rights reserved
How to identify your gene of interest (contd)
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Functional screening (expression cloning)
– similar to complementation
– if you have a functional assay expression cloning
may be appropriate choice
– strategy:
• Large pools (~10,000) of cDNAs tested for
presence of a particular function
– microinjection
– transfection
– receptor binding (panning)
• positive pools are subdivided and retested to
obtain pure cDNAs
• cycle is repeated until single clones obtained
– Advantages
• functional approach
• in vivo testing is possible
• can identify secreted proteins and receptors
– Disadvantages
• low throughput
• very tedious
• sensitivity issue due to pool size
• extensive retesting of pools is required
– applications:
• many receptors and transporters cloned this way
BioSci 203 lecture 20
page 14
©copyright
Bruce Blumberg 2001. All rights reserved
How to identify your gene of interest (contd)
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Antibody screening of cDNA expression libraries
– let’s say you have an antibody in hand and want the
corresponding cDNA
– requirements
• antibody must recognize denatured epitope, i.e.,
should work in a western blot
– many monoclonals recognize 3-D or sugar
epitopes
• affinity purified antibodies work best
• cDNA expression library, e.g., λgt11 series
– approach
• plate library and induce replicate filters
• incubate with antibody
• wash and develop the filters
• repeat until a pure clone is obtained
– verification
• use phage fusion protein to affinity purify
antibody and verify that it reacts with original
protein
– advantages
• best choice if only antibody is available
– disadvantages
• λgt11 and relatives are painful to work with
• your antibody may not be suitable
– sugar directed
– structural epitope
BioSci 203 lecture 20
page 15
©copyright
Bruce Blumberg 2001. All rights reserved
How to identify your gene of interest (contd)
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A partial amino acid sequence?
– Purified protein of interest and have one or more
partial amino acid sequences
• make a peptide antibody and screen (slow)
• Oligonucleotide screening based on aa sequence
– multiple codons for most aa
• PCR between multiple primers
– three types of oligos in use
• long guess-mers - pick the wobble base
– relies on low stringency hybridization
• inosine - use inosine for multiple bases
– I:C >> others
• degenerate oligos (mixtures of all possible seqs)
– mixtures of < 1024 virtually always work
– approach
• pick an aa sequence that predicts a reasonable
probe complexity (avoid ser, leu, arg) WHY?
• synthesize fully degenerate mixture
• label and hybridize at low stringency (Tm-25 for
the most AT rich sequence possible)
• wash at high stringency in 3M
tetramethylammonium chloride
– TMAC stabilizes AT base pairs -> melting
temperature is a strict function of length
– works best for 21-23 mers
BioSci 203 lecture 20
page 16
©copyright
Bruce Blumberg 2001. All rights reserved
How to identify your gene of interest (contd)
•
A partial amino acid sequence (contd)
– degenerate oligo and TMAC
• advantages
– degenerate oligos always work
– fast
– only requires a single sequence
• disadvantages
– TMAC method requires strict adherence to
technique
– aa sequence may not predict a good oligo
» e.g., too many leu, ser or arg
– PCR
• advantages
– very fast
– almost anyone can manage
• disadvantages
– requires 2 good sequences
– Stoped here
BioSci 203 lecture 20
page 17
©copyright
Bruce Blumberg 2001. All rights reserved