BioSci 203 lecture 20 page 1 © copyright Bruce Blumberg 2001. All rights reserved Bio Sci 203 Lecture 20 - cDNA library screening Bruce Blumberg ([email protected])

BioSci 203 lecture 20 page 1 ©copyright Bruce Blumberg 2001. All rights reserved

Bio Sci 203 Lecture 20 - cDNA library screening

• Bruce Blumberg ([email protected])

– office - 4203 Bio Sci II

– 824-8573

– lab 5427 (x46873), 5305 (x43116)

– office hours Wednesday 1-2.

• http://blumberg-serv.bio.uci.edu/bio203-w2002/index.htm

• http://blumberg.bio.uci.edu/bio203-w2002/index.htm

• Link is also main class web site

• Today

– wrap up cDNA library screening

– characterization of clones obtained from screening

– Protein protein binding assays

http://blumberg-serv.bio.uci.edu/bio203-w2002/index.htm










http://blumberg.bio.uci.edu/bio203-w2002/index.htm









mRNA frequency and cloning

• mRNA frequency classes

– classic references

• Bishop et al., 1974 Nature 250, 199-204

• Davidson and Britten, 1979 Science 204, 1052-1059

– abundant

• 10-15 mRNAs that together represent 10-20% of the total RNA mass

• > 0.2%

– intermediate

• 1,000-2,000 mRNAs together comprising 40-45% 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

• How does one go about identifying genes that might only occur at a few copies per cell?


Normalization and subtraction

• 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

• 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


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



• Subtraction - removing cDNAs (mRNAs) expressed in two populations leaving only differentially expressed

– Sagerström et al. (1997) Ann Rev. Biochem 66, 751-783

• +/- 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?



• 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



– 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



• 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



• 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


How to identify your gene of interest

• Screening methods depend on what type of information you have in hand.

– Related gene from another species?

– A piece of genomic DNA?

– A mutant

– A functional assay?

– An antibody?

– A partial amino acid sequence?

– A DNA element required for expression of an interesting gene?

– An interacting protein?

– A specific tissue or embryonic stage?

• Low stringency hybridization

• Hybridization

• Complementation

• Positional cloning

• Expression screening

• Expression library screening

• Oligonucleotide screening

• Various binding protein strategies

• Interaction screening

• Subtracted or +/- screening


How to identify your gene of interest (contd)

• If you wish to identify a cDNA, what is the most important piece of information you need?

• 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

– Information on where the mRNA is expressed

• either what tissue or

• what time during development

– such information is indispensable!!



• 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



• 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



• 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



• 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



• 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



• 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

Documents

BioSci 203 lecture 20 page 1 © copyright Bruce Blumberg 2001. All rights reserved Bio Sci 203 Lecture 20 - cDNA library screening Bruce Blumberg ([email protected])