BioSci 203 lecture 20 page 1 © copyright Bruce Blumberg All rights reserved Bio Sci 203 Lecture 20 - cDNA library screening Bruce Blumberg –office Bio Sci II – –lab 5427 (x46873), 5305 (x43116) –office hours Wednesday Link is also main class web site Today –wrap up cDNA library screening –characterization of clones obtained from screening –Protein protein binding assays

BioSci 203 lecture 20 page 2 © copyright Bruce Blumberg All rights reserved mRNA frequency and cloning mRNA frequency classes –classic references Bishop et al., 1974 Nature 250, Davidson and Britten, 1979 Science 204, –abundant mRNAs that together represent 10-20% of the total RNA mass > 0.2% –intermediate 1,000-2,000 mRNAs together comprising % of the total % 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?

BioSci 203 lecture 20 page 3 © copyright Bruce Blumberg All rights reserved 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, –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 C o t =5.5 –driver C o t value is critical for success of normalization –5-10 is optimal –higher values are not better

BioSci 203 lecture 20 page 4 © copyright Bruce Blumberg 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 5 © copyright Bruce Blumberg All rights reserved Normalization and subtraction (contd) Subtraction - removing cDNAs (mRNAs) expressed in two populations leaving only differentially expressed –Sagerström et al. (1997) Ann Rev. Biochem 66, /- screening St. John and Davis (1979) Cell 16, –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 6 © copyright Bruce Blumberg 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 7 © copyright Bruce Blumberg 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 8 © copyright Bruce Blumberg All rights reserved Normalization and subtraction (contd) Subtractive screening - Sargent and Dawid (1983) Science 222, –Make 1st strand cDNA from a tissue and then hybridize it to excess mRNA from another larger C o t is best –remove double stranded materials -> common seqs –make a probe or library from the remaining single stranded cDNA – fold more sensitive than +/- screening

BioSci 203 lecture 20 page 9 © copyright Bruce Blumberg All rights reserved Normalization and subtraction (contd) 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 10 © copyright Bruce Blumberg All rights reserved 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

BioSci 203 lecture 20 page 11 © copyright Bruce Blumberg All rights reserved 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!!

BioSci 203 lecture 20 page 12 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) 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 13 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) 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 14 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) 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 15 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) 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 16 © copyright Bruce Blumberg All rights reserved How to identify your gene of interest (contd) 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 (T m -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 mers

BioSci 203 lecture 20 page 17 © copyright Bruce Blumberg 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