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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 BioSci 203 lecture 20 page 1 ©copyright Bruce Blumberg 2001. All rights reserved mRNA frequency and cloning • 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 • 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 • 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 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) • • 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) • 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) • 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 • 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) • 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!! • 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) • 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) • 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) • 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) • 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) • 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