Bio Sci 203 Lecture 20 - cDNA library screening.ppt

1、BioSci 203 lecture 20 page 1 copyright Bruce Blumberg 2001. All rights reserved,Bio Sci 203 Lecture 20 - cDNA library screening,Bruce Blumberg (blumberguci.edu) 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/

2、index.htm http:/blumberg.bio.uci.edu/bio203-w2002/index.htm Link is also main class web siteToday wrap up cDNA library screening characterization of clones obtained from screening Protein protein binding assays,BioSci 203 lecture 20 page 2 copyright Bruce Blumberg 2001. All rights reserved,mRNA freq

3、uency 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

4、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 cellHow does one go about identifying genes that might only occur at a few copies per cell?,BioSci 203 lecture 20 page 3 co

5、pyright 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 probeNormalization - process of reducing the frequen

6、cy 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 fo

7、ld 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 no

8、t better,BioSci 203 lecture 20 page 4 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 digeste

9、d 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 l

10、ibraries 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,B

11、ioSci 203 lecture 20 page 5 copyright Bruce Blumberg 2001. All rights reserved,Normalization and subtraction (contd),Subtraction - removing cDNAs (mRNAs) expressed in two populations leaving only differentially expressed Sagerstrm et al. (1997) Ann Rev. Biochem 66, 751-783 +/- screening St. John and

12、 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 liverColonies or plaques that are expressed in target tissue (tumor) compared with co

13、ntrol are picked Why arent all colonies labeled in normal tissue?,BioSci 203 lecture 20 page 6 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

14、for absence of a signal example - hybridize a tumor cDNA library with probe prepared from normal liverselect 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 2001. All rights reserved,Normalization

15、and subtraction (contd),Advantages Relatively simple approach Doesnt 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 st

16、andard 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 2001. All rights reserved,Normali

17、zation 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 sing

18、le stranded cDNA 10-100 fold more sensitive than +/- screening,BioSci 203 lecture 20 page 9 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

19、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

20、 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 specifi

21、c genes,BioSci 203 lecture 20 page 10 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?A piece of genomic DNA?A mutantA functional assay?An antibody?A part

22、ial 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,

23、Various binding protein strategies,Interaction screening,Subtracted or +/- screening,BioSci 203 lecture 20 page 11 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

24、?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 conditi

25、ons 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 -

26、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 Bruc

27、e 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 i

28、n 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 2

29、03 lecture 20 page 13 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 li

30、brary 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

31、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 2001. All rights reserved,How to identify your gene of

32、 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 (pannin

33、g) 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 exten

34、sive retesting of pools is required applications: many receptors and transporters cloned this way,BioSci 203 lecture 20 page 15 copyright Bruce Blumberg 2001. All rights reserved,How to identify your gene of interest (contd),Antibody screening of cDNA expression libraries lets say you have an antibo

35、dy 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., gt11 series approach plate library and induce

36、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 gt11 and re

37、latives are painful to work with your antibody may not be suitable sugar directed structural epitope,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? Purified protein of interest and have one

38、 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 -

39、 use inosine for multiple bases I:C others degenerate oligos (mixtures of all possible seqs) mixtures of melting temperature is a strict function of length works best for 21-23 mers,BioSci 203 lecture 20 page 17 copyright Bruce Blumberg 2001. All rights reserved,How to identify your gene of interest

40、 (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 sequencesStoped here,