BioSci 203 lecture 21 page 1 © copyright Bruce Blumberg 2001. All rights reserved Bio Sci 203 Lecture 21 - cDNA library screening &sequence characterization

BioSci 203 lecture 21 page 1 ©copyright Bruce Blumberg 2001. All rights reserved

Bio Sci 203 Lecture 21 - cDNA library screening &sequence characterization

• Bruce Blumberg ([email protected])

– office - 4203 Bio Sci II

– 824-8573

– lab 5427 (x46873), 5305 (x43116)

– office hours Wednesday 1-2.

• This week

– Protein protein binding assays

– Characterization of Selected DNA Sequences

• DNA sequence analysis

– mRNA Analysis

• Techniques to detect and quantitate mRNA

– Northern

– RNase protection

– RT-PCR

– in-situ hybridization

– Transcript mapping


How to identify your gene of interest (contd)

• Two hybrid screening

– originally used in yeast, now other systems possible

– prepare bait - target protein fused to DBD (GAL4) usual

• stable cell line is commonly used

– prepare library as fusion proteins with a known activation domain



• Two hybrid screening (contd)

– approach

• transfect library into cells and either select for survival or activation of reporter gene

• purify and characterize positive clones

– advantages

• seems simple and inexpensive on its face

– in materials

• functional assay

– disadvantages

• fusion proteins bias the screen against full-length cDNAs.

• Binding parameters not manipulable

• bait must not have activation function

• Difficult or impossible to detect interactions between proteins and complexes.

• Doesn’t work for secreted proteins

• Many months to screen

– savings in materials are eaten up by salaries

– avg grad student costs $30k/year

– avg postdoc or tech costs $40k/year

• MANY false positives



• In vitro interaction screening

– based on in vitro expression cloning (IVEC)

• transcribe and translate cDNA libraries in vitro into small pools of proteins (~100)

• test these proteins for their ability to interact with your protein of interest

– EMSA

– co-ip

– FRET

– SPA

– advantages

• functional approach

• smaller pools increase sensitivity

• automated variant allows diversity of targets

– proteins, protein complexes, nucleic acids, protein/nucleic acid complexes, small molecule drugs

– very fast

– disadvantages

• can’t detect heterodimers unless 1 partner known

• expensive consumables (but cheap salaries)

– typical screen will cost $10-15K

• expense of automation


Current generation binding assays

• scintillation proximity assay

– Target is bound to solid phase - bead or plate

– radioactive protein or ligand is added and allowed to reach equilibrium

• 35S, 125I, 3H work best

– radioactive decay is quenched in solution, only detected when in “proximity” of the solid phase, e.g. when bound to target

– applications

• ligand-receptor binding with 3H small molecules

• protein:protein interaction

• protein:DNA


Current generation binding assays

• SPA (contd

– advantages

• homogeneous, equilibrium assay

– washing is not necessary!

• Can detect weak interactions,

• insensitive to high off rate

• large dynamic range

– disadvantages

• radioactive

• equipment

• in vitro only


Current generation binding assays (contd)

• FRET - fluorescent resonance energy transfer– based on the transfer of energy from one fluor to

another that is not normally excited at that wavelength

– Many types of fluorescent moieties possible

• rare earth metals europium cryptate

• fluorescent proteins

– GFP and variants

– allophycocyanin

• Tryptophan residues in proteins

– application

• very commonly used for protein:protein interaction screening in industry

• FRET microscopy can be used to prove interactions between proteins within single cells

– Roger Tsien



• FRET (contd)

– advantages

• can be very sensitive

• may be inexpensive or not depending on materials

• non-radioactive

• equilibrium assay

• single cell protein:protein interactions possible

• time resolved assays possible

– disadvantage

• poor dynamic range - 2-3 fold difference full scale

• must prepare labeled proteins or ligands

• tunable fluorometer required (we have one here)



• AlphaScreen - amplified luminescent resonance proximity

– Bind proteins to two types of beads

• donor bead accepts laser light at 680 nm and emits singlet oxygen

• acceptor bead receives singlet oxygen and emits light at 520-620

– principle is that singlet oxygen can only diffuse one bead diameter before decaying

• only closely association between donor and acceptor gives a signal

– applications

• receptor:ligand binding

• protein:protein binding

• discovery of peptide ligands for proteins



• AlphaScreen (contd)

– advantages

• very sensitive, equilibrium assay

• very fast

– disadvantages

• requires instrument ~$100K

• cost of beads

• must bind proteins to beads

• single source for beads and instrument

• completely in vitro assay



• BRET2 (Bioluminescence Resonance Energy Transfer)– based completely on bioluminescent reaction from

Renilla reniformis• can make lots of money by copying nature!

– Renilla luciferase emits blue light in presence of its substrate coelenterazine

– If GFP is nearby, it accepts this blue light and emits green light

• make two fusion proteins, one to rluc the other to GFP

• mix everything together

• if proteins interact then green light is detected

• if not, only blue light



• BRET2 (contd)

– applications

• protein protein interactions in solution

• protein:protein interactions within a single cell

– advantages

• fairly sensitive

• no laser required to excite

– minimal equipment required

• works in living cells (substrate is permeable)

– disadvantage

• need to make fusion proteins

• single source for reagents



• Eletrochemiluminescent assays – Origen system IGEN– based on a molecule that emits light when stimulated in an

electrical field• ruthenium derivative

– capture a molecule with magnetic beads, if binding occurs, you can elicit light

– Applications

• becoming widely used in clinical diagnostics as a radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA) substitute

• some applicability to protein:protein and protein:DNA binding



• Eletrochemiluminescence (contd)

– advantages

• rapid and sensitive compared with RIA or ELISA

• good quantitation

– disadvantages

• requires instrument

• proprietary reagents

• completely in vitro assay



• Biacore (surface plasmon resonance)

– surface plasmon waves are excited at a metal/liquid interface

– Target bound to a thin metal foil and test sample flowed across it

– Foil is blasted by a laser from behind

• SPR alters reflected light intensity at a specific angle and wavelength

• Binding to target alters refractive index which is detected as change in SPR

• Change is proportional to change in mass and independent of composition of binding agent



• Biacore (contd)

– Advantages

• Can use any target

• Biological extracts possible

• Measure kinetics

• Small changes detectable with correct instrument

– 360 d ligand binding to 150 kd antibody

• Can use as purification and identification system

– Disadvantages

• Machine is expensive (we have two)

• “high throughput” very expensive

• Not trivial to optimize


Analysis of genes and cDNAs

• Characterization of cloned DNA

– what things do we want to know about a new gene?

• Complete DNA sequence

– cDNA sequence

– genomic sequence?

– Restriction enzyme maps?

• Where are introns and exons?

– Particularly if knockouts are coming

• where is the promoter(s)?

– Alternative promoter use?

– Mapping transcription start(s)

• where and when is mRNA expressed?

– How abundantly is it expressed in each place?

– Is there any association between expression levels and putative function?

• What is the function of this gene?

– Loss-of-function analysis decisive

» knockout

» antisense

» mutant mRNA e.g. dominant negative

– gain of function may be helpful

» transgenic

» mutant mRNA - constitutively active transcription factor


Analysis of genes and cDNAs (contd)

• Landmarks in DNA sequencing

– Sanger, Nicklen and Coulson. Sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. 74, 5463-5467 (1977).

– Sanger, F. et al. The nucleotide sequence of bacteriophage ΦX174. J Mol Biol 125, 225-46. (1978).

– Sutcliffe, J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol 43, 77-90. (1979).

– Sanger et al., Nucleotide sequence of bacteriophage lambda DNA. J Mol Biol 162, 729-73. (1982).

– Messing, J., Crea, R. & Seeburg, P. H. A system for shotgun DNA sequencing. Nucl.Acids Res 9, 309-21 (1981).

– Anderson, S. et al. Sequence and organization of the human mitochondrial genome. Nature 290, 457-65 (1981).

– Deininger, P. L. Random subcloning of sonicated DNA: application to shotgun DNA sequence analysis. Anal Biochem 129, 216-23. (1983).

– Baer et al. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature 310, 207-11. (1984). (189 kb)

– Innis et al. DNA sequencing with Taq DNA polymerase and direct sequencing of PCR-amplified DNA Proc. Natl. Acad. Sci. 85, 9436-9440 (1988)



• Landmarks in DNA sequencing (contd).

– 1995 - Haemophilus influenzae (1.83 Mb)

– 1995 - Mycoplasma genitalium (0.58 Mb)

– 1996 - Saccharomyces cerevisiae genome (13 Mb)

– 1996 - Methanococcus jannaschii (1.66 Mb)

– 1997 - Escherichia coli (4.6 Mb)

– 1997 - Bacillus subtilis (4.2 Mb)

– 1997 - Borrelia burgdorferi (1.44 Mb)

– 1997 - Archaeoglobus fulgidus (2.18 Mb)

– 1997 - Helicobacter pylori (1.66 Mb)

– 1998 - Treponema pallidum (1.14 Mb)

– 1998 - Caenorhabditis elegans genome (97 Mb)

– 1999 - Deinococcus radiodurans (3.28 Mb)

– 2000 - Drosophila melanogaster (120 Mb)

– 2000 - Arabidopsis thaliana (115 Mb)

– 2001 - Escherichia coli O157:H7 (4.1 Mb)

– 2001 - Human “genome”



– 1995 - Haemophilus influenzae (1.83 Mb)

• first bacterium sequenced, human pathogen

– 1995 - Mycoplasma genitalium (0.58 Mb)

• smallest free living organism

– 1996 - Saccharomyces cerevisiae genome (13 Mb)

– 1996 - Methanococcus jannaschii (1.66 Mb)

• first Archaebacteria

– 1997 - Escherichia coli (4.6 Mb)

– 1997 - Bacillus subtilis (4.2 Mb)

– 1997 - Borrelia burgdorferi (1.44 Mb)

• Lyme disease

– 1997 - Archaeoglobus fulgidus (2.18 Mb)

• first sulfur metabolizing bacterium

– 1997 - Helicobacter pylori (1.66 Mb)

• first bacteria to cause cancer

– 1998 - Treponema pallidum (1.14 Mb) syphillus

– 1998 - Caenorhabditis elegans genome (97 Mb)

– 1999 - Deinococcus radiodurans

• resistant to radiation, starvation, ox stress

– 2000 - Drosophila melanogaster (120 Mb)

– 2000 - Arabidopsis thaliana (115 Mb) (plant)

– 2001 - Escherichia coli O157:H7 (4.1 Mb)

– 2001 - Human “genome”


The human genome

• In Feb 12 2001, Celera and Human Genome project published “draft” human genome sequencs

– Celera -> 39114

– Ensembl -> 29691

– Consensus from all sources ~30K

• Number of genes

– C. elegans – 19,000

– Arabidopsis 25,000

• Predictions had been from 50-140k human genes

– What’s up with that?

– Are we only slightly more complicated than a weed?

– How can we possibly get a human with less than 2x the number of genes as C. elegans

– Implications?

• UNRAVELING THE DNA MYTH: The spurious foundation of genetic engineering, Barry Commoner, Harpers Magazine Feb, 2002


The human genome

• The answer – Sloppy science

– Gene sets don’t overlap completely

– Floor is 42K and likely total 50K or more

• 96k UniGene clusters from ESTs

= 42113


DNA Sequence analysis

• Complete DNA sequence

– complete sequence is desirable but takes time

• how long depends on size and strategy employed

– which strategy to use depends on various factors

• how large is the clone?

– cDNA

– genomic

• How fast is sequence required?

• sequencing strategies

– primer walking

– cloning and sequencing of restriction fragments

– progressive deletions

• bidirectional

• unidirectional

– Shotgun sequencing

• whole genome

• with mapping

– map first (C. elegans)

– map as you go (many)


DNA Sequence analysis (contd)

• Primer walking - walk from the ends with oligonucleotides

– sequence, back up ~50 nt from end, make a primer and continue



• Primer walking (contd)

– advantages

• very simple

• no possibility to lose bits of DNA

– restriction mapping

– deletion methods

• no restriction map needed

• best choice for short DNA

– disadvantages

• slowest method

– about a week between sequencing runs

• oligos are not free (and not reusable)

• not feasible for large sequences

– applications

• cDNA sequencing when time is not critical

• targeted sequencing

– verification

– closing gaps in sequences



• Cloning and sequencing of restriction fragments

– once the most popular method

• make a restriction map

• subclone fragments

• sequence

– advantages

• straightforward

• directed approach

• can go quickly

• cloned fragments often useful otherwise

– RNase protection

– nuclease mapping

– in situ hybridization

– disadvantages

• possible to lose small fragments

– must run high quality analytical gels

• depends on quality of restriction map

– mistaken mapping -> wrong sequence

• restriction site availability

– applications

• sequencing small cDNAs

• isolating regions to close gaps



• nested deletion strategies - make sequential deletions from one end of the clone

– cut, close and sequence

• make restriction map

• digest with enzymes that cut in polylinker and insert

• religate and sequence from end with restriction site

• repeat until sequence is finished, filling in gaps with oligos



• nested deletion strategies (contd)

– cut, close and sequence

• advantages

– fast

– simple

– efficient

• disadvantages

– limited by restriction site availability in vector and insert

– need to take the time to make a restriction map

– BAL31 mediated deletions (archaic)

• digest insert from both ends with BAL31

• repair, subclone and sequence

• advantages

– was once the only way to make progressive deletions

• disadvantages

– bidirectional

– can’t protect -> must reclone

• applications

– no longer used

– superseded by ExoIII-mediated deletion cloning



• nested deletion strategies (contd)

– Exonuclease III-mediated deletion -



– Exonuclease III-mediated deletion (contd)

• cut with polylinker enzyme

– protect ends -

» 3’ overhang

» phosphorothioate

• cut with enzyme between first cut and the insert

– can’t leave 3’ overhang

• timed digestions with Exonuclease III

• stop reactions, blunt ends

• ligate and size select recombinants

• sequence

• advantages

– unidirectional

– processivity of enzyme gives nested deletions



• Nested deletion strategies

– Exonuclease III-mediated deletion (contd)

• disadvantages

– need two unique restriction sites flanking insert on each side

– best used successively to get > 10kb total deletions

– may not get complete overlaps of sequences

» fill in with restriction fragments or oligos

• applications

– method of choice for moderate size sequencing projects

» cDNAs

» genomic clones

– good for closing larger gaps

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BioSci 203 lecture 21 page 1 © copyright Bruce Blumberg 2001. All rights reserved Bio Sci 203 Lecture 21 - cDNA library screening &sequence characterization