Like Illumina, but immobilized templates are SS DNA molecules (~200 nt)

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Last updated 11/03/10 100 AM
http//www.helicosbio.com/Technology/TrueSingleMol
eculeSequencing/tabid/64/Default.aspx
Like Illumina, but immobilized templates are SS
DNA molecules (200 nt) Each cycle adds one
base,records, and then cleaves the fluorescent
group and washes it away. Several billion
single molecule spots per slide.
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Helicos paired end sequencing
1 2 3
4 5 6
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Helicos virtual terminator Inhibits DNA Pol once
incorporated (so 1 base at a time) Cleavable via
the S-S bond (reduce it)
Free 3 OH never blocked
dU-3P,5P
dUTP
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Quanti?cation of the yeast transcriptome by
single-molecule sequencing Lipson et al. NATURE
BIOTECHNOLOGY 27 652, 2009
Tail 3 end with A via terminal transferase,
adding dT to terminate
Make cDNA via oligo dT
Add Cy5-labeled special nucleotide tri-Ps DNA
Pol. Wash. Record image.
Hybridize to surface-linked oligo dTs
Add next Cy5-labeled special nucleotide tri-Ps
DNA Pol. Wash. Record image.
Cleave dye from incorporated nt. Wash.
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smsDGE digital gene expression via Helicos
sequencing and counting MA microarray data
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QPCR (Quantitative PCR) Q-RT-PCR (Quantitative
reverse transcription-PCR)
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Distribution of yeast transcripts
mRNA
Est. copies/cell 0.5 5 50 500
TSS position relative to ATG
TSS transcription start site t.p.m.
transcripts per million
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http//www.pacificbiosciences.com
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ZMW zero mode waveguide
10 zl volume seen (1 zeptoliter 10-21 L.)
Add template and special phospho nucleotides.
One DNA Pol molecule per ZMW
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Cleaved when incorporated
Other technologies
Phospho-linked fluorescently-labeled nucleoside
triphosphates
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Emission
Excitation
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Use a circular template to get redundant reads
and so more accuracy.
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• Pacific Biosciences
• 3000 ZMWs, but density expected to climb
• Each ZMW capable of 400,000 bases per day
• 6 days X 3000 X 400,000 7.2 x 109 (at1X
  coverage)
• Predict by 2014 will sequence a human genome in
  15 min.Predict by 2014 will sequence a human
  genome for low hundreds of
• Exact number of ZMWs per chip thousands,
  perhaps 3000 as of 2010

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Applications of deep sequencing
Also definition and discovery of cis-acting
regulatory motifs in DNA and RNA
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Detection of methylated C (all in CpG
dinucleotides)
cytosine
----CmpG--- gt
----CpG-- gt
----CmpG--- gt
lt ---G p Cm---
DS DNA
Na bisulfite Heat
Na bisulfite Heat
----CmpG--- gt
----UpG-- gt
PCR
----TpG-- gt
----CpG-- gt
lt--GpC---
lt--ApC---
All NON-methylated Cs changed to T
uracil
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Definition and discovery of cis-acting regulatory
motifs in DNA and RNA
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Definition of sequences (6-mers) that affect
pre-mRNA splicing (Ke and Chasin, unpublished).
Order an equal mixture of all 4 bases at these
positions
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Rank 6-mer score ( -1 to 1) 1 AGAAGA 1.0339
2 GAAGAT 0.9918 3 GACGTC 0.9836 4
GAAGAC 0.9642 5 TCGTCG 0.9517 6 TGAAGA
0.9434 7 CAAGAA 0.9219 8 CGTCGA
0.8853 4086 TAGATA
-0.8609 4087 AGGTAG -0.8713 4088 CGTCGC
0.8850 4089 CTTAAA -0.8786 4090 CCTTTA
-0.8812 4091 GCAAGA 0.8911 4092 TAGTTA
-0.8933 4093 TCGCCG 0.9113 4094 CCAGCA
-0.8942 4093 CTAGTA -0.9251 4094 TAGTAG
-0.9383 4095 TAGGTA -0.9965 4096 CTTTTA
-1.0610
Best exonic splicing enhancers
Worst exonic splicing enhancers, best exonic
splicing silencers
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Constitutive exons
Alternativexons
Pseudo exons
Composite exon (from 100,000)
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Experiment 1 1 1 2
2 12 2
2 1 2
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OUTLINE OF NEXT LECTURE TOPICSExpression and
manipulation of transgenes in the laboratory

• In vitro mutagenesis to isolate variants of your
  protein/gene with desirable properties
• Single base mutations
• Deletions
• Overlap extension PCR
• Cassette mutagenesis
• To study the protein Express your transgene
• Usually in E. coli, for speed, economy
• Expression in eukaryotic hosts
• Drive it with a promoter/enhancer
• Purify it via a protein tag
• Cleave it to get the pure protein
• Explore protein-protein interaction
• Co-immunoprecipitation (co-IP) from
  extracts
• 2-hybrid formation
• surface plasmon resonance
• FRET (Fluorescence resonance energy
  transfer)
• Complementation readout

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Site-directed mutagenesis by overlap extension
PCR
PCR fragment subsequent
cloning in a plasmid
Ligate into similarly cut vector
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Cut with RE 1 and 2
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Cassette mutagenesis random mutagenesis but in
a limited region 1) by error-prone PCR
Original sequence coding for, e.g., a
transcripiton enhancer region
--------------------------------------------------
-------------------------------------------------
-------------------
PCR fragment with high Taqpolymerase and Mn2
instead of Mg2 ? errors
-------------------------------------------
--------------------------------------------
-----------------
Cut in primer sites and clone upstream of a
reporter protein sequence.
Pick colonies Analyze phenotypes Sequence
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Cassette mutagenesis random mutagenesis but in
a limited region 2) by doped synthesis
Target e.g., an enhancer element
--------------------------------------------------
-------------------------------------------------
-------------------
Original enhancer sequence
---------------------------------------------
-------- ----------------------------------
-----------------
Buy 2 doped oligos anneal OK for up to 80 nt.
Clone upstream of a reporter.
Doping e.g., 90 G, 3.3 A, 3.3 C,
3.3 T at each position
Pick colonies Analyze phenotypes Sequence
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• Got this far

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E. coli as a host

• PROsEasy, flexible, high tech, fast, cheap
  but problems
• CONs
• Folding (can misfold)
• Sorting -gt can form inclusion bodies
• Purification -- endotoxins
• Modification -- not done (glycosylation,
  phosphorylation, etc. )
• Modifications
• Glycoproteins
• Acylation acetylation, myristoylation
• Methylation (arg, lys)
• Phosphorylation (ser, thr, tyr)
• Sulfation (tyr)
• Prenylation (farnesyl, geranylgeranyl on cys)
• Vitamin C-Dependent Modifications (hydroxylation
  of proline and lysine)
• Vitamin K-Dependent Modifications (gamma
  carboxylation of glu)
• Selenoproteins (seleno-cys tRNA at UGA stop)

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Some alternative hosts

• Yeasts (Saccharomyces , Pichia)
• Insect cells with baculovirus vectors
• Mammalian cells in culture (later)
• Whole organisms (mice, goats, corn)
  (not discussed)
• In vitro (cell-free), for analysis only(good for
  radiolabeled proteins)

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Yeast Expression Vector (example)
Saccharomyces cerevisiae(bakers yeast)
2 mu seq yeast ori oriE bacterial ori Ampr
bacterial selection LEU2, e.g. Leu
biosynthesisfor yeast selection
2 micron plasmid
Complementation of an auxotrophy can be used
instead of drug-resistance
Your favorite gene(Yfg)
Auxotrophy state of a mutant in a biosynthetic
pathway resulting in a requirement for a nutrient
GAPD the enzyme glyceraldehyde-3 phosphate
dehydrogenase
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Yeast - genomic integration via homologous
recombination
HIS4
t
p
Yfg
FunctionalHIS4 gene
DefectiveHIS4 gene
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Double recombination Yeast (integration in Pichia
pastoris)
P. pastoris-tight control-methanol induced
(AOX1)-large scale production (gram
quantities)
Alcohol oxidase gene
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PROTEIN-PROTEIN
INTERACTIONS Yeast 2-hybrid system to discover
proteins that interact with each other Or to test
for interaction based on a hypothesis for a
specific protein.
(bait)
?
Y e.g., a candidate protein being tested for
possible interaction with X
Or Y e.g., a cDNA library used to discover a
protein that interacts with X
?
(prey)
BD (DNA) binding domain AD activation
domain
http//www.mblab.gla.ac.uk/maria/Y2H/Y2H.html
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No interaction between X and Y no reporter
expression
Yes, interaction between X and Y reporter
protein is expressed
Y e.g., a cDNA library used to discover a
protein that interacts with X Recover the Y
sequence from reporter colonies by PCR to
idenify protein Y
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Fusion library
Bait protein is the known target proteinfor whom
partners are sought
prey
and/or
Two different assays help, as there are often
many false positives.
BD DNA binding domain TA transactiavting
domain
http//www.mblab.gla.ac.uk/maria/Y2H/Y2H.html
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3-HYBRID select for proteins domains that bind
a particular RNA sequence
Prey
Bait
Prey could be proteins from a cDNA library
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Yeast one-hybrid Insert a DNA sequence
upstream of the selectable or reporter Transform
with candidate DNA-binding proteins (e.g., cDNA
library) fused to an activator domain.
Each T one copy of a DNA target sequence
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Indirect selection using a yeast 3-hybrid
systema more efficient glycosynthase enzyme
Directed Evolution of a Glycosynthase via
Chemical Complementation Hening Lin, Haiyan Tao,
and Virginia W. Cornish J. AM. CHEM. SOC. 2004,
126, 15051-15059
Turning a glycosidase into a glyco-synthase Glyco
sidase Glucose-Glucose (e.g., maltose) H2O
? 2 Glucose
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Indirect selection using the yeast 3-hybrid
system(one of the hybrid moelcules here is a
small molecule)
e.g., from a mutated library of enzyme
glycosynthase genes
glucose
Leu2 gene
Leu2 gene
Transform a yeast leucine auxotroph. Provide
synthetic chimeric substrate molecules. Select
in leucine-free medium.
DHFR dihydrofolate reductase GR
glucocorticoid receptor (trancription factor
) MTX methotrexate (enzyme inhibitor of
DHFR) DEX dexamethasone, a glucocorticoid
agonist, binds to GR AD activation domain, DBD
DNA binding domain
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Selection of improved cellulases via the yeast
2-hybrid system
Survivors are enriched for cellulase genes that
will cleave cellulose with greater efficiency
(kcat / Km)
Yeast cell
Cellobiose (disaccharide)
URA-3 (toxic)
cellulase
Directed Evolution of Cellulases via Chemical
Complementation. P. Peralta-Yahya, B. T. Carter,
H. Lin, H. Tao. V.W. Cornish. JACS 2008, 130,
1744617452
x
x
x
x
Library of cellulase mutant genes (one per cell)
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Substrate
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Pathway to pyrimidine nucleotides
How does the URA-3 system work?
analog
5-fluoroorotic acid

• URA-3 gene for orotidine phosphate (OMP)
  decarboxylase

5-Fluoro-OMP
URA-3 decarboxylation (pyr-4)
5-Fluoro-UMP
Uridine kinase
Exogenousuridine
Thymidylate Synthetase inhibition
RNA
Death
Ura3 is FOA sensitive ura3- is FOA resistant
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Measuring protein-protein interactions in
vitro Xone protein Y another protein
Pull-downs Binding between defined purified
proteins, at least one being purified. Tag each
protein differently. Examples His6-X HA-Y
Bind to nickel ion column, elute (his), Western
with HA Ab GST-X HA-Y Bind to glutathione ion
column, elute (glutathione), Western with
HA Ab His6-X 35S-Y (made in vitro)
Bind Ni column, elute (his), gel
autoradiography.
No antibody needed.
(HA influenza virus flu hemagglutinin)
glutathione Gamma-glutamyl-cysteinyl-glycine.
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Example of a result of a pull-down experiment
Also identfy by MW (or mass spec)
Total protein no antibody or Western (stained
with Coomassie blue or silver stain)
Antibody used in Western
Compare pulled down fraction (eluted)with loaded
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Western blotting
To detect the antibody use a secondary antibody
against the primary antibody. The secondary
antibody is fusion protein with an enzyme
activity (e.g., alkaline phosphatase). The
enzyme activity is detected by its catalysis of a
reaction producing a luminescent compound.
http//www.bio.davidson.edu/courses/genomics/metho
d/Westernblot.html
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Detection of antibody binding in western blots
Antibody to protein on membrane
Alkaline phosphatase fusion
Non-luminescent substrate-PO4
Y
Y
Luminescent product PO4
Secondary antibody-enzyme fusion(e.g., goat
anti-rabbit IgG)
Detect by exposing to film
Protein band on membrane
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Far western blotting to detect specific
protein-protein interactions. Use a specific
purified protein as a probe instead of the
primary antibody
To detect the protein probe use an antibody
against it. Then a secondary antibody, a fusion
protein with an enzyme activity. The enzyme
activity is detected by its catalysis of a
reaction producing a luminescent compound.
protein
protein
http//www.bio.davidson.edu/courses/genomics/metho
d/Westernblot.html
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Expression via in vitro transcription followed by
in vitro translation
T7 RNA polymerase binding site (17-21 nt)
VECTOR
cDNA
.ACCATGG..
Radioactively labeled protein
1. Transcription to mRNA via the T7 promoter T7
polymerase
2. Add to translation system rabbit
reticulocyte lysate or wheat
germ lysate
Or E. coli lysate (combined transcription
translation) All commerically available as kits
Add ATP, GTP, tRNAs, amino acids, label
(35S-met), May need to add RNase
(Ca-dependent) to remove endogenous mRNA In
lysate
NOTE Protein is NOT at all pure (100s of lysate
proteins present), just radio-pure
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• Co-immunoprecipitation
• Most times not true precipitation, which
  requires about equivalent concentrations of
  antigen and antibody
• Use protein A immobilized on beads (e.g.,
  agarose beads)
• Protein A is from Staphylococcus aureus binds
  tightly to Immunoglobulin G (IgG) from many
  species.

Does X interact with Y in the cell or in vitro?
incubate

anti-X IgG
Or cell extract
Protein A

Wash by centrifugation (or magnet) Elute with
SDS Detect X, Y in eluate by Western blotting
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Surface plasmon resonance (SPR) The binding
events are monitored in real-time and it is not
necessary to label the interacting biomolecules.
glass plate
http//home.hccnet.nl/ja.marquart/BasicSPR/BasicSp
r01.htm
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Expression in mammalian cells Lab
examples HEK293 Human embyonic kidney (high
transfection efficiency) HeLa Human cervical
carcinoma (historical, low RNase) CHO Chinese
hamster ovary (hardy, diploid DNA content,
mutants) Cos Monkey cells with SV40 replication
proteins (-gt high transgene copies) 3T3 Mouse or
human exhibiting regulated (normal-like)
growth various others, many differentiated to
different degrees, e.g. BHK Baby hamster kidey
HepG2 Human hepatoma GH3 Rat pituitary
cells PC12 Mouse neuronal-like tumor
cells MCF7 Human breast cancer HT1080 Human with
near diploid karyotype IPS induced pluripotent
stem cells and Primary cells cultured with a
limited lifetime. E.g., MEF mouse embryonic
fibroblasts, HDF Human diploid
fibroblasts Common in industry NS1 Mabs Mouse
plasma cell tumor cells Vero vaccines
African greem monkey cells CHO Mabs, other
therapeutic proteins Chinese hamster ovary
cells PER6 Mabs, other therapeutic proteins Human
retinal cells
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