Last updated 11/03/10 1:00 AM
http://www.helicosbio.com/Technology/TrueSingleMoleculeSequencing/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
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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
dUTP
dU-3’P,5’P
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Quantification of the yeast transcriptome by single-molecule sequencing
Lipson et al. NATURE BIOTECHNOLOGY 27: 652, 2009
Make cDNA
via oligo dT
Hybridize to
surface-linked
oligo dTs
Cleave dye
from
incorporated nt.
Wash.
Tail 3’ end with A
via terminal
transferase,
adding dT to
terminate
Add Cy5-labeled
special
nucleotide tri-Ps
+ DNA Pol.
Wash.
Record image.
Add next Cy5labeled special
nucleotide tri-Ps
+ DNA Pol.
Wash.
Record image.
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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 = transcription start site
t.p.m. = transcripts per million
TSS position relative to ATG
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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.)
One DNA Pol
molecule per
ZMW
Add template
and special
phospho
nucleotides.
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Cleaved when
incorporated
Other technologies
Phospho-linked fluorescentlylabeled nucleoside triphosphates
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Excitation
Emission
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Use a circular
template to get
redundant reads and
so more accuracy.
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Pacific Biosciences
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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
cytosine
Detection of methylated C (~all in CpG dinucleotides)
----CmpG--- >
----CpG-- >
----CmpG--- >
< ---G p Cm--DS DNA
Na bisulfite
Heat
Na bisulfite
Heat
----CmpG--- >
----UpG-- >
PCR
----TpG-- >
<--ApC--uracil
All NON-methylated
Cs changed to T
----CpG-- >
<--GpC---
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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
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6-mer
AGAAGA
GAAGAT
GACGTC
GAAGAC
TCGTCG
TGAAGA
CAAGAA
CGTCGA
:
4086
4087
4088
4089
4090
4091
4092
4093
4094
4093
4094
4095
4096
TAGATA
AGGTAG
CGTCGC
CTTAAA
CCTTTA
GCAAGA
TAGTTA
TCGCCG
CCAGCA
CTAGTA
TAGTAG
TAGGTA
CTTTTA -1.0610
score (~ -1 to +1)
1.0339
0.9918
0.9836
0.9642
0.9517
Best exonic splicing enhancers
0.9434
0.9219
0.8853
:
-0.8609
-0.8713
0.8850
-0.8786
-0.8812
0.8911
Worst exonic splicing enhancers,
-0.8933
= best exonic splicing silencers
0.9113
-0.8942
-0.9251
-0.9383
-0.9965
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Constitutive exons
Alternativexons
Pseudo exons
Composite exon (from ~100,000)
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Sequence of 36
Quality code
CGCACTGTGCTGGAGCTCCCGGGGTTAACTCTAGAA abU^Vaa`a\aaa]aWaTNZ`aa`Q][TE[UaP_U]
TACACTGTGCTGGAGCTCCCAACGGCAACTCTAGAA a`P^Wa`[`Wa^`X_X_XWVa^NSP]_]S^X_T\X^
CGCACTGTGCTGGAGCTCCCATGGAGAACTCTAGAA aTa`^b``baaaa^aab^YaTQLOHIa`^a``TX]]
TACACTGTGCTGGAGCTCCCCTCCCAAACTCTAGAA I_`aaaa`aaaaaaa_a_^[KZIGIGZ`U`\^P^^`
CGCACTGTGCTGGAGCTCCCAATAGTAACTTTAGAA aY_\abb[T\abaaa`a`bZ[HXXIZa_`_LGMS[`
TATACTGTGCTGGAGCTCCCGACGTAAACTCTAGAA aba]^aa_a]`aa]_]`XWSMFGGIPX[P]X`V_Y^
TACACTGTGCTGGAGCTCCCTGGTAAAACTCTAGAA a_^a^aa`aYaaa_aY`Y_^[I]VY\`]V]R\W]VV
TACACTGTGCTGGAGCTCCCAATAAAAACTCTAGAA XZababa`aZaaaaaYaYXX`baa``\\TaUa\aW`
Variable region
Constant regions
(peculiar to our expt.)
2 nt barcode (TA or CG)
Experiment:
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1+2
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OUTLINE OF NEXT LECTURE TOPICS
Expression and manipulation of transgenes in the laboratory
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In vitro mutagenesis to isolate variants of your protein/gene with desirable properties
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To study the protein: Express your transgene
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Single base mutations
Deletions
Overlap extension PCR
Cassette mutagenesis
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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RS1
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RS2
Site-directed mutagenesis by
overlap extension PCR
PCR
fragment
subsequent cloning
in a plasmid
RS1
RS2
Ligate into similarly cut vector
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 Taq
polymerase and Mn+2 instead of Mg+2  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
-----------------------------------------------------------*------------------------*-*-*------------*------------*-------*--------*--*-**---------------*-----------*--*------
Clone upstream of a reporter.
Pick colonies
Analyze phenotypes
Sequence
Buy 2 doped oligos; anneal
OK for up to ~80 nt.
Doping = e.g.,
90% G,
3.3% A,
3.3% C,
3.3% T
at each position
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Got this far
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E. coli as a host
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PROs:Easy, flexible, high tech, fast, cheap;
but problems
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CONs
Folding (can misfold)
Sorting -> can form inclusion bodies
Purification -- endotoxins
Modification -- not done (glycosylation, phosphorylation, etc. )
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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
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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
(baker’s yeast)
2 mu seq:
yeast ori
oriE = bacterial ori
Ampr = bacterial selection
LEU2, e.g. = Leu biosynthesis
for yeast selection
Complementation of
an auxotrophy can
be used instead of
drug-resistance
2 micron plasmid
GAPDterm
Your
favorite
gene
(Yfg)
LEU2
Auxotrophy = state of a mutant
in a biosynthetic pathway
resulting in a requirement for a
nutrient
Ampr
GAPDprom
oriE
GAPD = the enzyme glyceraldehyde-3 phosphate dehydrogenase
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Yeast - genomic integration via homologous
recombination
t
p
Vector
DNA
gfY
HIS4
Genomic
DNA
Genomic
DNA
HIS4 mutation-
t
p
Yfg
Functional
HIS4 gene
Defective
HIS4 gene
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Double recombination Yeast (integration in Pichia pastoris)
P. pastoris
-tight control
-methanol induced (AOX1)
-large scale production AOX1t
(gram quantities)
HIS4
Vector
DNA
Yfg
3’AOX1
AOX1p
Genomic
DNA
Alcohol oxidase gene
AOX1 gene (~ 30% of total protein)
Genomic
DNA
Yfg
AOX1p
AOX1t
HIS4
3’AOX1
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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
?
?
BD = (DNA) binding domain
(prey)
Or: Y = e.g., a cDNA
library used to
discover a protein
that interacts with X
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 protein
for 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 system:
a 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
Glycosidase: 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)
Cellobiose
(disaccharide)
URA-3 (toxic)
cellulase
x
x
x
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Library of cellulase mutant genes
(one per cell)
Directed Evolution of
Cellulases via Chemical
Complementation. P. PeraltaYahya, B. T. Carter, H. Lin, H.
Tao. V.W. Cornish.
JACS 2008, 130, 17446–17452
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Substrate
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How does the URA-3
system work?
Pathway to pyrimidine nucleotides:
5-fluoroorotic acid
5-Fluoro-OMP
URA-3
URA-3 = gene for
orotidine phosphate
(OMP) decarboxylase
decarboxylation
(pyr-4)
5-Fluoro-UMP
RNA
Death
Thymidylate
Synthetase
inhibition
Exogenous
uridine
Ura3+ is FOA sensitive; ura3- is FOA resistant
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Measuring protein-protein interactions in vitro
X=one 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)
Antibody used in Western
Total protein: no antibody or Western
(stained with Coomassie blue
or silver stain)
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/method/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 =
Luminescent product + PO4=
Secondary antibody-enzyme fusion
Detect by exposing to film
(e.g., goat anti-rabbit IgG)
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.
protein
protein
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.
http://www.bio.davidson.edu/courses/genomics/method/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?
B
Y
X
Y
Y
incubate
C
X
D
X
A
A
X
Y
A
A
A
Wash by centrifugation
(or magnet)
Elute with SDS
Detect X, Y in eluate by
Western blotting
Y
+ Protein A
X
A
A
A
D
A
A
X
A
C
Y
A
A
+
X
Or cell extract
Y
B
A
A
A
A
A
B
+
C
X
Y
D
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/BasicSpr01.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 (-> 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
Vero
vaccines
CHO
Mabs, other therapeutic proteins
PER6 Mabs, other therapeutic proteins
Mouse plasma cell tumor cells
African greem monkey cells
Chinese hamster ovary cells
Human retinal cells
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