Last updated 10/26/11
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.
1
2
3
Helicos paired end sequencing
1
2
3
4
5
6
7
4
Helicos virtual terminator
Inhibits DNA Pol once incorporated (so 1 base at a time)
Cleavable via the S-S bond (reduce it)
Fluorescent tag
Free 3’ OH
never blocked
dUTP
dU-3’P,5’P
5
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
Note: no amplifications
or ligations
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 triPs (A)
+ DNA Pol.
Wash.
Record image.
6
smsDGE = digital gene expression via Helicos sequencing and counting
MA = microarray data
7
QPCR = quantitative PCR, real time PCR
Exponential phase
Nonexponential
plateau
phase
CT value
Threshold line
Bio-rad
8
QPCR (Quantitative PCR)
Q-RT-PCR (Quantitative reverse transcription-PCR)
Run 96 samples simultaneously
9
Some data produced: 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
10
Complete Genomics
(DNA nanoballs)
AcuI: a type IIS restriction enzyme
RCR = rolling circle replication
Rolling circle DNA synthesis (Φ29 polymerase)
11
12
Complete Genomics
13
Complete Genomics\”CPAL
Probes degenerate at all
but one position, colored for
the base at that position.
5 probe sets for positions +1
to +5 relative to anchor end
Hybridize, wash, ligate, wash,
image.
Second anchor set extends 5 nt
(degenerate reach).
Repeat10 nt sequenced.
Repeat with anchors on the
other side of the adaptor.
Repeat for the other 3 adaptors.
Total 70 nts sequenced
(theor. = 80)
14
Complete Genomics
Est. 1 billion spots (reads) per slide
Lower cost
200 human genomes sequenced
Business plan: sell sequencing service, not machines
15
http://www.pacificbiosciences.com
16
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.
17
Cleaved when
incorporated
Other technologies
Phospho-linked fluorescentlylabeled nucleoside triphosphates
18
Excitation
Emission
19
20
Use a circular
template to get
redundant reads and
so more accuracy.
21
Pacific Biosciences
•
50,000 ZMWs (Aug., 2011), and density may climb
•
Long reads (e.g., full molecule analysis for splicing isoform)
•
Direct RNA sequecning possible.
•
DNA methylation detectable
22
DNA methylation detection by bisulfite conversion
23
Agilent SureSelect RNA
Target Enrichment
Capture a subgenomic
region of interest for
economy and speed of
sequencing:
E.g.,
the entire exome (all exons
w/o introns or intergeneic
regions)
hundreds of cancer genes
a particular genomic locus
Alternative: hybridize to a
custom microarray.
Agilent
24
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---
25
26
DEEP SEQUENCING (Next generation sequencing, High throughput sequencing,
Massively parallel sequencing) applications:
Human genome re-sequencing (mutations, SNPs, haplotypes, disease associations,
personalized medicine)
Tumor genome sequencing
Microbial flora sequencing (microbiome)
Metagenomic sequencing (without cell culturing)
RNA sequencing (RNAseq; gene expression levels, miRNAs, lncRNAs, splicing isoforms)
Chromatin structure (ChIP-seq; histone modifications, nucleosome positioning)
Epigenetic modifications (DNA CpG methylation and hydroxymethylation)
Transcription kinetics (GROseq; nascent RNA, pulse labeled RNA)
High throughput genetics (QUEPASA; cis-acting regulatory motif discovery)
Drug discovery (bar-coded organic molecule libraries)
27
Ke et al, and Chasin, Quantitative evaluation of all hexamers as exonic splicing
elements. Genome Res. 2011. 21: 1360-1374 ).
Order an equal mixture
of all 4 bases at these 6 positions
28
29
Rank
1
2
3
4
5
6
7
8
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
ESRseq 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
30
Constitutive exons
Alternativexons
Pseudo exons
Composite exon (from ~100,000)
31
31
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:
1
1
1
2
2
1+2
2
2
1
2
32
Next generarion method:
Use custom oligo libraries to construct minigene libraries (40,000, up to 60 nt
long):
E.g., for saturation mutagenesis to identify all exonic bases contributing to splicing
(or transcription or polyadenylation, …..)
Use bar codes to detect sequences missing from the selected molecules
E.g., Nat Biotechnol. 2009 27:1173-5. High-resolution analysis of DNA regulatory
elements by synthetic saturation mutagenesis. Patwardhan RP, Lee C, Litvin O,
Young DL, Pe'er D, Shendure J.
Long (200-mer) synthetic oligo library
OUTLINE OF NEXT LECTURE TOPICS
Expression and manipulation of transgenes in the laboratory
•
In vitro mutagenesis to isolate variants of your protein/gene with desirable properties
–
–
–
–
•
To study the protein: Express your transgene
–
–
–
–
–
•
•
•
•
•
•
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
33
33
RS1
34
34
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
1
2
35
35
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
36
36
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
37
Got this far
38
38
E. coli as a host
•
PROs:Easy, flexible, high tech, fast, cheap;
but problems
•
•
•
•
•
CONs
Folding (can misfold)
Sorting -> 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)
39
39
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)
40
40
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
41
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
42
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
43
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
44
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
45
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
46
3-HYBRID: select for proteins domains that bind a particular RNA sequence
Prey
Bait
Prey could be proteins from a cDNA library
47
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
48
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
49
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
50
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
x
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
51
Substrate
52
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
53
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.
54
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
55
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
56
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
57
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
58
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”
59
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
60
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
61
62