BEAMing: PCR on beads compartmentalized in a wateroil emulsion. Second view.

1
BEAMing PCR on beads compartmentalized in a
water-oil emulsion. Second view.
2
BEAMing beads, amplification, emulsion,
magnetics cloning DNA molecules via PCR on
beads Each bead is provided with a single DNA
template and that seqeunce is amplified to yield
many copies of the complement immobilized on the
bead. (Dressman et al., Vogelstein lab. PNAS
2003.)
Aqueous microspheres
No template or bead
Had one template
Had another template
No template
No bead
Remove oil
Or other ways to read out (e.g., CCD camera,
laser scanner)
E.g., hybridization to a complementray probe
 Dressman, D., Yan, H., Traverso, G., Kinzler,
K.W., and Vogelstein, B. 2003. Transforming
single DNA molecules into fluorescent magnetic
particles for detection and enumeration of
genetic variations. Proc Natl Acad Sci U S A 100
8817-8822.
3
FACS Fluorescence-activated cell sorter
Impart a charge on the recognized cell
Less than one cell or particle per droplet. Thus
the most that most droplets contain is one
particle.
Can be used purely anaytically without the
sorting capability. Then called flow cytometry,
or also called FACS anyway.
Charged plates attract droplets containing a
particle of the opposite charge
Cells remain viable if treated with care.
4
Histogram-type display
No fluorescence (background)
No. of cells
Red stained
Having this much fluorescence
5
Scatter plot display
Analysis on 2 colors
One cell
Amount of green fluorescence (log)
You decide on the positions of of demarcations
Say, want high reds but low greens Instruct the
FACS to deflect cells in this quadrant. Collect
and grow or analyze further.
Amount of red fluorescence (log)
6
Beaming bead FACS analysis
Analysis of beads representing the humn genome
using allele-specific hybridization probes and
the FACS
A. Flow cytometry data 2-D plots where each
point represents one particle. Then contour lines
plotted around the point density. Here light
scattering (irrespective of wavelength) is
measured. Instrument can be set to reject data
from 2-bead doublets that scatter light more.
Both signals
Red signal
B-D. Amplified beads hybridized to 2 probes, one
specific to the S allele of a certain gene and
one specific to the L allele. The beads carry
the amplified PCR products corresponding to this
region from 3 human individuals. The blue
points come from micrspheres that contained both
tyes of PCR products from both alleles, despite
the high dilution.
Neither signal
Green signal
Log plot
7
Massively parallel DNA sequencing technology
3 Sequencing by ligation (The sequencing by
oligonucleotide ligation and detection, SOLiD,
ABIApplied Biosystems, Inc.) Uses beamed beads,
hybridizing degenerate oligomers with fluorescent
labels. Complex technology will not be discussed
here. Advantage reads each addition twice, so
more accurate. Off-shoot of polony technology
pioneered by the George Church lab. Polony PCR
colony, a colony of DNA molecules founded by a
single DNA molecule. Orignally in a gel, not on a
bead. Refs. Webinar (also good for a general
beaming explanation) http//appliedbiosystems.cnpg
.com/lsca/webinar/rhodes/chemistry/20070618/ Shen
dure, J., Porreca, G.J., Reppas, N.B., Lin, X.,
McCutcheon, J.P., Rosenbaum, A.M., Wang, M.D.,
Zhang, K., Mitra, R.D., and Church, G.M. 2005.
Accurate multiplex polony sequencing of an
evolved bacterial genome. Science 309 1728-1732.
8
PROTEOMICS
NaOH
H3PO4
--ampholyte mixture --
-

• 2-dimensional gel electrophoresis
• 1. ISOELECTRIC FOCUSING
• Isoelectric focusing
• pH gradient established via a series of
  ampholytes in gel
• Tight bands form at isoelectric point of each
  protein (pH at which net charge)
• Equilibrium method, not migration rate
• 2. SDS-PAGE
• - Molecular weight

-
SDS

Ampholytes small organic buffers with basic and
acidic groups (I,e, zwitterions e.g., amino acids
9
PROTEIN IDENTIFICATION Mass spectrometry
MALDI-TOF (Matrix assisted laser desorption
time of flight)
Liquid chromatography
1-D or 2-D Gel electrophoresis
Separate proteins with 1-D or 2-D
electrophoresis. Or use 1-D high-performance
liquid chromatography (HPLC) column, where the
output is automatcally fed into the mass
spectrometer). Protein ID digest with trypsin.
Determine composition of peptides from masses.
Compare to translated human genome (peptide
predictions). Find the unique protein that could
rise to those peptides.
Find a unique protein that can yield these
peptides
10
Mammalian cell
genetics Introduction Genetics as a subject
(genetic processes that go on in somatic
cells that replicate, transmit, recombine,
and express genes) Genetics as a tool. Most
useful the less you know about a process. 4
manipulations of genetics 1- Mutation in
vivo (chance selection, usually) targeted gene
knock-out or alteration in vitro site
directed or random cassette 2- Mapping
Organismic mating ?segregation, recombination
(e.g., transgenic mice) ultimateseq. change
Cell culture cell fusion segregation
radiation hybrids FISH 3- Gene juxtaposition
(complementation) Organisms matings ?
phenotypes of heterozygotes Cell culture
cell fusion ? heterokaryons or hybrid cells 4-
Gene transfer transfection
11
Mammalian cell genetics cont. Advantages of
cultured cells (vs. whole organism) numbers,
homogeneity Disadvantages of cultured mammalian
cells limited phenotypes limited
differentiation in culture (but some phenotypes
available) no sex (cf. yeast)
Mammalian cell lines Most genetic manipulations
use permanent lines, for the ability to do
multiple clonings Primary, secondary cultures,
passages, senescence. Crisis, established cell
lines, immortality vs. unregulated growth. Most
permanent lines immortalized, plus
"transformed, (plus have abnormal
karyotypes) Popular cell lines 3T3, HeLa,
L-cells, HEK293, CHO, BHK, etc.
12
Mutation in cultured mammalian cells Problem of
epigenetic change Variants vs. mutants Stable
heritable alterations in phenotype that are not
due to mutations heritable switches in
gene regulation (?) DNA CpG methylation,
histone acetylation / de-acetylation Diploidy.
Heteroploidy. Haploidy. The problem of diploidy
and heteroploidy (cf. e.g., yeast, or C. elegans,
Dros.) F2 ? homozygotes) Recessive mutations
(most knock outs) are masked.
13
Solutions to diploidy problem Dominant
mutations only (too limited) Haploid cells
hasn't worked just modest chromosome reductions
in CHO Use haploid genes (XY) or functionally
haploid (XX, allelic exclusion)
restrictive Heterozygous loci (rare, despite
CHO reputation) Double mutants (incl. mutation
segregation, or mutation homozygosis(rare but
can be done) Heavy mutagenesis,
mutants/survivor increases but mutants/ml
decreases. How hard is it to get mutants?
What are the spontaneous and induced mutation
rates? Measurement of spontaneous mutation
rates. Rate vs. frequency (freq includes
accumulation). Spont 10-7/cell-generation
Induced as high as 10-3 /cell (EMS,
UV) Note Same considerations for creation of
recessive tumor suppressor genes in cancer
requires a double knockout EMS ethyl
methanesulfonate ethylates guanine UV (260 nm)
induces pyrimidine dimers between two adjacent
pyrimidines on the same DNA strand
14
L
R
R
L
Loss of heterozygosity (LOH)by mitotic
recombination between homologous chromosomes
(rare)
-
-
-


2 heterozygotes again
L
R
L
R
or
L
R
R
L
-
-


-
-


Paternal Chr. 4, say
Maternal Chr. 4
Recombinant chromatids
-
-


Heterozygote
After homologous recombination (not sister
chromatid exchange)
1 homozygote /1 homozygote -/-
Recessive phenotype is unmasked
(one mechanism of homozygosis of recessive tumor
suppressor mutations in cancer development)
15
Mutagenesis (induced general mutations, not site
directed) Chemical and physical agents
MNNG point mutations (single base
substitutions) EMS
Bleomycin small
deletions UV mostly point mutations but also
large deletions Ionizing radiation (X-,
gamma-rays) large deletions, rearrangements Exp
erimental variables Mutagen Dosage kill 90
usually, as more killing leaves too few
survivors, including mutant survivors
Expression period dilute out WT molecules
(pre-existing protein and mRNA) Metabolic
cooperation WT toxic product can be transferred
cell to cell. Therefore it may be necessary to
plate at low density. Dominant vs. recessive
mutations Dom. rare (subtle), but expression
easily observed, Recessives easier to get, but
their expression is is masked.
16

• Categories of cell mutant selections
• Exploitable metabolic pathways
• Purine and pyrimidine biosynthesis auxotrophs
• (auxotrophs require a nutrient in the medium that
  the WT doesnt)
• Auxotrophs BUdR (BrdU) Kao and Puck. Kill
  growing cells. General method.
• Analogous to penicillin selection in
  prokaryotes.
• Many auxotrophs in amino acid or nucleotide
  biosynthetic pathways have been isolated (most
  in CHO)
• 2. Drug resistance see sheet on nucleotide
  metabolism
• A. Mutant lacks toxifying enzyme (a major
  class)
• e.g., HPRT (TGR), APRT (DAPR, 8-azaAR), TK
  (BrdUR)
• B. Enzyme target becomes a better discriminator
  
• (ouabain NaK ATPase pump a-amainitin
  RNA Pol II)
• C. Permeation changes influx blocked or efflux
  increased. (MDR, P-glycoprotein)
• D. Improved de-toxification via chelation,
  covalent modification,
• or overproduction of target (dhfr
  MTX-resistance)

17
Got this far
18
in italics
19
Growth pattern examples
GHT glycine, hypoxanthine, and thymidine A
adenine G guanine
in italics

-
-
-


-





-
-
-
-
-

-
-
-


-
20
HAT hypoxanthine amethopterin thymidine
-


-
-
-


-

-

21
Cell mutant types cont.

• 3. Temperature-sensitive mutants cell cycle
  mutants.
• Tritiated amino acid suicide (aa-tRNA
  synthetases)
• 4. Antibodies. Lysis with complement. Targets
  cell surface constituents mostly (e.g., MHC)
• 5. Visual inspection at colony level
• A. Sib selection (G6PD)
• B. Replica plating (LDH)
• C. Secreted product (Iganti-Ig IP)
• FACS fluorescence-activated cell sorter.
• 1-D and 2-D fluorescence displays (cell
  surface Ag)
• Brute force
• (clonal biochemical analysis, e.g.,
  electrophoretic variants (e.g., Ig, isozymes)
• MHC major histocompatability locus or proteins
  G6PD glucose-6-phosphate dehydrogenase LCH
  lactate dehydrogenase Ig immunoglobulin

22
Cell fusion (for gene juxtaposition, mapping,
protein trafficking, ) Fusogenic agents PEG,
Sendai virus (syncytia promoting, as
HIV). Heterokaryons (2 nuclei), no cell
reproduction (limited times). (e.g., studied
membrane fluidity, nuclear shuttling, gene
activation (myoblasts) Hybrids (nuclei fuse,
some cells (minority) survive and reproduce).
Small of heterokaryons. Complementation (e.g.,
auxotrophs with same requirement) allos
selection Dominance vs. recessiveness can be
tested. Chromosome loss from hybrids ? Mapping
chromosome assignment. Synteny. Radiation
hybrids linkage analysis (sub-chromosomal
regional assignments).
PEG polyethylene glycol, (1000 to 6000 MW)
23
Cell fusion

Hprt, TK-
Parental cells
Hprt-, TK
HAT-
HAT-
PEG (polyethylene glycol, mw 6000 Sendai virus,
inactivated
Cell fusion
Heterokaryon (or, alternatively, homokaryon)
HAT medium
Hprt-, TK, Hprt TK-
HAT
Cell cycle, Nuclear fusion, Mitosis, survival
Hprt-, TK, Hprt TK-
membrane dynamics (lateral diffusion
Edidin), shuttling proteins (hnRNP A1
Dreyfuss), gene regulation (turn on myogenesis
Blau)
Hybrid cell
gene mapping (synteny Ruddle) gene regulation
(extinction Weiss) Complementation (pyrimidine
path Patterson)
24
Complementation analysis
Parental cells
Parental cells


gly-
gly-
gly-
gly-
Cell fusion
Cell fusion
glyA- glyA-
glyA- glyB-
Hybrid cell
Hybrid cell
Glycine-free Medium No growth, no
complementation, ?same gene (named glyA)
Glycine-free Medium Yes, growth, Yes,
complementation, ?different genes genes (named
glyA and glyB)
25
Mapping genes to chromosomes
Hybrid cell
Reduced hybrid
Spontaneous chromosome loss (human
preferentially)
Hprt-, TK, Hprt TK-
Hprt-, TK, Hprt TK-
Correlate identified chromosome loss With loss of
phenotypic trait (isozyme, DNA sequence, etc.)
26
DNA transfection
Transfection agents DEAE-dextran (toxic, OK for
transient) CaPO4 (co-precipitate) Electroporation
(naked DNA, high quick voltage ? transient
holes) Lipofection (multilamellar
liposomes) Polybrene (detergent?) Ballistic
(DNA-coated gold particles) Must traverse
cytoplasm. Much engulfed in lysosomes.
Inhibition of lysosomal function often helps
(chloroquin). Co-integration of high MW DNA .Can
2000 KB. Separate plasmids transfected
together ? same site (co-integration). Separate
transfections ? separate locations Random or
semi-random (many) integration sites (unless
targeted) Low but real homologous recombination
rate. History mammalian cell transfection
developed for practical use at Columbia (PS
Wigler, Axel and Silverstein)
DEAE diethyl-amino-ethyl (positively charged)
27
Mike Wigler
Richard Axel
Saul Silverstein
History discovered for practical use at Columbia
(PS Wigler Axel and Silverstein)
28
Transient transfection vs.
permanent cloned genes Unintegrated DNA
chromosomally
integrated. Unnatural?
position effects
? Super-physiological expression (so
average many) levels (per transfected cell)
? Transient -gt 10-50 transfection efficiency
(stain) Permanents more like 0.001
transfectants per µg DNA per cell (high). i.e.,
106 -gt 1000 colonies could be much less for
certain types of cells
29
One the most dramatic first applications of gene
transfection from total DNA Transfer of the
growth-transformed phenotype from one cell to
another ability to grow in multilayers or in
suspension in soft agar (Weinberg, Wigler) DNA
from tumor transfected into growth controlled
mouse 3T3 cells. Looked for foci (focus). Made
a library from growth-transformed
transfectant. Screened for human Alu repeat.
Verified cloned DNA yielded high frequency of
focus-forming transfectants. Isolated cDNA by
hybridization. Sequence ?identify gene a
dominant oncogene. Ras, a signaling protein in
transducing pathway for sensing growth factors
30
Recombination gene targeting Mitotic
recombination between homologous chromosomes
relation to cancer through the loss of tumor
suppressor genes LOH loss of homozygosity WT
/ ? mutation ? /- (WT phenotype) ? (LOH
via homologous recombination in G2 or chromosome
loss and duplication)? -/- (mutant phenotype
revealed) Recombination of transfecting
genes homologous (rare) vs. non-homologous
(common) recombination.
31
Gene knockouts via homologous recombination.
ES cells and transgenic mice. Selection for
homologous recombinants via the loss of HSV TK
genes (Capecchi) tk homol. region YFG
homol. region tk (YFG your favorite
gene) Allele replacements in cultured cell lines
(e.g., APRT). Most work in ES cells ? mice ?
homozygosis via F1 breeding Little work in
cultured lines Myc double sequenctial K.O.
viable, sick (J. Sedivy) Splicing factor (ASF)
double K.O. in chick DT40 lymphoid cells (high
rate of homologous recombination (J. Manley)
Would be lethal, but cover with inducible human
ASF gene (tet-off). Then add tet to analyze
effects of gene product removal.
32
Double knockout of the ASF gene, a vital gene, by
homologous recombination Coverage of a vital gene
by adding an inducible transgene.
First disrupted gene
(diploid)
Chicken DT40 cells
Homology regions

ASF-
neo
Tet-off promoter
pur
neo
ASF-
neo
tet
pur
ASF-
pur
X
Second disrupted gene
Cell dies without ASF
cell viable
Wang, Takagaki, and Manley, Targeted disruption
of an essential vertebrate gene ASF/SF2 is
required for cell viability. Genes Dev. 1996 Oct
1510(20)2588-99.