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Karel Klepárník
Oddělení bioanalytické instrumentace
Ústav analytické chemie
Akademie věd České republiky
Brno
Moderní analytická instrumentace pro genetický
výzkum, lékařskou diagnostiku a molekulární
identifikaci organizmů
Capillary electrophoresis
CE
detection
system
outlet
electrode
chamber
mobilityelectrophoretic
electroosmotic
B)+
mobility
electrophoretic
electroosmotic
A)-
high
voltage
inlet
electrode
chamber
purge pressure
detection
window detail
injection point
separation capillaryBGE BGE
capillary effective length (LD)
capillary total length (LC)
Capillary electrophoresis scheme Why capillary electrophoresis?
T
L
R


4
22
0
RE
TTT R

solid – solidair – solid
LrdrUUdIQ J
/2
2

dr
dT
rLQ C
2
T0
TR
ΔT
Miniature capillary: low R => fast separation
1) high resistivity  low current at high voltage  low heat production
2) efficient heat transport  low temperature difference inside the capillary
DNA electromigration
K. Klepárník, P. Boček, DNA diagnostics by Capillary Electrophoresis
Chemical Reviews 107, 5279 – 5317, 2007.
DNA primary structure
Homogeneous polyelectrolyte
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DNA electromigration regimes in sieving media
Size separations of homogeneous polyelectrolytes are impossible in free solutions
Short DNA fragments
Low concentration of media
Long DNA fragments
High concentration of media
Ogston regime
Ogston (1958): * distribution of spaces in a random network of rigid rods
available to a spherical molecule
* penetration probability
)
3
4
2(exp
3
2 r
LrP rD

 
ν average density of number of fibers
2L fiber length
D „pore“ radius
r particle radius
Rodbard Chrambach (1970): )(exp
00
cK
V
V
P r
a
rD



Ferguson plot (1964): cK r
 0
loglog 
31
)(


n
r
drK
µ el. mobility
µ0 free electrolyte el. mobility
Va accessible volume
V0 void volume
Kr retardation coef.
c gel concentration
d gel fibre radius
Fergusson plot
Ferguson plots of DNA molecules in agarose gels. The logarithm of the mobility, extrapolated to
zero electric field strength at each gel concentration, is plotted as a function of agarose
concentration, %A.
cK r
 0
loglog 


r
q
6
0

kbp
Biased Reptation Model
M
1

2
2
L
hq x










3
1
3
2



N
q
ε <<1
Tk
qEa
B
2

L tube legth
ξ friction inside
x field direction
N reptation segments
ε scaled el. field
q segment charge
a segment length
1/(3N) N<<ε-2
µ/µ0 ~
ε2/9 N>> ε-2
log M
log
Ogston sieving
reptation without stretching
reptation with stretching
Rs  m
  1/M
Rs < m
Rs > m
a b c
0


Dependence of DNA electrophoretic mobility on molecular mass
Polymerase chain reaction
PCR amplification
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PCR amplification scheme
DNA template
DNA
dissociation
90 ºC
Primer
annealing
62 ºC
DNA
synthesis
72 ºC
Correct copies
N=2n+1 – 2(n+1)
1st cycle: n=1
22 – 2∙2 = 0
2nd cycle: n=2
23 – 2∙3 = 2
3rd cycle: n=3
24 – 2∙4 = 8
DNA
primer
DNA
primer
Human Genome Project
J. CRAIG VENTER, Ph.D., PRESIDENT, CELERA GENOMICS
REMARKSAT THE HUMAN GENOME ANNOUNCEMENT
THE WHITE HOUSE
MONDAY, JUNE 26, 2000
Mr. President, Honorable members of the Cabinet, Honorable members of Congress, distinguished guests.
Today, June 26, 2000 marks an historic point in the 100,000-year record of humanity. We are announcing today that for
the first time our species can read the chemical letters of its genetic code. At 12:30 p.m. today, in a joint press
conference with the public genome effort, Celera Genomics will describe the first assembly of the human
genetic code from the whole genome shotgun sequencing method. Starting only nine months ago on
September 8, 1999, eighteen miles from the White House, a small team of scientists headed by myself, Hamilton O.
Smith, Mark Adams, Gene Myers and Granger Sutton began sequencing the DNA of the human genome using a novel
method pioneered by essentially the same team five years earlier at The Institute for Genomic Research in Rockville,
Maryland. The method used by Celera has determined the genetic code of five individuals....
…There would be no announcement today if it were not for the more than $1
billion that PE Biosystems invested in Celera and in the development of the
automated DNA sequencer that both Celera and the public effort used to
sequence the genome…
DNA sequencing
Analysis of Sanger sequencing fragments DNA sequencing strategy
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Fluorescence chemistry
N C S
O -
O
C
N +
(C H 3
)2O
(C H 3
)2
N
N H 2
-R
N O 2
N H (C H 2
)5 C
O
O N
O
O
N H 2
-R
C H 2
C H 2
F F
B
N N
H 3
C
H 3
C
C
O
O N
O
O
N H 2
-R
O
3
S S O 3
-
N
+ N
O
O
O
O
N
N H 2
R
n
n = 1 : C y 3
n = 2 : C y 5
n = 3 : C y 7
N N +
O
SO O
C l
N H 2
-R
N C S
C
O
O H
OOH O
N H 2
-R
Fluorescein Rhodamine
Texas Red
NBD
BODIPY
Cy3,5,7
Fluorescent lebels
Cy3
488
nm
610
nm
ROX
ACCEPTOR
DONOR
PRIMER SEQUENCE
Sequencing primer attached to Fluorescence Resonance Energy Transfer
NH
5'-TTTTCCCAGTCACGACG-3'
(CH)2(CO) NH (CH2)6
C
O
COOH
O N+
N
O
O
N
N N
N
O
NH2
O
O -O P
O
(CH2)6
NH
O C
(CH2)5
N+
CH3
C2H5
O
CH CH CH
O
N
CH3
N (C H 3
)2(C H 3
)2
N O
C O 2
C
l
C l
O
N H
O
N H
OO
-
O
O
2
C
O
H
N
O
O
N H
ON
O
3 H
O 9
P 3
O
A C C E P T O R
D O N O R
d d T T P T E R M IN A T O R
595 nm
488 nm
Dideoxy terminator attached to Fluorescence Resonance Energy Transfer
LIF detection
Ar-ion
laser
40 mW
separation
capillary
ID 50 mm
objective
40x; 0.65
blocker
520 nm
beam
splitter
band pass
610 nm
PMT blocker
520 nm
band pass
540 nm
band pass
590 nm
band pass
570 nm
50% 488 nm
50% 514 nm
lens
Four channel LIF detection arrangement
Spectral filtering
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SENSOR
LASER
PINHOLE
OPTICS
BEAM
SPLITTER
MICROSCOPE
OBJECTIVE
FOCUS
SCHEME OF CONFOCAL DETECTOR
Space filtering
excited
sample
laser
beam
polymer filled
capillaries
sheath-flow
cuvette
open tubings
electrode chamber
electrode chamber
Sheath-flow
cuvette
DNA sequencing record
DNA sequencing over 1000 bases in 1.5 hour
Separation matrix: LPA 2.0% (w/v) 5.5 MDa
E: 150 V/cm, T: 50 °C
DNA sequencing up to 1300 bases in 2 hours
Separation matrix: LPA 2.0% (w/w) 17 MDa, 0.5% (w/w) 270 kDa
E: 125 V/cm, T: 70 °C
DNA mutation analysis
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Restriction (amplification) fragment legth polymorphism
RFLP (AFLP)
Size based separation of ds or ss DNA fragments
Resolution:
ss > 1000
ds > 400
Single Strand Conformation Polymorphism
SSCP
wild type
point mutation
native dsDNA denatured ssDNA native environment
Principle of SSCP technique
dsDNA
ssDNA
dsDNA
relativeabsorbanceat260nm
a) health homozygote
time
ssDNA
SSCP analysis
Detection of point mutation C > T in
phenylalanine hydroxylase gene on
chromosome 12
Separation conditions:
2% solution of agarose SeaPrep in 1xTBE
with 10% formamide
T - 30 °C
LC - 55 cm
LD - 50 cm
E – a) 183 V/cm, b) 135 V/cm.
Phenylketonuria
b) heterozygote
Single nucleotide primer extension
Minisequencing
SNuPE
Next generation
sequencing
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Helicos
The HeliScope™
Sequencer
2 . 109 b/day
109 reads/run
25 – 55 bp read lengths
Genome Sequencer
FLX System
3 . 108 b/day
100 Mb/7.5 hour run
400 000 reads/7.5 hour
200 – 300 bp read lengths
Solexa
Illumina Genome Analyzer
6 . 108 b / day
3 . 109 b / 5 days run
50 . 106 oligo clusters
36 – 50 bp read lengths
Parallel single molecule sequencing by synthesis Photocleavable dideoxy nucleotides
Pacific Biosciences
Single molecule real time sequencing
SMRTTM
www.pacificbiosciences.com
DNA sequencing – DNA polymerase
RNA sequencing – reverse transcriptase
Codone-resolved translation elongation by single ribosomes
Tens of nucleotide peaks in 1 sec
Read length 1 – 15 kb
80 000 detection points
15 min/genome: 50 n/s * 80 000 points * 15 min * 60 s = 3.6 Gb
DNA polymerase 529 processivity 20 kB – 400 b/s
Some enzymes are not processive
$ 100/genome
Ion Torrent
The Ion Personal Genome Machine (PGM™) sequencer
Different templates in microwells
Washing steps by individual nucleotides G, C, T, A
The world's smallest solid-state pH meter
Digital output
http://www.iontorrent.com/