Note: Descriptions are shown in the official language in which they were submitted.
6 TIIE 15 ~ F.~ .3l;~ 08"3 RID~ T ~ M.~BEE
WO 95f286~6 2 1 8 7 7 ~3 0 PCTISE~1~10039G
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NETHOD ANl~ APPAR~T~S FO~ EI~ECTROPHO~:TIC ~LYSIS
The presen~ invention relates to electrophoretic
analysis, and more part~cularly to a method and an
apparatus for electrophoretic separation of fluorescence-
labelled mixtures of substa~ces, particularly nucleic acid
fragments obtained in sequencing reactions.
Since some time past there are two basic methods for
DNA sequence determina~ion, ~iz. the chemic~l degrada~ion
method (Maxam and Gilbert, Proc. nat. Acad. Sci. U.S.A.,
Vol. 74, p. 560-564 (1977)) and the chain termination
method ~Sanger et al., Proc. nat. Acad. Sci. U.S.A., Vol.
74, p. 5~63-5467 ~1977)).
In the chemical degr~dation method, the D~A strand ~o
be analy~ed is labelled at one end with a detectable ~ag.
The sample is divided into ~our parts, each of these parts
being treated with ~ respective reagent capable of cle~ing
specifically at one of the four b~ses. The reac~ion
conditions are adapted to obtain approximately one or a few
cleava~es per molecule. Each re~ction mixture will ~hen
contain a mixture of a number of fragments of different
lengths, among them end-labelled fragments of lengths
corresponding to all possible cleavage sites, i.e. the base
that is specifically cleaved by the respective reagen~ By
separating ~he four reac~io~ mixtures according co frag~ent
size in parallel in four lanes on an electrophoretic gel, a
detectable ladder of labelled bands representing the
relative positions o~ one o~ the four bases is ob~ained.
~rom these ~ra~ment ladders the DNA sequence in question
3 0 m~y then dir~ctly be read.
In the chain termination ~ethod, which is the one most
used today, the DNA fragment to be analyse~ is instead used
as a template for DNA synthesis in ~our different reaction
mixtures by means of a star~er sequence, or so-called
primer, hybridised to the 5~-en~ of the strand, and DNA
polymerase in the presence of the four deoxynucleoside
triphosphates. Each reaction mixture con~ains a low
concentration of ~ chain terminator in the form of a
11 19 ~)6 TIIE 15:111 F.~ 36" 11~').3 Rll)~ T ~ M.~BEE ~ 115
WO 9512863G 2 1 8 7 72~) 0 P~'/SEg5/0039~
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respective one of the four dideoxynu~leoside triphoshate
analogues which upon incorporation in ~he growing chain
preve~s continued growth. Ei~her the primer, one or more
of the deoxynucleoside triphosphates or the te~minators are
labelled with a detec~able tag. In each reaction mixture is
then obtained ~ population of partially synthesized
labelled DNA molecules having a common 5'-end but varying
in length to a base-specific 3'-end. Electrophoretic
separation of the four different reac~ion mixtures side by
side on a gel as in the Maxa~-Gilbert method gives four
fragment ladders from which the desired DNA sequence thus
m~y be read.
While originally, radioactive phosphorus was used as
l~bel and the fragment ladders obtained after the
electrophoresis were imaged on autoradiograms, the use of
fluorescent labels has m~de possible more or less automatic
analysis with continuous detectio~ of the fragment bands
that migrate i~ the different lanes using fluorescence
detectors.
US-A-4,675,095 desc~ibes an advantageous
electrophoretic apparatus for this purpose where the
exciting light is introduced sideways between the two gl~ss
plates tha~ hold the electrophoretic gel between them, a~d
~he emitted fluorescence is detected on one side of ~he gel
~5 p~rpendicularly to the excitation light path By such an
arrangement a sing~e light source may be us~d for a n~mbe~
of electrophoresis l~nes, simultaneously as background
light caused by fluorescence and light scattering in the
glass pl~tes themselves are avoided.
A commercial automated development of this type of
apparatus for DNA seq~ence analysls based on the chain
termina~ion method and using labelling with one and the
same fluorescent tag, or fluorophore, is marketed ~y
Pharmacia Biotech AB, Uppsala, Sweden, under ~he trade name
A.L.F. DNA Sequencer~ (where A.L.F. stands for ~Au~oma~ed
Laser Fluorescent"), That apparatus h~s 40 electrophoresis
lanes where the excitation is effec~ed by lase~ liyh~ and
the light emitted from the fluorophore-containing DNA
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WO gsr2863~ 2 1 8 7 / ~3 0 PCI/SE;95~003~G
fragment bands is detected by an equal number of sep~rate
fixed detec~ors (photodiodes), one for each lane. ~fter the
samples have been lo~ded onto the gel, the detection
signals ~re collected automatically and sent to a comp~ter
S for storing and processing. Since each sample requires four
lanes, i.e. one lane for each of the four different base--
related termina~ors, 10 different D~A samples may thus be
analysed at the same t ime.
The object of the present invention is to increase ~.h~
capacity of D~A sequencin~ apparatus of this type by making
it possible to analyse two or more samples simul~aneously
in one and the s~me electrophoretic gel lane. According to
the invention this is achieved by labelling these samples
with diff~rent fluoropho~es e~cited ~t different
w~velengths by means of separate excitation so~rces. By
having the excitation/dete~tion of the respective
fluorophores t~ke place at differen~ times, the differen~
sample-related sign~ls ma~ be distinguished from one
another.
The inventive concept is, however, not re~tricted to
DNA sequencing bu~ may equally well be applied to a~y
electrophoretic separ~tion, not only of nucleic acids but
also of other types of subst~nces, such as e.g proteins.
One aspect of the invention therefore relates tO a
~ethod o~ electrophore~ically analysing a mixture of
fluorophore-labelled substances by dete~tinq the su~stances
in an electrophoresis lane as they are sep~rated and pass
photometric detector means ~ixed relative to the
electrophoresis lane, which method is charac~erize~ by
~0 simultaneously analysing in the same ele~trophoresis lane
~wo or optionally more di~ferent substance mix~ures which
are labelled by different fluorophores capable of being
excited at different wavelengths or wavelength bands h~
alternately illumina~in~ the ~etection area wi~h light of
these different wavelengths, ~nd relatin~ a detected
fluorescent light emission to the respective fluorophore on
the basis of the time for the excitation thereof.
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21 87780
w09st~636 4 PCT/SE95/00396
~ no~her aspect of the invention relates to ~ system
for electrophoretic analysis of fluorophore-labelled
substance mixtures, comprising an electrophoresis zone
having at leas~ one lane, and photometric detector mea~s
fixed relative to this lane for detec~ing separated
fluorophore-labelled substances as they pass the detector
means, which system is characterized hy further comprisinq
means for al~ernately emitting to said lane light of
different wavelengths or wavelength bands corresponding to
the excitation wa~eleng~hs of two or more different
fluorophores, and synchronisation means for rela~ing a
de~ected fluorescent light emission to a respective
excitation wavelength.
In a p~eferred embodimen~ the method and system of the
invention are adapted to nucleic acid sequencing. While the
"samples n labelled wi~h di~ferent fluorophores in ~he
present case are prim~rily meant to consist of fragment
populations obtained by one and the same base-specific
sequencing reaction o~ the a~ove-mentioned type on several
different DN~ or RNA fragments to be analysed, the
differently labelled samples in question may, o~ course,
also he two or more diffe~en~ fragment pop~lations obtained
by base-specific sequencing reactions on one and the s~me
DNA or RNA fragment to be analysed. Also so-called fragment
analysis may ad~antageously be per~ormed with the method
and system according to the invention.
In the following the invention will be descri~ed in
more detail wit~ regard to a specific, non-limiting
embodimen~, reference being made to the accompanying
drawing~, wherein
Fig. 1 is a schematic illustration of a prior art
system for nucleic acid sequencing,
Fig. 2 is a schematic illustration of an embodiment of
a system according to t~e invention, and
Fig. 3 is a schematic diagram which shows the filter
characteristic of a filter for filtering ou~ fluorescent
light from the de~ec~or in the system of Fig. 2.
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21 87780
WO 9512863G PCI'/SE95/0039
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The prior art analytical system shown in Fig. 1 is of
the type mentioned above with A.L.F. DNA Sequencer~
(Pharmacia BioteCh AB, Uppsala, Sweden) as a commercial
example and intended for a fluorescent label. It operates
~ith a fixed laser beam and a number of ~ixed detectors
arranged across the separation gel. To ~his end the
illustrated system has An electrophoresis ~nit 1 having a
gel 2 with a num~er of lanes, here as example four lanes,
each represented by a detection zone 3 monitored by a
respective photodetec~or 4 (only one shown) in the form of
a photodiode. A laser S introduces a light beam 6 of
wavelength ~1 into the ~el plane 2 ~or excitation of ~he
fluorescent label of the DNA fragments ~ha~ pass the
detec~ion zones ~. Emitted fluorescence is detected
perpendicularly to the gel plane by the photodiodes 4. An
optical filter 7 prevents the excitation light from
reaching the detectors 4 while simultaneously ~llowing
emitted fluorescence to pass through. ~he detectors 4 ar-e
meant to be co~nected to a computer-~ased da~a collecting
system so tha~ the DNA sequence determined in each case may
be presen~ed in char~ form.
In Fig. 2, the system of Fig. 1 has been modified
according ~o the invention for the use of two different
fluorophores, the capacity of the system therehy being
doubled. In comparison with the system in Fig. 1 this
modified system has in addition to the laser 5 an extra
laser 8 which emits light a~ another waveleng~h ~2 adap~ed
to excite the additional fluorophore.
Further, a rotating chopper di~ 9, or "chopper", is
dis~osed in the light path of the beams ~rom the two lasers
5, 8, so that the beams are chopped up by alternately being
allowed to pass through the chopper disc. A dichroic mirror
10 arranged to re~lect the beam from the laser 5 of
wavelength ~1 and transmi~ the beam fro~ the laser 8 o~
~avelength ~2 is placed between the chopper disc 9 and the
elPctrophoretic apparatus 1. Hereby the two laser beams are
combined and aligned to the same ~hysical path, i.e. the
same optical axis, fo~ a co~mon incidence into the gel ~
ll l9 i9~ T~IE 15 :12 F.~ .3~2 1.~2.3 RIr)t-l~TT ~ M~BEE @1~
WO 95/28~i36 2 1 8 7 7 8 0 PCl'lSE95tO039G
For the above-mentione~ automatic data collecting syste~ to
know when the respective lasers 5 and 8 ~re active, the
detector electronics is synchronized with the laser
chopping ~hrou~h sync pulses from the chopper disc 9.
The chopping or pulsing of the laser beams may, of
course, be accomplished in other ways. For example, the
rotating disc and the dichroic mirrors may be combined to a
single component that provides for both chopping and be~n
combination by replacing the rotating disc ~y a mechanical
component o~ equivalent construction where mirrors have
been substituted fo~ che solid parts of the chopper dis~.
Further, i~ order to avoid movin~ mechanics, acous~o-
optical or electro-optical modul~tors may ~e used instead
of the-~otating disc. It is, of course, also possible to
lS accomplish an analogous function wi~h solid state
components.
In the modified sys~em according to ~ig. 2, the
optical filter 7 in Fig. 1 is replaced by a filter 11
capable of blocking both laser wavelengths ~1 and ~2
instead of only the wavelen~th ~1 One embodiment o~ such a
filter consists of a combination of a glass ~ype absorption
filter and an interference filter ~thin film ~ilter~, the
absorption filter functioning as a s~bs~rate for the
interference fil~er An example of a filter characteristic
2S obtained hereby ~transmission versus wavelength) is shown
in ~ig. 3. As a speci~ic example, such a filter with
damping at las~r wavelengths 488 nm ~argon laser) and 633
nm (He-Ne-laser or l~ser diode) h~s been obtained from
Omega Optical Inc., U.S.A., which on behalf of applicant
deposited an ~B ~rejection band) type interference filter
- on an OG 515 ~ilter glass with high damping for 488 nm to
also ~btain a high damping for a band around 633 nm.
Exemplary of a suitable fluorophore combi~a~i~n for these
laser wavelengths is fluo~escein, which exhibits.a ligh~
absorption maximum at a wavelength of about 490 nm and a
light emission maxi~um at a wavelength of about 530 nm, and
CyS (Biological Detection Systems, Ino., Pittsburgh, PA,
U.S.A.), which is a carbocyanine-based dye containlng a
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WO 95128636 7 PC:~T/sE95too396
reactive succinimidyl ester and which exhibits a light
absorption maxi~um at a wavelength of about 650 ~m and a
light emission maximum at about 670 n~
With ~he analytical system shown in Fig. 2, a doubled
capacity is thus oh~ained in comparison with the prior art
syste~ according to Fig. 1. The capacity may, o~ course, be
fu~ther increased if three, or optionally eve~ more
lasers~fluorophores are used.
~he invention is, of co~rse, not restricted to the
embodimen~s specifically descri~ed above and shown in the
drawings, but many variations and modi~ications may be made
within the scope of the general inventive concept as
defined in the following claims.
