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Description  |
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BACKGROUND OF THE INVENTION
This invention pertains to apparatus for fluorescence detection in
electrophoresis systems, and particularly to such apparatus for automatic
sequencing of nucleic acids.
The structural analysis of DNA plays an increasingly important role in
modern molecular biology. About 4.times.10.sup.6 bases of DNA have been
sequenced since the introduction of the enzymatic, or dideoxy, method of
rapid sequencing developed by Sanger and his coworkers (Sanger, et al,
Proc. Natn. Acad. Sci. U.S.A. 74,5463-5467 (1977), A. J. H. Smith, Meth.
Enzymol. 65,560-580 (1980)) and the chemical method developed by Maxam and
Gilbert (S. M. Maxam and W. Gilbert, Meth. Enzymol. 65,499-559 (1980)).
Typically, four separate reactions are performed on the particular DNA
segment to be analyzed. In the enzymatic method, these reactions produce
DNA fragments terminating in either adenosine (A), cytosine (C), quanosine
(G), or thymidine (T). In the chemcial method, typically, fragments
terminating in G, G+A, C+T, or C are produced. In both cases the four sets
of reaction products are electrophoresed in adjacent lanes of a
high-resolution polyacrylamide gel. An autoradiographic image of the gel
is produced, and the autoradiogram is examined to determine the relative
lengths of the DNA fragments generated in each of the four reactions. The
DNA sequence is inferred directly from that information.
Both of these techniques are very effective but they are also highly
labor-intensive, relatively expensive, and involve the use of
radioisotopes; values of approximately three to ten thousand bases
sequenced per person-year at a cost of one to five dollars per base are
representative. For these reasons and since much DNA remains to be
sequenced (there are 3.times.10.sup.9 bases in the human genome alone),
there has been much recent activity directed toward development of an
automated and non-isotopic method of DNA sequence analysis.
One of the more successful attempts has been carried out by Lloyd Smith and
coworkers in the laboratory of Leroy Hood at California Institute of
Technology (see Bio/Technology, Vol. 3, May 1985). In that approach, four
fluorescent dyes with different colored tags are used instead of
radioactive labels. Each color corresponds to a different nucleoside so
that if the samples are co-separated electrophoretically, the "ladder" of
DNA fragments produced during sequencing is segregated into fluorescent
multi-colored rungs, each color corresponding to one of the bases A, G, C,
or T. As the length of the column is scanned by a fluorescence sensor, the
order of the colored bands corresponds to the specific gene sequence. The
specific fluorophores selected by Smith, et al, were fluorescein
isothiocyanate (FITC) with an emission peak at 520 nm,
4-chloro-7-nitrobenzo-2-oxa-1-diazole (NBD chloride) emitting at 550 nm,
tetramethylrhodamine isothiocyanate (TMRITC) emitting at 580 nm, and Texas
Red emitting at 610 nm. These emission peaks make the dyes look green,
green-yellow, orange-red, and red, respectively.
The specific method used by Smith, et al, was an adaptation of the dideoxy
(enzymatic) method of Sanger, which generally involves cloning the gene of
interest in the single-stranded DNA phage M13. A primer sequence
complementary to the phase sequence adjacent to the cloned gene is used to
initiate a DNA synthesis that copies a portion of the gene. In the scheme
devised by the Cal Tech group, a single molecule of fluorescent label is
linked to each primer. The cloned genes and primers are then placed in
four separate DNA synthesis reaction mixtures, each containing all four
nucleosides. A small amount of a dideoxy form of a nucleoside, ddATP,
ddCTP, ddGTP, or ddTTP is added to each batch. When a dideoxy triphosphate
randomly replaces a conventional nucleoside and is incorporated into the
developing DNA strand in the synthesis reaction, the nascent DNA copy
immediately stops growing. As a result, all strands in the batch with
ddATP terminate at a location where adenosine appears in the sequence.
Site-specific stops at the C, G, and T positions occur in the other three
reaction batches as well.
To distinguish the four bases, a different fluorescent label is used in
each reaction mixture. To achieve that, all DNA copies that end in A are
labeled with the green-colored FITC; those terminating in C are labeled
with the green-yellow NBD chloride, those terminating in G are labeled
with the orange-red TMRITC tag, and copies terminating in T are labeled
with Texas Red.
In the Cal Tech automated system, aliquots from all four reaction mixtures
are electrophoresed through a single polyacrylamide tube gel that sorts
the various length fragments by size. Positioned at the bottom of the
electrophoresis gel is an argon ion laser that sequentially illuminates
each band as it migrates through the gel. When excited by laser light the
fluorophores emit at their characteristic wavelength, and the emissions
are detected and identified by a sensor. The sequence of emission colors
is converted by the machine into a nucleotide sequence.
Although the Cal Tech group has been able to automate the sequencing
process, bringing what used to require four lanes into one lane, and have
substantially eliminated problems with mobility differences between bases,
significant problems still remain to be solved. First, the detection
system design is less than optimal in sensitivity. Second, and more
importantly, the apparatus can handle only one column at a time, whereas
when using autoradiographs many lanes on a slab gel can be sequenced at
the same time.
What is needed is a high throughput, real time, fluorescence detection
apparatus that can perform nucleic acid sequencing on many lanes of a gel
simultaneously. Furthermore, the apparatus should have a high sensitivity
but the detection system should not require frequent attention by trained
personnel.
SUMMARY OF THE INVENTION
A real-time, automated, nucleic acid sequencing apparatus is provided that
offers high speed, definitive sequencing on many samples at the same time.
The apparatus permits more than one clone to be sequenced at a time, thus
vastly decreasing the time required to sequence longer fragments and
reducing sequencing costs accordingly. Furthermore, the detection system
at the heart of the apparatus is designed to eliminate costly alignment
procedures and, concomitantly, to eliminate the need for constant
attention by highly trained personnel.
In its broadest sense, and in accordance with preferred embodiments of the
invention, an apparatus is provided for detecting electromagnetic
radiation from a plurality of lanes in an electrophoresis system wherein
the plurality of lanes are arranged in a planar array. The apparatus
includes an optical system for detecting the radiation at a plurality of
wavelengths emanating from the plurality of lanes. To accomplish that
function, the optical system is made up of a collection for focussing the
radiation, a filter for selectively transmitting the plurality of
wavelengths received from the collection element, and a detection system
for measuring intensity of the radiation received from the filter means.
The apparatus also has a translational stage for mounting the optical
system and for moving the optical system in a direction parallel to the
planar array of electrophoresis lanes in order to move the collection
element to receive radiation from the lanes, one lane at a time. The
apparatus also includes a computer system for controlling the filter and
the stage. The computer system also receives intensity data from the
detector and correlates the intensity data with the corresponding lane and
corresponding wavelengths transmitted by the filter in real time.
To use the apparatus for sequencing nucleic acids, the nucleic acids are
prepared according to the enzymatic method and labeled as per Smith, et
al. Nucleic acids are then electrophoresed in the apparatus and the
computer is used to sort the intensity data into time and wavelength
information for each lane, thereby arriving at a sequence for each lane.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates an electrophoresis apparatus and enclosure according to
the invention.
FIG. 2A shows a schematic representation of the invention in a horizontal
cut through the optical system.
FIG. 2B is a schematic representation of the invention in a vertical cut
through the optical system.
FIGS. 3A and 3B illustrate the construction of wells in the electrophoresis
apparatus.
FIG. 4A illustrates a focussing telescope according to the invention.
FIGS. 4B and 4C show details of lenses used in the focussing telescope.
FIG. 5A illustrates a collector lens and Fabry lens group according to the
invention.
FIGS. 5B, 5C and 5D show details of the lenses illustrated in FIG. 5A.
FIG. 6 is a table showing detailed dimensions for the lenses and optical
system.
FIGS. 7A-7D illustrate the mechanical configuration of the optical system
in four views.
FIG. 7E is a table of dimensions for the mechanical configuration shown in
FIGS. 7A-7D.
FIG. 8 is a schematic illustration of a computer system according to the
invention.
FIG. 9 is a flow chart illustrating the computer logic to arrive at a
nucleic acid sequence from the electrophoresis and timing data.
FIGS. 10A-10D are plots of intensity data taken with the apparatus of the
invention for each of four wavelengths.
FIG. 11 is a superposition of the plots of FIGS. 10A-10D.
DETAILED DESCRIPTION OF THE INVENTION
Shown in FIG. 1 and FIGS. 2A and 2B is a schematic representation of a
preferred embodiment of the invention which is made up of an
electrophoresis apparatus 100 having a polyacrylamide slab gel 104, a
laser 300 for providing an intense source of electromagnetic radiation,
and an optical detection system 200 that directs the laser light onto the
slab gel and detects the fluorescence of dyes attached to the materials
being electrophoresed in the gel. During operation, the electrophoresis
apparatus 100, the laser 300 and the optical detection system are enclosed
in a light-tight environment, such as a box (not shown).
The electrophoresis apparatus 100 has a transparent front panel 106 and a
transparent back panel 105 with gel 104 sandwiched therebetween. Also
included is lower buffer chamber 101 and an upper buffer chamber 109 which
are designed to provide communication between the gel 104 and the buffer
solutions contained in the two chambers. The buffer chambers and the
plates 105 and 106 are held in a fixed relationship to each other by a
frame 107 having clamps 113 and 115. The electrophoresis apparatus also
includes a beam dump 111 for stopping direct light from laser 300 from
traversing a path beyond the electrophoresis apparatus. In addition, each
buffer chamber includes a banana plut (112 and 114) for connecting a
voltage source across the gel (typically 1000-1500 volts). In one
preferred mode, the gel 104 is 8% by weight of acrylamide monomer and is
prepared according to techniques well known in the art (see Maniatis, et
al, Molecular Cloning, page 478). In order to introduce samples to be
sequenced into the electrophoresis apparatus, a series of wells such as 91
through 97 is created at the top of the gel as illustrated schematically
in FIGS. 3A and 3B. First, spacers 98 and 99 are placed on plate 106 to
define the desired thickness of the gel 104 and plate 105 is affixed to
plate 106 creating a cavity therebetween. Typical materials for spacer 98
and 99 are nylon or Delrin.TM. strips. The gel is then poured into the
cavity, the comb is placed on top, and the gel is allowed to solidify. The
comb 102 is then removed, leaving the series of wells at the top of the
gel. Samples can then be injected directly into the wells. Because the
samples are suitably segregated by the walls of the wells, and because the
diffusion coefficient is very low in the gel, well defined lanes such as
lanes 110 and 108 illustrated in FIG. 2A appear during electrophoresis,
one lane for each well. Although only seven lanes have been shown, in the
preferred mode sixteen are typically used for a 10-inch wide gel. The
depth and width of the wells are both typically in the range of 0.5 cm to
1 cm, and spacers 98 and 99 are usually about 1 cm wide. Gel thickness is
typically in the range of 0.5 mm to 1.0 mm, and the preferred height of
the gel is 15.75 inches. The plates 105 and 106 are typically 3.0 mm to
5.0 mm thick and are constructed of a clear, non-fluorescent material such
as pyrex borosilicate glass. A particularly suitable material is a Schott
glass known in the art by the tradename TEMPAX.TM. which has a low
fluorescence, an index of refraction close to that of the gel (n=1.47),
and which can withstand high temperatures and thermal shock. (TEMPAX.TM.
is produced by the Schott Optical Company.) It should also be noted that
there is a cut-out 90 in the top of plate 106 in order for the buffer
solution in buffer chamber 109 to communicate with the gel during
electrophoresis. In another preferred mode, the top 2 cm to 4 cm of the
gel immediately below the comb is poured with 5% by weight of acrylamide
monomer, with the balance of the gel being 8% acrylamide monomer. This
combination of concentrations appears to enhance the speed and the amount
of the sample materials which enter the gel initially. Yet another
approach is to mix agarose with the acrylamide gel in the top 2 cm to 4
cm.
As illustrated in FIG. 1, the electrophoresis apparatus is designed to fit
onto a shelf 117 of an enclosure 370. The shelf is configured to slide
into the enclosure 370 bringing the plate 106 into contact with a heat
transfer plate 119, in order to equalize and dissipate heat generated
during the electrophoresis process. A slot 123 in plate 119, permits light
to pass through the plate 119 for causing fluorescence of the various dyes
and for permitting detection of that fluorescence. The enclosure 370
includes a base plate 241 that is located above the shelf 117 so that the
buffer chamber 101 can slide thereunder. Structural integrity of the
enclosure is provided by liner 121 that fits around the perimeter of the
electrophoresis apparatus 100 when it is in place on the shelf.
The optical detection system 200 is attached to a plate 239 which rides on
a translational stage 231 attached to base plate 241 via a guide rail 233.
The stage is translated horizontally back and forth by a screw 252 driven
by a DC motor 237, and position of the stage is monitored by a shaft
encoder 238. Optical sensors 242 and 244 (not shown) are used to monitor
end of travel for the stage. Although there are several different
translational stages that can be used, a particularly useful one because
of its size and smooth operation is bearing track assembly part number RSR
5WUU, available from THK, Co. Ltd., located in Elk Grove Village, Ill.
As indicated earlier, the source of electromagnetic radiation to cause
fluorescence is laser 300. In the preferred embodiment, an Argon ion laser
is used which is operated in a mode providing two lines, one at 488.0
nanometers and one at 514.5 nanometers, with both lines having about equal
power, about 7 mW for each line, and a total power of 20 mW. Unlike the
optical detection system 200, laser 300 is held fixed. Light 250 emanating
from the laser first impinges on a 45.degree. mirror 253, then on a
45.degree. mirror 251 so that it again becomes parallel to the direction
of translation of the optical detection system. Thus, motion of the
optical detection system does not effect the incidence angle of the light
into the optical detection system.
The optical detection system 200 is made up of a focussing telescope 260
having lenses 257 and 259 to decrease the size of the incident beam and to
focus the light onto the gel lanes. The path of the light from the
focussing telescope is diverted toward the gel by a Brewster angle mirror
255 through a window 125, the window and the mirror being fixed relative
to the stage 231. The incidence angle at mirror 255 was chosen at the
Brewster angle in order to minimize the polarized laser light scatter that
interferes with fluorescence detection. The individual lanes are accessed
by moving the stage back and forth along guide rail 233. As light from
laser 300 strikes a dye in a lane, the dye fluoresces as indicated for
lane 108. A collector lens 221 collects a portion of the fluorescent light
and directs it toward a filter wheel 223 which is made up of four color
filters arranged as quadrants of the wheel. As the filter wheel is
rotated, the pass bands of the filters selectively transmit particular
wavelengths of the fluorescent light, one at a time, to a Fabry lens group
226, made up of lenses 225 and 227.
The Fabry group is located at the focus of the collector lens 221 and is
configured to image the collector lens onto the active area of a side-on
photomultiplier tube 229, such as an R928 available from Hamamatsu, rather
than to image the lanes themselves. Generally, the photocathode output
signal varies with the location of the light signal on the active area. By
imaging the collector lens instead of the lanes, the location of the light
on the photomultiplier is stable even when the location of illumination on
the gel is changed. Hence, one does not see spurious variations in the
photomultiplier output signal if the illumination location on the gel
should be changed for some reason e.g. from imperfect alignment. Another
purpose of using a pair of lenses for the Fabry group is to further
desensitize the system to aberration components arising from alignment
errors. In order to focus the collector lens onto the photomultiplier tube
within a reasonable distance, the Fabry group must have a relatively high
power. To use one lens to achieve high power results in field curvature
and geometric distortion which, if not corrected, would cause the image on
the active surface to move in and out of focus if the area of illumination
varies laterally during a measurement sweep e.g. if it were not properly
aligned or if the gel plate should have ripples. Hence, a two lens group
is used, the Fabry lens 227 being an aspheric, so that together field
curvature and geometric distortion are removed. Hence, even with
misalignment or ripples, the image on the photomultiplier is exceedingly
stable and does not change in size.
The particular color filters used with system vary with the selection of
dyes. In the preferred mode, each dye is selected from a separate one of
four sets of dyes. For those dyes, the filters chosen have nominal center
wavelengths of 540 nm, 560 nm, 580 nm and 610 nm, all with a band pass of
10 nm (as measured at the 50% transmission point). Such filters are
available, for example, from OMEGA Optical of Battlesborough, VT. The four
sets of dyes are as follows. Set I consists of fluorescein
mono-derivatized with a linking functionality at either the 5 or 6 carbon
position (as determined by the Color Index numbering system). Illustrative
examples of set I members include
fluorescein-5-isothiocyanate,
fluorescein-6-isothiocyanate (the -5- and -6-forms being referred to
collectively as FITC),
fluorescein-5-succinimidylcarboxylate,
fluorescein-6-succinimidylcarboxylate,
fluorescein-6-succinimidlylcarboxylate,
fluorescein-5-iodoacetamide,
fluorescein-6-iodoacetamide, fluorescein-5-maleimide, and
fluorescein-6-maleimide.
These examples of members of set I are available commerically, e.g.
Molecular Probes, Inc. (Junction City, OR), or can be synthesized using
standard techniques. Set II consists of
2',7'-dimethoxy-4',5'-dichlorofluorescein mono-derivatized with a linking
functionality at the 5 or 6 carbon position (the carbons being identified
in accordance with the Color Index numbering system). Set II members can
be obtained by standard modifications of
2,7-dimethoxy-4,5-dichloro-9-(2',4'-dicarboxylphenyl)-6-hydroxy-3H-xanthen
-3-one and
2,7-dimethoxy-4,5-dichloro-9-(2',5'-dicarboxyphenyl)-6-hydroxy-3H-xanthen-
3-one (IUPAC notation) disclosed in U.S. Pat. No. 4,318,846. For example,
the 4' and 5' carboxys of these compounds can be condensed with
N-hydroxysuccinimide using dicyclohexylcarbodiimide to form an
amine-selective linking functionality, e.g. as illustrated by examples 6
and 8 of the above-referenced patent (Col. 28-29). Kasai et al., Anal.
Chem., Vol. 47, pgs. 34-37 (1975), discloses the basic technique for such
condensations. Examples of members of Set II dyes are
2',7'-dimethoxy-4',5'-dichlorofluorescein-5-succinimidylcarboxylate and
2',7'-dimethoxy-4',5'-dichlorofluorescein-6-succinimidylcarboxylate (the
-5- and -6- forms being referred to collectively as DDFCS). Set III
consists of tetramethylrhodamine monoderivatized with a linking
functionality at either the 5 or 6 carbon position. Illustrative examples
of set III members include
tetramethylrhodamine-5-isothiocyanate,
tetramethylrhodamine-6-isothiocyanate (the -5- and -6- forms being referred
to collectively as TMRITC),
tetramethylrhodamine-5-iodoacetamide,
tetramethylrhodamine-6-iodoacetamide,
tetramethylrhodamine-5-succinimidylcarboxylate,
tetramethylrhodamine-6-succinimidylcarboxylate,
tetramethylrhodamine-5-maleimide, and
tetramethylrhodamine-6-maleimide.
These exemplary dyes are available commercially, e.g. Molecular Probes,
Inc., or can be synthesized using standard techniques. Set IV consits of
rhodamine X derivatives having a disubstituted phenyl attached to the
molecule's oxygen heterocycle, one of the substituents being a linking
functionality attached to the 4' or 5' carbon (IUPAC numbering) of the
phenyl, and the other being an acidic anionic group attached to the 2'
carbon. Illustrative examples of set IV members include Texas Red
(trademane of Molecular Probes, Inc.), rhodamine X-5-isothiocyanate,
rhodamine X-6-isothiocyanate, rhodamine X-5-iodoacetamide, rhodamine
X-6-iodoacetamide, rhodamine X-5-succinimidylcarboxylate, rhodamine
X-6-succinimidylcarboxylate, rhodamine X-5-maleimide, and rhodamine
X-6-maleimide. Most of these exemplary dyes are availabe commercially,
e.g. Molecular Probes, Inc., or can be synthesized using standard
techniques. For example, in the case of Texas Red it can be synthesized
according to the procedure disclosed in Titus et al., "Texas Red, a
Hydrophilic, Red-Emitting Fluorophore for Use with Fluorescein in Dual
Parameter Flow Microfluorometric and Fluorescence Microscopic Studies," J.
Immunological. Methods, Vol. 50, pgs. 193-204 (1982).
The details of the optics assemblies making up the optical detection system
are provided in FIGS. 4A through 4C and FIGS. 5A through 5C, which show
the relative positions of the various lens elements and the specifications
for the lenses. The lens dimensions are provided in a Table in FIG. 6.
Shown in FIGS. 7A through 7D are the mechanical details of the optical
detection system 200. A housing 701 holds the focussing telescope 260, and
the Brewster angle mirror 255, and contains the collector lens 221 and the
slot 125. Light from the collector lens is directed down a tube 703 to the
filter wheel 223 which is contained in a wheel housing 705. The Fabry
group 226 is contained in a housing 707 adjacent the photomultiplier tube
229. A power supply 709 for the photomultiplier tube is located at one end
of the tube and a circuit board 711 provides the electronic controls for
the photomultiplier tube and the filter wheel. A stepper motor 713 is used
to drive the filter wheel via a drive belt 716 entrained over pulleys 715
and 717, pulley 715 being used to drive shaft 714 which is connected to
the filter wheel. An index wheel 719 is used to encode the filter wheel
position via an optical encoder 721. FIG. 7E provides a Table having the
relevant mechanical dimensions of the optical detection system 200.
Shown in FIG. 8 is a schematic representation of a computer system 400 for
controlling operation of the optical detection system and for performing
the required data analysis. The system 400 includes a central computer 801
such as an IBM pc which is coupled to a process control computer 802 such
as the Z80 based microcomputer used by Applied Biosystems of Foster City,
Calif., in their 381 DNA synthesizer. The process control computer 802 is
coupled to an interface 803 for communicating with the elements of the
optical system which require monitoring of control. Those elements include
the stepper motor 713, the encoder 721 for the filter wheel, the output
from the photomultiplier tube 229, the drive motor 237 for the stage 231,
the shaft encoder 238 and the limit switches 242 and 244 for stage 231.
Analysis of intensity information obtained during the sequencing process
is performed by the central computer 801.
METHOD OF OPERATION
First, samples to be sequenced are prepared according to the Sanger
enzymatic method described earlier in the Background of the Invention. The
buffer chambers are filled with an appropriate buffer and the
electrophoresis apparatus is loaded onto the sliding shelf 117. The gel is
then pre-electrophoresed for about one-half hour to remove any fluorescent
impurities. The samples are then injected into the wells at the top of the
gel and a high voltage is connected between the buffer chambers to start
the electrophoresis process.
To start the detection portion of the operation, the laser is turned on and
the computer 801 indexes the filter wheel to a first filter and causes the
stage 231 to move the optical detection system across the 16 vertical
lanes of the gel 104. Each lane corresponds to a separate sequencing
operation using all four dyes, in the same manner as the single column
used by Lloyd Smith, et al. The stage is moved across the gel in about 1
second, and 192 light intensity measurements are made during the scan.
Each measurement of the light intensity is an average taken over a
distance of approximately 0.8 mm and over a time of 0.005 seconds, these
measurements hereinafter referred to as channels. At the end of the first
scan, the computer causes a second filter to be rotated into position
(about 0.5 sec.) in the path of the light being detected. The direction of
the stage 231 is then reversed and the optical detection system 200
resumes detection on this reverse scan, again measuring 192 channels. This
process is then repeated for the third and fourth filters.
In the preferred mode, each lane in the gel is designed to be about 4 mm
wide, with each lane being separated by about 4 mm, as determined by the
comb design when preparing the gel. With this design, each lane spans 5
channels. To increase signal to noise ratio, intensity data associated
with the channels corresponding to a particular lane are summed. The four
passes past a particular lane then provide four color data for that lane
at that time. During each 6 second period (4 filters.times.(1 sec/scan+0.5
sec/filter change), a four color datum point is recorded for each lane.
This four color data can then be analyzed using multicomponent analysis to
provide the desired sequence information as will be described later. A
flow chart illustrating the above method is shown in FIG. 9.
Compared to the speed at which the sample DNA moves down a lane, the six
second time required to obtain the four color data is nearly simultaneous.
In effect, a four wavelength emission spectrum is measured for each time
unit during the electrophoresis at a fixed distance down the gel. By
multicomponent analysis, the data for these four wavelengths yields
information about the four relative concentrations of the dye-labeled DNA
pieces moving down a lane. Peak concentrations of a particular dye label
then correspond to a particular base in the DNA sequence. The four plots
of concentration versus time, are overlayed and the peaks determined, the
matching of the peaks with the DNA bases yeilding the sequence.
Analytically, the multicomponent analysis amounts to solving four equations
in four unknowns. The general formula for the analysis is:
##EQU1##
where Aij is the standard fluorescence of dye j at filter wavelength i,
and Cj is the concentration of dye j, Fi is the fluorescence intensity
measured through filter i. Solving the above equations yields a unique set
of concentrations at each point in time. For completely automated
analysis, standard noise reduction and peak finding algorithms can be used
to call the sequence, or a trained individual can inspect the set of
concentrations to arrive at a sequence. For convenience during operation,
the four components of concentration are plotted simultaneously on a color
monitor on computer system 801 while the gel is being scanned during
electrophoresis.
The standard fluorescence coefficients Cij are determined by measuring the
fluorescence with each filter when a known unit concentration of each dye
is present in the gel, one dye at a time. For such measurements, one can
use a single band of dye-labelled primer. Generally these coefficients are
a function of the laser wavelength and intensity, the gel characteristics,
the optical filter, and photomultiplier response.
UTILITY OF THE INVENTION
FIGS. 10 and 11 illustrate the result of a DNA sequencing run made with the
invention on bases 120 through 190 of the cloning vector mp8 of the
bacteriohpage M13 using DNA polymerase, Klenow fragment. The results shown
correspond to one lane of the sixteen, the other lanes providing the same
kind of information for the particular samples sequenced there. The
specific dyes corresponding to this run are fluorescein-5-isothiocyanate;
2',7'-dimethoxy-4'-5'-dichlorofluorescein;
tetramethylrhodamine-5-isothiocyanate, and Texas Red. FIG. 10 shows the
relative intensities recorded for each base during sequencing run as a
function of time. FIG. 11 shows the four different intensities plotted one
on top of the other, which makes the sequence somewhat easier to call,
since the relationship between peaks becomes more apparent. This
relationship is even clearer when using a color monitor on the computer
801 so that the intensity for each base is plotted in a different color,
one on top of the other. As indicated, in most instances, the call is
unequivocal. However, certain low signal peaks do occur. For example, see
bases 174 and 181. In an unknown sample, such bases in the sequence might
be overlooked. However, as is typical even in radiographic sequencing,
those skilled in the art often do more than one sequencing run, typically
with other sequencing emzymes to eliminate ambiguous base calls caused by
the particular chemistry chosen. For example, it is well known to those
skilled in the art that the cytosine sequence has large variations in
amplitude. Hence, one typically also performs a sequencing run on the
complementary strand to check the results of the cytosine call. Another
approach is to use a different enzyme completely, for example, reverse
transcriptase.
Hence, the automated method and apparatus of the invention can be used to
provide high speed, definitive sequencing analysis on many samples at
once. This permits more than one clone to be sequenced at a time, vastly
decreasing the time required to sequence larger fragments, and reducing
costs accordingly. In addition, as designed, once the system is
constructed, little if any further alignment by trained personnel is
required at the operating end. Further, as compared to the radiographic
method, the real time sequencing method has many other benefits. First,
sequencing is performed with all four bases in one lane, rather than four
separate lanes, thus avoiding mobility variation problems between lanes.
Second, no radioactive materials are required, and the materils that are
used have a considerably longer shelf life th | | |
