Multiple tag labeling method for DNA sequencing

A DNA sequencing method described which uses single lane or channel electrophoresis. Sequencing fragments are separated in said lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radio-isotope labels.

BRIEF DESCRIPTION OF THE INVENTION 
This invention relates generally to a multiple tag labeling method for DNA 
sequencing and more specifically to a capillary array electrophoresis 
apparatus that uses a two-channel fluorescence detection system employing 
multiple dye labeling. 
BACKGROUND OF THE INVENTION 
Current automated DNA sequencing methods use fluorescence detection of 
labeled DNA sequencing fragments. One method is to form four sets of DNA 
sequencing fragments terminating in G, A, T or C where each set is labeled 
with the same fluorophore, and then mn the sequencing fragment sets in 
adjacent lanes in a slab gel electrophoresis apparatus. Various apparatus 
have been suggested for scanning the gel to monitor said fragments as or 
after they move through the gel. In copending application Ser. No. 
07/531,900 filed Jun. 1, 1990, U.S. Pat. No. 5,091,652, incorporated 
herein by reference, there is described a laser-excited confocal 
fluorescence gel scanner which provides enhanced detection of 
fluorescently labeled DNA sequencing fragments separated on a slab gel. 
The detection system uses an epi-illumination format where the laser power 
is focused on the sample by a microscope objective followed by confocal 
detection. However, lane-to-lane variations in the migration velocity of 
the DNA fragments make it difficult to deduce the correct alignment of the 
bands in the four sequencing lanes. The throughput is reduced because of 
the need for running four lanes to detect the four sets of DNA sequencing 
fragments terminating in G, A, T and C. 
A solution proposed to overcome these drawbacks is to label each sequencing 
fragment set with a different fluorophore, and then to perform the 
electrophoresis operation in only one lane. This requires a multi-color 
detection system and dyes that do not alter the mobility of the fragments 
relative to one another. A method and apparatus for sequentially scanning 
four colors in multiple lanes in a slab gel is described in U.S. Pat. No. 
4,811,218 and by Smith, et al., Nature 321,674 (1986). An alternative 
method using four different dye labeled dideoxy terminators along with 
two-color detection has been described in U.S. Pat. No. 4,833,332 and by 
Probet et al. Science 238, 336 (1987). 
Capillary electrophoresis is emerging as a high-speed DNA sequencing 
method. In copending application Ser. No. 07/840,501 filed Feb. 24, 1992, 
U.S. Pat. No. 5,274,240, there is described an automated sequencing 
apparatus which employs an epi-illumination format where a laser is 
focused to a small volume by a microscope objective and fluorescence 
emitted from said volume is gathered by the same objective followed by 
confocal detection. An array of side-by-side parallel capillaries is 
sequentially and periodically moved past the focal volume or vice versa to 
cause and detect fluorescence in labeled DNA sequencing fragments within 
the capillaries. The capillary array electrophoresis scanner is described 
in said application for use in a one-color, single-channel detection 
system where each set of DNA sequencing fragments is separated in a 
separate capillary or in a four-color, four-channel detection system where 
each set of fragments is labeled with a different fluorophore for 
separation and detection in only one capillary. 
It is very difficult, in practice, to find four dyes of exactly the same 
electrophoretic shift. Therefore, it becomes necessary to perform 
complicated shift corrections before the sequence can be read. Four-color 
detection has been described in connection with capillary electrophoresis 
by Smith and coworkers using a simultaneous four-color detection system 
where the signal is split between each of four channels (Nucleic Acids 
Research 18, 4417-4421 (1990)). This is satisfactory, but the 
signal-to-noise ratio is reduced because the signal is split between four 
different channels, and the problem of maintaining equal band shifts for 
each of the sets of labeled sequencing fragments using different dyes must 
still be resolved. 
OBJECTS AND SUMMARY OF THE INVENTION 
It is an object of this invention to provide an improved DNA sequencing 
apparatus and method. 
It is another object of this invention to provide a DNA sequencing method 
in which each set of sequencing fragments is labeled with two different 
fluorescent dyes. 
It is yet another object of this invention to provide a DNA sequencing 
method in which the sets of sequencing fragments are labeled with 
fluorescent dyes having substantially the same mobility shift. 
It is another object of the invention to provide a sequencing method in 
which the sequencing fragments are labeled with different fluorescent dyes 
and the ratio of the fluorescent signals in two different detection 
wavelength regions is employed to detect the fragments. 
The foregoing and other objects of the invention are achieved by a 
multi-color electrophoresis scanning apparatus which employs labeling 
selected DNA sequencing fragments with different mole fractions of 
fluorophores, electrophoresing the labeled sequencing fragments in a 
single lane to cause separation by sizes and determining the position of 
said fragments in the overall sequence by detecting the ratio of intensity 
of fluoresence at two detection wavelengths from said labeled fragments.

DETAILED DESCRIPTION OF DRAWINGS 
A suitable, two-color capillary array electrophoresis scanner for detecting 
DNA sequencing is shown in FIG. 1. It consists of two confocal detection 
channels that are coupled into the optical system using a dichroic 
beamsplitter. One channel detects the yellow-green emission from labeled 
fragments, and the other detects the red emission from labeled fragments. 
The DNA fragments are labeled with only two dyes which have been selected 
so that they have the same mobility shift during electrophoresis. In one 
example, the fluorescently labeled primers used in the production of the 
Sanger sequencing fragments were supplied by Applied Biosystems Inc. 
(Foster City, Calif.). The 5'-end of these primers are covalently attached 
to one of the two different fluorophores (FAM or JOE) which have similar 
absorption spectra and different emission spectra. The exact structure of 
these dyes is proprietary. However, the dyes fluorescein and NBD (Smith, 
et al., Nature, 321,674, 1986) have optical properties that are similar to 
those of FAM and JOE, respectively. Fragments labeled with JOE and FAM had 
substantially the same mobility shift in the capillary electrophoresis 
separation media. 
The two-color capillary array electrophoresis scanner is shown in detail in 
FIG. 1. Light (488 nm; 6 mW) from an argon ion laser (Spectra-Physics 
2020, Mountain View, Calif.), not shown, is reflected off a long-pass 
dichroic beamsplitter 11 (96% reflection for s-polarization at 488 nm, 
Omega Optical 480 DM, Brattleboro, Vt.) and directed through a 
32.times.0.40 N.A. infinite conjugate objective 12 (Carl Zeiss LD 
Plan-Achromat 440850, Thornwood, N.Y.). The input beam diameter, 5 mm, is 
selected to give a 9 .mu.m diameter spot in a given capillary. The 
micrometer adjustment 13 of the objective z-position is used to center the 
focused beam in the capillary. The fluorescence emission induced in the 
fluorophore by the laser beam is collected by the objective and passed 
back through the first beamsplitter (.about.92% transmission) to a second 
beamsplitter 14 (Omega Optical 565 DRLP) which separates the JOE and FAM 
emissions (fluorescence emission peaks at 557 nm and 530 nm, 
respectively). The two resulting beams are separately focused with 100 mm 
focal length achromat lens 17, 18 (Melles-Griot, Irvine, Calif.) through 
400 .mu.m diameter spatial filters 21, 22 (Melles-Griot) to effect 
confocal detection of the fluorescence emission. A bandpass discrimination 
filter with a transmission window of 525.+-.5 nm (Omega Optical, 525ODF10) 
and 488 nm rejection band filter (Omega Optical, 488 RB) shown at 23, are 
placed in front of the photomultiplier 26 (RCA 31034A) dedicated to FAM 
detection while a bandpass discrimination filter, shown at 24, with a 
transmission window of 590.+-.17 nm (Omega Optical, 590DF35) was placed in 
front of the photomultiplier 27 (RCA 31034A) dedicated to JOE detection. 
The outputs of the cooled phototubes 26, 27 are terminated (1 M.OMEGA.), 
amplified and filtered (bandwidth .about.DC to 300 Hz) with a low-pass 
filter-amplifier and digitized with a 12-bit ADC (Metra Byte DASH16-F, 
Taunton, Mass.) in an IBM PS2 computer. A computer-controlled dc servo 
motor-driven translation stage 31 (DCI4000, Franklin, Mass.) with a 6" 
travel and 2-5 .mu.m resolution is used to translate the capillary array 
past the laser beam. Scanning of the capillary array is accomplished with 
periodic sweeps (1.4 s) of the array while sampling data at 1,500 samples 
per second per channel. With a line-scan rate of 2 cm/s, the physical 
dimension of the pixels acquired represent 13.3 .mu.m. The computer is 
used to control the translation stage and to acquire and display images in 
a split screen format for the output of each detector. The fluorescence 
images are displayed in real time in pseudo-color and stored for 
processing. 
Selected mole fractions of each of said sequencing fragment sets are 
synthesized using a primer labeled with a first fluorophore and different 
selected mole fractions of each of said fragment sets are synthesized 
using a primer labeled with a second fluorophore and the combined mole 
fractions are electrophoresed in a single capillary or lane and the 
fluorescence intensity is detected as a function of time as the DNA 
fragments move down the capillary. 
One method for coding the sequencing fragments is binary coding. This is 
shown schematically in FIG. 2. The fragments terminating in A are 
synthesized using a 50/50 mixture of primers, half labeled with the red 
emitting (JOE) and half labeled with the green emitting (FAM) dye, and 
thus, carry the code (1,1). The G-fragments are synthesized using a primer 
that is just labeled with the red dye and carry the code (0,1), the 
T-fragments are synthesized with just the primer labeled with the green 
dye and carry the code (1,0) and the C-fragments are not labeled at all, 
carrying the code (0,0). 
Binary coded DNA sequencing fragments were prepared through the following 
procedure: M13mp18 DNA sequencing fragments were produced using a 
Sequenase 2.0 kit (U.S. Biochemical Corp., Cleveland, Ohio). Commercially 
available FAM and JOE-tagged primers (400 nM, Applied Byosystems, Foster 
City, Calif.) were employed in the primer-template) annealing step. Three 
annealing solutions were prepared: 
1. 4 .mu.l of reaction buffer, 13 .mu.l of M13mp18 single-stranded DNA, and 
3 .mu.l of FAM; 
2. 6 .mu.l of reaction buffer, 20 .mu.l of M13mp18 DNA, 1.5 .mu.l of FAM, 
and 3 .mu.l of JOE; 
3. 6 .mu.l of reaction buffer, 20 .mu.l of M13mp18 DNA, and 4.5 .mu.l of 
JOE. 
The tubes were heated to 65.degree. C. for 3 minutes and then allowed to 
cool to room temperature for 30 minutes. When the temperature of the 
annealing reaction mixtures had dropped below 30.degree. C., 2 .mu.l of 
0.1 M DTT solution, 4 .mu.l of reaction buffer, and 10 .mu.l of ddT 
termination mixture were added in tube 1; 3 .mu.l of DTT solution, 6 .mu.l 
of reaction buffer and 15 .mu.l of ddA termination mixture were added in 
tube 2; and 3 .mu.l of DTT, 6 .mu.l of reaction buffer and 15 .mu.l of ddG 
termination mixture were added in tube 3. Diluted Sequenase 2.0 (4 .mu.l) 
was added in tube 1, and 6 .mu.l of diluted Sequenase were added in tubes 
2 and 3. The mixtures were incubated at 37.degree. C. for 5 minutes. 
Ethanol precipitation was used to terminate the reaction and recover the 
DNA sequencing sample followed by resuspension and pooling in 6 .mu.l of 
80% (v/v) formamide. The sample was heated at 90.degree. C. for 3 minutes 
and then placed on ice until sample injection. 
An example of the sequencing of DNA fragments employing this coding in the 
apparatus of FIG. 1 is shown in FIG. 3 where the outputs for the 
wavelengths in the above examples are overlapped. The sequence can be 
easily read off by examining the ratio of the green to the red signal 
intensity to determine the fragments G, A, T or C. When the red, 
represented by dotted curve, is largest, the fragment terminates in G; 
when the green, represented by the solid curve, is much bigger than the 
red, it is a T; when the red and green are the same, it is an A; and when 
there is a gap, it is a C. 
The advantages of this labeling or coding method are: (1) The instrument 
design is simplified. Since there are only two optical detection channels, 
the optical efficiency is increased, giving a better signal-to-noise 
ratio. (2) With just two carefully selected dyes, there is no mobility 
shift of one set of base fragments relative to the other. This is clearly 
seen in FIG. 3, where the precise registration of the peaks in the red and 
green channels shows that the fragment migrations are essentially 
identical. (3) In the foregoing example, only two dye-labeled primers are 
needed. Thus, the number of labeled primers that must be synthesized is 
reduced. (4) Since the ratio of the signal in the green and red channels 
is used to identify the base, the base calling is not sensitive to changes 
in the optical alignment, laser intensity, or to the amount of the DNA 
fragments that migrate in a particular band. The latter point is very 
important since the termination reaction has different efficiency 
depending on where the termination occurs in the sequence. FIG. 4 is a 
plot of the fluorescence intensity in the green channel divided by that in 
the red channel for approximately 300 bases in an M13mp18 DNA sequencing 
run. The T fragments were labeled solely with FAM, the G fragments were 
labeled solely with JOE, and the A fragments were labeled with FAM and 
JOE. The ratio was calculated based on peak maxima. The diamonds represent 
labeled T fragments, the triangles represent labeled G fragments and the 
dots represent labeled A fragments. It is seen that the ratio provides an 
excellent determination of the identity of the fragments. 
Of course it is to be realized that the labeling of the sets of DNA 
sequencing fragments can also be performed by labeling the dideoxy 
nucleotide terminator used in the sequencing reactions with a fluorescent 
label (Prober et al., Science 238, 336 (1987)) as opposed to labeling the 
primer. In this case, selected mole fractions of each of said sequencing 
fragment sets are synthesized using a terminator labeled with a first 
fluorophore and different selected mole fractions of each of said fragment 
sets are synthesized using a terminator labeled with a second fluorophore 
and the combined mole fractions are electrophoresed in a single lane or 
capillary, and the fluorescence intensity is detected at the 
characteristic emission wavelengths of the first and second fluorophores. 
The ratio of the intensities at the two wavelengths then determines the 
identity and sequence of the DNA. It is obvious that the different coding 
methods developed with labeled primers can also be implemented using 
dye-labeled terminators. 
Of course, a variety of coding algorithms can be used along with this ratio 
detection. For example, some workers might object to the binary coding 
since the C-fragments are not explicitly detected. This can be resolved in 
several ways. 
1. A second sequencing run can be done on the same DNA strand where the 
binary coding is simply permuted. Then A would be (0,0); G would be (0,1); 
T would be (1,0) and C would be (1,1). Since we can run a very large 
number of lanes on the capillaries, determining the sequence twice is not 
a problem. 
2. One could sequence the complementary strand using the binary coding 
algorithm in FIG. 2. The presence of a C on the original strand would now 
be detected as a G on the complementary strand using the (0,1) coding. 
3. Finally, one could use a modified labeling algorithm where all the 
fragments are labeled with a dye, but the relative amounts of the two dyes 
are adjusted to give four distinctive ratios for the green to the red 
channel. As depicted in FIG. 5, this coding would be specified by, for 
example, A (1,0); G (1,1); T (1,2); and C (0,1). In this case, the A 
fragments would only be labeled with the green dye; half of the G 
fragments would be labeled with the green and half with red; 1/3 of the T 
fragments would be labeled with the green dye and 2/3 with the red dye; 
finally, all the C fragments would be labeled with the red dye. 
Since the ratio of the signals is used, three dyes that have the same 
mobility shift effect as one another can be used and various mixtures of 
three dyes can be used to label the primers to produce four sets of DNA 
sequencing fragments. The two-channel detection method with a ratio read 
out would still be used but by using three dyes in mixtures. The important 
point here is the concept of using ratio detection to code for all four 
base fragments on one capillary using only two detector channels. 
Finally, it is clear that this method is not limited to fluorescence. For 
example, different ratios of two or more different isotopic labels could 
be employed. That is, the DNA sequencing fragments terminating in G, A, T 
and C can be coded by labeling the fragments with different ratios of 
isotopes. The labeled fragments could then be detected through 
measurements of radioactive emissions at two different energies, if the 
isotopes were radioactive, or by using mass spectrometer detection of 
ratios of stable isotopes.