U.S. patent application number 13/342471 was filed with the patent office on 2012-08-30 for pulsed-multiline excitation for color-blind fluorescence detection.
Invention is credited to Robert F. Curl, Carter Kittrell, Michael Metzker, Graham B. I. Scott.
Application Number | 20120219029 13/342471 |
Document ID | / |
Family ID | 25476036 |
Filed Date | 2012-08-30 |
United States Patent
Application |
20120219029 |
Kind Code |
A1 |
Scott; Graham B. I. ; et
al. |
August 30, 2012 |
Pulsed-Multiline Excitation for Color-Blind Fluorescence
Detection
Abstract
The present invention provides a technology called
Pulse-Multiline Excitation or PME. This technology provides a novel
approach to fluorescence detection with application for
high-throughput identification of informative SNPs, which could
lead to more accurate diagnosis of inherited disease, better
prognosis of risk susceptibilities, or identification of sporadic
mutations. The PME technology has two main advantages that
significantly increase fluorescence sensitivity: (1) optimal
excitation of all fluorophores in the genomic assay and (2)
"color-blind" detection, which collects considerably more light
than standard wavelength resolved detection. Successful
implementation of the PME technology will have broad application
for routine usage in clinical diagnostics, forensics, and general
sequencing methodologies and will have the capability, flexibility,
and portability of targeted sequence variation assays for a large
majority of the population.
Inventors: |
Scott; Graham B. I.;
(Houston, TX) ; Kittrell; Carter; (Houston,
TX) ; Curl; Robert F.; (Houston, TX) ;
Metzker; Michael; (Houston, TX) |
Family ID: |
25476036 |
Appl. No.: |
13/342471 |
Filed: |
January 3, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12388358 |
Feb 18, 2009 |
8089628 |
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13342471 |
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11248910 |
Oct 12, 2005 |
7511811 |
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12388358 |
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09941165 |
Aug 28, 2001 |
6995841 |
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11248910 |
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Current U.S.
Class: |
372/38.02 ;
359/831 |
Current CPC
Class: |
G01J 3/4406 20130101;
G01N 21/6428 20130101; G01N 2021/6419 20130101; G01N 21/6458
20130101; G01J 3/10 20130101; G01N 2021/6441 20130101; G01J 3/021
20130101 |
Class at
Publication: |
372/38.02 ;
359/831 |
International
Class: |
H01S 3/00 20060101
H01S003/00; G02B 5/04 20060101 G02B005/04 |
Claims
1. A device comprising: (a) one or more lasers having two or more
excitation lines; (b) one or more beam steering mirrors wherein
said excitation lines each strike said mirrors; (c) a first prism,
wherein said two or more excitation lines strike one surface and
exit from a second surface of said first prism; and (d) a second
prism at an angle relative to said first prism, wherein said two or
more excitation lines strike one surface of said second prism after
exiting said first prism and exit said second prism, wherein said
two or more excitation lines are substantially colinear or coaxial
after exiting said second prism.
2. The method of claim 1, wherein said two or more excitation lines
are substantially coaxial after exiting said second prism.
3. The method of claim 1, wherein said two or more excitation lines
are substantially colinear after exiting said second prism.
4. A method of illuminating a sample comprising: (a) steering two
or more excitation lines onto a first surface of a first prism; (b)
steering two or more excitation lines from the second surface of
said first prism to a first surface of a second prism; wherein said
second prism is angled about 45.degree. from said first prism; (c)
steering said two or more excitation lines onto a sample after
exiting second surface of said second prism, wherein said two or
more excitation lines are substantially colinear or coaxial after
exiting said second prism.
5. The method of claim 4, wherein said two or more excitation lines
are substantially coaxial after exiting said second prism.
6. The method of claim 4, wherein said two or more excitation lines
are substantially colinear after exiting said second prism.
7. A method of controlling a sequence of excitation lines
comprising: obtaining a TTL circuit comprising an electronic
stepper wherein said circuit is operationally connected to one or
more lasers having two or more excitation lines; and controlling
the sequential firing of the one or more lasers having two or more
excitation lines with a clock pulse from the circuit, wherein the
frequency of firing one laser is equivalent to the frequency of
firing a second laser, but phased shifted so that one or more
lasers having two or more excitation lines can be sequentially
pulsed.
8. The method of claim 7, wherein the cycle time of one clock pulse
is from 1 second to 5 seconds.
9. The method of claim 7, wherein the length of time a first laser
produces an excitation line is similar to the length of time a
second laser produces an excitation line.
10. The method of claim 7, wherein between 2-to-16 excitation lines
are sequentially pulsed.
11. The method of claim 10, wherein between 2-to-8 excitation lines
are sequentially pulsed.
12. A method of controlling a sequence of excitation lines
comprising: obtaining a TTL circuit comprising an electronic
stepper wherein said circuit is operationally connected to two or
more lasers; and controlling the sequential firing of the two or
more lasers with a clock pulse from the circuit, wherein the
frequency of firing a first laser is different from the frequency
of firing a second laser.
13. The method of claim 12, comprising between 2-to-16 lasers.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of co-pending U.S. patent
application Ser. No. 12/388,358, filed Feb. 18, 2009; which is a
divisional of U.S. patent application Ser. No. 11/248,910, filed
Oct. 12, 2005, and now U.S. Pat. No. 7,511,811; which is a
continuation of U.S. patent application Ser. No. 09/941,165, filed
Aug. 28, 2001, and now U.S. Pat. No. 6,995,841; the contents of
which applications are incorporated herein by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] I. Field of the Invention
[0003] The present invention relates generally to the fields of
high throughput genetic analysis applications and fluorescence
spectroscopy. More particularly, it provides a variety of
compositions and methods for use in high-throughput DNA sequence
identification.
[0004] II. Description of Related Art
[0005] The Human Genome Project (HGP) holds tremendous promise for
discoveries of the molecular mechanisms that trigger the onset of
many common diseases over the next several decades. The initial HGP
goals underway will provide or have provided the complete and
accurate genome sequences of human and multiple well-studied
genetic model organisms, such as mouse, rat, fruit fly, nematode,
yeast and numerous bacteria. From this foundation of reference
genome sequences, the elucidation of complete gene sets, coupled
with comparative cross-species studies, are expected to assist
significantly in the assignment to specific human genes of protein
function and disease associations. Other technologies complement
the assignment of biological functions: gene and protein expression
profiling, mouse gene-knockouts, and techniques that measure
protein-protein interactions. The elucidation of gene
structure-protein function relationships are key to understanding
how genomic sequence variation between individuals can cause
increased risk or predisposition to certain complex diseases or are
even the etiologic agents responsible for the onset of particular
diseases. However, the use of genetic variation in clinical
practice is only beginning and technology to facilitate its use is
greatly needed.
[0006] The most commonly observed form of human sequence variation
is single nucleotide polymorphisms (SNPs), which occur at a
frequency of approximately 1-in-300 to 1-in-1000 base pairs. In
general, 10%-to-15% of SNPs will affect either protein function by
altering specific amino acid residues, or will affect the proper
processing of genes by changing splicing mechanisms, or will affect
the normal level of expression of the gene or protein by varying
regulatory mechanisms. Several recent examples are the associations
of mutations with the NOTCH4 gene and schizophrenia (Wei et al.,
2000), peroxisome proliferator-activated receptor gamma
(PPAR.gamma.) gene and severe insulin resistance (Deeb et al.,
1998), and melanocortin-4 receptor (MC4R) gene and inherited
obesity (Yeo et al., 1998).
[0007] The identification of informative SNPs will lead to more
accurate diagnosis of inherited diseases, better assessment of risk
susceptibilities, and could be assayed in specific tissue biopsies
for sporadic mutations. An individual's SNP profile could be used
to offset and significantly delay the progression of disease by
helping in the choice of prophylactic drug therapies. A SNP profile
of drug metabolizing genes could be used to prescribe a specific
drug regimen to provide safer and more efficacious results. To
accomplish goals like these, genome sequencing will move into the
resequencing phase of not just a handful of individuals, but
potentially the partial sequencing of most of the population.
Resequencing simply means sequencing in parallel specific regions
or single nucleotides that are distributed throughout the human
genome to obtain the SNP profile for a given complex disease.
[0008] For this technology to be applicable and practicable for
routine usage in medical practice, it must be robust, easy-to-use,
highly sensitive, flexible, portable, and the results should be
accurate and rapidly obtained. While current technologies at large
genome centers are robust and results are accurate, they are
inadequate and inflexible for resequencing millions of individuals
in routine clinical practice. It is therefore advantageous to
develop a DNA sequencing instrument, which meets these needs.
Miniaturization of this technology is also advantageous because
smaller instruments potentially require less sample and reagents
and can be more readily transported and located in areas such as
clinics or doctors' offices.
[0009] Ideally, DNA sequencing technology would have the
sensitivity for direct assays without DNA amplification, and be
simple and portable for routine usage in basic, applied, and
clinical laboratories. Currently, DNA sequencing technology for
high-throughput analyses are specialized and centralized in large
genome centers and require numerous molecular biology manipulations
that take days or weeks of preparation before DNA sequence analysis
can be performed. Thereafter, the state-of-the-art technology
involves the attachment of four different fluorescent dyes or
fluorophores to the four bases of DNA (i.e., A, C, G, and T) that
can be discriminated by their respective emission wavelengths, the
electrophoretic separation of the nested set of dye-labeled DNA
fragments into base-pair increments, and the detection of the dye
fluorescence following irradiation by a single argon-ion laser
source. Current instrumentation for electrophoretic separation
comprises a 96-capillary array that disperses the different
fluorescent signals using a prism, diffraction grating,
spectrograph, or other dispersing element and images the four
colors onto a charged-coupled device (CCD) camera. The throughput
of each 96-capillary instrument is approximately 800 DNA samples
per day, and the success of the HGP in large-scale genomic
sequencing has been attributed to the use of hundreds of these
machines throughout the world. The main disadvantages of the
current technology are the laborious cloning or amplification steps
needed to provide sufficient DNA material for analyses, the
relatively large size of the instruments (roughly the size of a
4-foot refrigerator), and the inadequate sensitivity of detection
(i.e., inefficient excitation of fluorescent dyes with absorption
maxima far from the laser excitation wavelength).
[0010] Although the resolution of spectral emission wavelengths is
the mainstream technology used in commercial and academic prototype
instruments, several groups have explored other physical properties
of fluorescence as a method for discriminating multicolor systems
for DNA sequence determination. Recently, Lieberwirth et al. (1998)
described a diode-laser based time-resolved fluorescence confocal
detection system for DNA sequencing by capillary electrophoresis.
In this system, a semiconductor laser (630 nm) was modulated using
a tunable pulse generator at a repetition rate of 22 MHz (454 psec
pulses) and focused by a microscope objective. The fluorescence was
collected by the same objective and imaged on a single photon
counting module APD (Lieberwirth et al., 1998).
[0011] The Luryi group at SUNY Stony Brook have proposed a multiple
laser excitation approach using different radio frequency (RF)
modulations and demodulations to discriminate a mixture of
fluorophores (U.S. Pat. Nos. 5,784,157 and 6,038,023). U.S. Pat.
No. 5,784,157 describes a 4-laser based fiber optic single
capillary monitoring device, which initially has a non-wavelength
component, but later the invention discusses the coupling of
spectral resolution for fluorophore discrimination. There are three
significant flaws apparent in this system relating to the enhanced
fluorescence cross-talk and laser scattered light, low sensitivity
detection, and a system that does not appear to scale beyond one
capillary.
[0012] As described, the target capillary is illuminated
simultaneously by all four lasers, which are modulated by different
RF signals. The different RF signals for all of the dyes are summed
together and the detector photodiodes are demodulated by additional
heterodyne RF signals. Interestingly, Gorfinkel and Luryi describe
the creation of Bragg reflectors to eliminate cross-talk modulation
for a given dye set. Fluorescence cross-talk, however, will not be
eliminated using this technique. Signal from the "wrong" dye, which
is weakly excited off-resonance by a particular laser, will be
encoded with the corresponding "wrong" frequency, decoded, and
added to the signal for the target dye. Moreover, scattered laser
light will also be modulated, and is likewise not rejected by the
heterodyne detection.
[0013] The simultaneous multi-modulation method also has a serious
shortcoming for the detection of low light levels, which is a
specific aim of the current invention. All the lasers are proposed
to operate simultaneously, followed by detection of substantially
all of the entire fluorescence, and conversion of the collected
fluorescence to an electrical signal. This design potentially
creates a correspondingly high quantum statistical noise level,
which should be distributed to all the detectors. The
demultiplexing process of RFs does not remove this excessive random
noise, even if the corresponding signal is small (Meaburn, 1976).
In comparison, the Pulse-Multiline Excitation (PME) system
described in the current invention exhibits noise levels in proper
proportion, so that a weak signal originating from a particular
laser pulse has a correspondingly low detected noise level during
that laser's sub-cycle. Optimizing the optical system for producing
low noise levels is essential in establishing the optimum contrast
between the presence and absence of a given dye.
[0014] Finally, U.S. Pat. No. 5,784,157 describes a rather
complicated array of optical fibers, combiners, splitters, and 4
heterodyne detectors with their associated spectral filters for a
single capillary channel. Scaling this system to a 2-capillary
system would entail doubling the mentioned detector components.
Unfortunately a CCD camera is not readily adapted for high
frequency RF modulation, as it is an "inherently discrete-time"
device. In a more recent document, U.S. Pat. No. 6,038,023, the
multiplicity of spectral filters has been replaced with a
dispersing prism spectrometer and a high speed one dimensional
array detector for use with a single capillary channel device; the
potential to scale up to a capillary array system is more feasible
as discussed by the Luryi group, but may require a multiplicity of
such spectrometer units.
[0015] The current invention comprises a novel fluorescence device,
which is capable of significant improvements in the limit of
detection of multi-color fluorescence reactions and may be applied
to direct measurement of such reactions from biological sources
(i.e., without the need for PCR or cloning amplifications).
Moreover, this technology, called Pulse-Multiline Excitation or
"PME" can be configured on a small work surface or in a small
instrument, compared to the current DNA sequencing instruments.
Thus, a DNA sequencer the size of a suitcase or smaller is
described.
[0016] The development of improved DNA sequencing chemistries will
likely improve the number of independent assays that can be run in
parallel. This technology will have broad application in both
general sequencing and forensic applications.
SUMMARY OF THE INVENTION
[0017] Thus, the present invention contemplates an apparatus and
method for use in high-throughput DNA sequence identification. An
aspect of the invention is a pulse-multiline excitation apparatus
for analyzing a sample containing one or more fluorescent species,
comprising: one or more lasers configured to emit two or more
excitation lines, each excitation line having a different
wavelength; a timing circuit coupled to the one or more lasers and
configured to generate the two or more excitation lines
sequentially according to a timing program to produce
time-correlated fluorescence emission signals from the sample; a
non-dispersive detector positioned to collect the time-correlated
fluorescence emission signals emanating from the sample; and an
analyzer coupled to the detector and configured to associate the
time-correlated fluorescence emission signals with the timing
program to identify constituents of the sample.
[0018] The detector and the analyzer may be integral. In one
embodiment, the two or more excitation lines intersect at the
sample, or the two or more excitation lines may be configured so
that they do not intersect in the sample. The two or more
excitation lines may be coaxial.
[0019] In one embodiment of the invention, the apparatus may
further comprise an assembly of one or more prisms in operative
relation with the one or more lasers and configured to render
radiation of the two or more excitation lines substantially
colinear and/or coaxial.
[0020] The apparatus may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16 or more excitation lines having 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more excitation
wavelengths, respectively. The sample may be comprised in 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, up to 20, up to 24,
up to 28, up to 36, up to 48, up to 64, up to 96, up to 384 or more
capillaries. A sheath flow cuvette may be used.
[0021] The timing program may comprise a delay between the firing
of each laser of between about 10 fs and about 5 s, between about 1
ms and about 100 ms, or between about 50 ps and about 500 ps. One
or more of the excitation lines is pulsed. The pulsed excitation
line may be controlled by transistor-transistor logic (TTL) or by
mechanical or electronic means. In one embodiment, the apparatus
may generate a sequence of discrete excitation lines that are
time-correlated with the fluorescence emission signals from the
sample.
[0022] The lasers may independently comprise a diode laser, a
semiconductor laser, a gas laser, such as an argon ion, krypton, or
helium-neon laser, a diode laser, a solid-state laser such as a
Neodymium laser which will include an ion-gain medium, such as YAG
and yttrium vanadate (YVO.sub.4), or a diode pumped solid state
laser. Other devices, which produce light at one or more discrete
excitation wavelengths, may also be used in place of the laser. The
laser may further comprise a Raman shifter in operable relation
with at least one laser beam. In one embodiment of the invention,
the excitation wavelength provided by each laser is optically
matched to the absorption wavelength of each fluorophore.
[0023] The detector may comprise a charged couple device, a
photomultiplier tube, a silicon avalanche photodiode or a silicon
PIN detector. The footprint of the device is preferably small, such
as less than 4 ft.times.4 ft.times.2 ft, less than 1 ft.times.1
ft.times.2 ft, and could be made as small as 1 in .times.3 in
.times.6 in.
[0024] Another aspect of the current invention comprises a method
of identifying sample components comprising: (a) preparing a sample
comprising sample components, a first dye and a second dye; (b)
placing the sample in the beam path of a first excitation line and
a second excitation line; (c) sequentially firing the first
excitation line and the second excitation line; (d) collecting
fluorescence signals from the samples as a function of time; and
(e) sorting the fluorescence by each excitation line's on-time
window, wherein the sample components are identified. It is an
aspect of the invention that the fluorescence signals are collected
from discrete time periods in which no excitation line is incident
on the sample, the time periods occurring between the firing of the
two excitation lines. This technique is known as "looking in the
dark." Yet another aspect of the present invention is that the
absorption maximum of the first dye substantially corresponds to
the excitation wavelength of the first excitation line. The
absorption maximum of the second dye may also substantially
corresponds to the excitation wavelength of the second excitation
line. In yet another aspect of the current invention there is a
third and fourth dye and a third and fourth excitation line,
wherein the absorption maxima of the third and fourth dyes
substantially correspond to the excitation wavelengths of the third
and four excitation lines, respectively. Similarly, there may be 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more dyes wherein the
absorption maxima of the dyes substantially corresponds to
excitation wavelengths of a 5.sup.th, 6.sup.th, 7.sup.th, 8.sup.th,
9.sup.th, 10.sup.th, 11.sup.th, 12.sup.th, 13.sup.th, 14.sup.th,
15.sup.th, 16.sup.th, or more excitation lines, respectively. The
dyes may be a zanthene, fluorescein, rhodamine, BODIPY, cyanine,
coumarin, pyrene, phthalocyanine, phycobiliprotein, Alexa,
squariane dyes, or some other suitable dye.
[0025] In one embodiment of the current invention, the sample
components enable the determination of SNPs. The method may be for
the high-throughput identification of informative SNPs. The SNPs
may be obtained directly from genomic DNA material, from PCR
amplified material, or from cloned DNA material and may be assayed
using a single nucleotide primer extension method. The single
nucleotide primer extension method may comprise using single
unlabeled dNTPs, single labeled dNTPs, single 3'-modified dNTPs,
single base-modified 3'-dNTPs, single alpha-thio-dNTPs or single
labeled 2',3'-dideoxynucleotides. The mini-sequencing method may
comprise using single unlabeled dNTPs, single labeled dNTPs, single
3'-modified dNTPs, single base-modified 3'-dNTPs, single
alpha-thio-dNTPs or single labeled 2',3'-dideoxynucleotides. The
SNPs may be obtained directly from genomic DNA material, from PCR
amplified material, or from cloned DNA materials and may be assayed
using Sanger sequencing.
[0026] In another embodiment of the current invention, analyzing
the signals is adapted for the accurate diagnosis of inherited
disease, better prognosis of risk susceptibilities, identification
of sporadic mutations, or prescribing tailor-made daily drug
regimens for individual patients. Analyzing the signals may be
adapted for routine usage in clinical diagnostics, forensics
applications or determining general sequencing methodologies.
[0027] Yet another aspect of the current invention is a method of
identifying sample components comprising: (a) obtaining a
biological sample; (b) labeling said sample with one or more
fluorophores; (c) separating components of said sample; and (d)
detecting said sample components with a device wherein said device
may comprise: one or more lasers configured to emit two or more
excitation lines, each excitation line having a different
excitation wavelength; a timing circuit coupled to the one or more
lasers and configured to fire the two or more excitation lines
sequentially according to a timing program to produce
time-correlated fluorescence emission signals from the sample; and
a non-dispersive detector positioned to collect the time-correlated
fluorescence emission signals; wherein said detector collects time
correlated data from said sample comprising fluorescent emissions
of the sample as a result of irradiation by the one or more
excitation lines.
[0028] The sample components may be nucleic acids, amino acids or
proteins. The separation may be by electrophoresis, chromatography
or mass spectrometry (MS) such as MALDI-TOF, quadrapole mass filter
or magnetic sector MS. The sample components may be addressed on
high density chip arrays.
[0029] In one embodiment, the method may further comprise: (e)
contacting said sample components on a surface comprising
immobilized oligonucleotides at known locations on said surface;
and (f) performing a single nucleotide incorporation assay or a
mini-sequencing assay. In yet another embodiment, the method may
further comprise rastering said surface or said excitation lines
such that said excitation lines contact said surface at multiple
locations.
[0030] Another aspect of the current invention is a device
comprising: (a) one or more lasers having two or more excitation
lines; (b) one or more beam steering mirrors wherein said
excitation lines each strike said mirrors; (c) a first prism,
wherein said two or more excitation lines strike one surface and
exit from a second surface of said first prism; and (d) a second
prism at an angle relative to said first prism, wherein said two or
more excitation lines strike one surface of said second prism after
exiting said first prism and exit said second prism, wherein said
two or more excitation lines are substantially colinear and/or
substantially coaxial after exiting said second prism. The angle of
the second prism relative to the first prism is dependent on the
optical material used. For example, for high dispersion flint
glass, the two prisms will be arranged such that the second prism
is angled at 45.degree. relative to the first prism. For quartz,
the angular displacement ranges from 30.degree. to 50.degree..
[0031] Another aspect of the current invention comprises a method
of illuminating a sample comprising: (a) steering two or more
excitation lines onto a first surface of a first prism; (b)
steering two or more excitation lines from the second surface of
said first prism to a first surface of a second prism; wherein said
second prism is angled about 45.degree. from said first prism; (c)
steering said two or more excitation lines onto a sample after
exiting second surface of said second prism, wherein said two or
more excitation lines are substantially colinear and/or
substantially coaxial after exiting said second prism.
[0032] Yet another aspect of the current invention comprises a
method of controlling a sequence of excitation lines comprising:
(a) obtaining a TTL circuit comprising an electronic stepper
wherein said circuit is operationally connected to one or more
lasers having two or more excitation lines; (b) and controlling the
sequential firing of the one or more lasers having two or more
excitation lines with a clock pulse from the circuit, wherein the
frequency of firing one laser is equivalent to the frequency of
firing a second laser, but phased shifted so that one or more
lasers having two or more excitation lines can be sequentially
pulsed. The cycle time of one clock pulse may be from 1 .mu.second
to 5 seconds, or from 100 .mu.second to 1 second. The length of
time a first laser produces an excitation line may be similar to
the length of time a second laser produces an excitation line. As
used herein, similar means within 20%, within 10%, or more
preferably within 5% of the time length. Between 2-to-16, or 2-to-8
excitation lines are sequentially pulsed.
[0033] Yet another method of the current invention comprises a
method of controlling a sequence of excitation lines comprising:
(a) obtaining a TTL circuit comprising an electronic stepper
wherein said circuit is operationally connected to one or more
lasers having two or more excitation lines; (b) and controlling the
sequential firing of the one or more lasers having two or more
excitation lines with a clock pulse from the circuit, wherein the
frequency of firing a first laser is different from the frequency
of firing a second laser. This method may be used to control 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 16 lasers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0035] FIG. 1. An example of a PME device where each laser is
regulated individually by its own power supply (A). The TTL stepper
or clock chip circuit (FIG. 3) chooses which power supply is turned
on at a specific time as it cycles through the five lasers. The
beam steering mirrors (B) allow various degrees of adjustment to
align the excitation lines so that once they go through a dual
prism assembly (FIG. 2) (C), they will become colinear and/or
coaxial. The beams enter the dark box (D) where scattered light is
reduced by the use of irises and a long cell. The dyes are detected
by the photomultiplier tube (E) through a collecting lens. The
signals are recorded by the oscilloscope (F) where digital pictures
can be analyzed.
[0036] FIG. 2. Inversion dispersion scheme using a dual prism
assembly to combine pulsed multiline excitation laser sources from
discrete locations. The beams all enter from the left hitting the
prism at varying angles and positions on the left side of the first
prism (upper left). As they hit the first prism, the laser beams
all bend approximately at a 45-degree angle. At this point the
beams are not yet colinear and/or coaxial but they are spatially
closer together than before. As they hit the second prism, they
once again hit at varying angles and positions, and become colinear
and/or coaxial as they exit the prism.
[0037] FIG. 3. TTL Circuit Clock Chip (74174). MR, when low, resets
the chip and sets all outputs to low; CP is the Clock Pulse Input.
Q0 through Q7 are outputs that connect to and signal each of up to
eight lasers to fire in sequence.
[0038] FIG. 4A, FIG. 4B and FIG. 4C. Photographic data from the
oscilloscope output. Two channels from the oscilloscope were set to
record the clock signal for firing the red laser (top line) and the
PMT detector output (bottom line). Arrows correspond to the red and
green laser pulses. Data on dilute aqueous solutions containing
both BODIPY 523/547 and BODIPY 630/650 dyes (FIG. 4A), BODIPY
630/650 dye only (FIG. 4B), or BODIPY 523/547 dye only (FIG. 4C)
were collected.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
I. The Present Invention
[0039] The present invention describes a novel device and approach
to fluorescence detection, which has general application for
genetic analysis methodologies with particular emphasis on DNA
sequencing technologies and high-throughput identification of
single nucleotide polymorphisms (SNPs). The PME technology has two
main advantages that significantly increase fluorescence
sensitivity: optimal excitation of all fluorophores in the genomic
assay and "color-blind" detection, which collects considerably more
light than traditional dispersive detectors. The fluorescence
detector can be designed to miniaturize DNA sequencing technology
with a sensitivity enabling direct detection of fluorescent DNA
assays from genomic DNA material. The PME is useful for clinical
diagnostic, forensic, and general sequencing.
II. Pulsed Multi-Line Excitation (PME) Detection
[0040] In the current invention, spectral dispersion or wavelength
discrimination of fluorescent dyes is eliminated, which increases
the amount of fluorescent signal detected. The sequential
pulsed-laser excitation system using multiple lasers emitting
specific wavelengths of light, which are matched for efficient
excitation of a given set of fluorophores, can determine
selectivity and sensitivity. By matching the absorption maximum of
each fluorophore, the PME technology excites each dye with the
highest quantum efficiency, thus considerably reducing the required
sample size (i.e., the number of fluorescent molecules required for
detection). At first glance, replacing one laser with four lasers
may appear counterintuitive to miniaturization. New solid state
lasers, however, such as diode pumped Nd:YAG sources or diode
lasers are much smaller (ca. 2'' long) than standard argon ion
lasers and are much more efficient requiring smaller power supplies
for operation. For example, the footprint of four solid-state
lasers together is approximately 20-fold smaller than a single
argon-ion laser system. Simply replacing the argon-ion laser, which
for a DNA sequencer relies on two excitation lines at 488 nm and
514.5 nm, with a equal power 532 nm Nd:YAG can reduce the laser
size, but would reduce the excitation/emission intensities of
shorter wavelength dyes, and still would not efficiently excite
longer-wavelength dyes that have absorption maxima far from the
laser wavelengths.
[0041] For the PME technology to discriminate four fluorescent
dyes, four excitation lines are combined by inverse dispersion,
which is illustrated but not limited to using a prism assembly or
diffraction grating. The resultant beam would look on average like
a "white light" laser beam. However, the solid-state lasers are
electronically controlled and are pulsed or fired sequentially as
discrete packages of wavelength specific light. Alternatively,
laser sources can be pulsed or fired using a synchronized shutter
system. Each dye brightly fluoresces when its matched laser source
is turned on, while it responds only weakly, if at all, to the
other three laser pulses. The fluorescence from each excitation
event is collected using a non-dispersed or "color blind" method of
detection. A non-dispersive detector is a detector in which the
incident radiation is not separated based on the emission
fluorescence wavelength of different fluorescent dyes. Thus, DNA
sequence is determined by the PME technology based on the time
correlation of detector response to specific wavelengths of
excitation light, and not spectral resolution of emission
wavelengths. Switching the solid-state lasers on a millisecond
timescale is straightforward, hence thousands of 4-laser excitation
cycles may be completed in the time scale for eluting a single base
of DNA by capillary electrophoresis.
[0042] Moreover, an advantage of the non-dispersed system is that
the detector (i.e., CCD) collects significantly more light, since
the fluorescent light is directly coupled to the detector.
Typically for the current DNA sequencer, a dispersive element
requires highly collimated light for effective wavelength
separation. Moving the collection lens closer to the sample can
increase the collected fluorescent light, but collimation is
lessened, and spectral selectivity is reduced. Similarly, reducing
the distance between the dispersing element and the detector
results in reduced spectral selectivity. For the non-dispersed
system, however, moving the collection lens much closer to the
sample or to the detector increases the collected light, inverse to
the square of the distance, but without sacrificing the selectivity
that is provided by four laser cycling. Thus, the miniaturization
process inherently delivers more fluorescent light to the
detector.
[0043] Typically, miniaturizing a system incurs inevitable
penalties in sensitivity and selectivity. For example, fluorescent
signal is lost as the laser source becomes smaller in size and
power, and selectivity is compromised because spectral dispersing
elements need physical space to separate emission wavelengths and
compressing the spectrometer portion of the detection sacrifices
spectral resolution. Consequently, the sample size is increased to
offset these losses, which tends to marginalize the benefits
derived by shrinking the conventional dispersive optical system.
The design described herein minimizes the losses in downsizing
instrumentation, but increases the sensitivity considerably by the
process of miniaturization. The current invention comprises a novel
detection system that allows the optical components to act
synergistically when miniaturized.
[0044] An additional advantage of non-dispersed detection is
enhanced signal-to-noise compared to the current 96-capillary DNA
sequencer. To obtain the wavelength spectrum for each DNA sequence
reaction, a large number of pixels are read out, and this
electronic readout process adds noise for every pixel read. For the
non-dispersed system, all pixels that receive light from a
particular capillary are "binned" and read out as a single unit,
considerably reducing the associated electronic noise.
III. Fluorophores
[0045] An advantage of using PME or any time-based detection as
opposed to wavelength discriminating detection is the increase in
the number of fluorophores, which can be used. At any given
excitation wavelength, there are often only about two or three
commercially available dyes that emit with narrow enough emission
bands with sufficiently separated wavelength maxima that can be
individually measured simultaneously (U.S. Pat. No. 6,139,800). If
three or more fluorophores can be found, there is still substantial
cross-talk or overlap of the emission spectra that will require
substantial deconvolution of the spectra with a corresponding
increase in the likelihood of error in identifying the species.
[0046] One solution to this problem has been the addition of a
second laser to allow for the simultaneous or sequential detection
of up to approximately six dyes (U.S. Pat. No. 6,139,800). However,
this solution still has the problem of substantial overlap in the
spectra and the need for signal intensities great enough to be
detected after spectral dispersion of the signal.
[0047] Optimally, the way to obtain the highest emission signal
possible is to optically match an excitation source with the
absorption maxima of a dye with a high molar extinction
coefficient. This is done for every fluorophore. However, the
excitation source need not match the absorption maxima exactly,
instead, it is important to obtain laser-dye combinations where
each dye has an absorption maxima which substantially corresponds
with one source wavelength with concomitant emission, coupled with
minimal absorption/emission (cross-talk) from the non-matched laser
sources used in the assay.
[0048] A system with four fluorophores used to detect the 4 DNA
bases is preferred. However, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, or 16 different fluorophores may be used with the PME
system.
[0049] A non-limiting list of dyes that may be used in the current
invention include BODIPY dyes (BODIPY 630/650, BODIPY 650/665,
BODIPY 589/616 or BODIPY-TR, BODIPY 581/591 BODIPY 523/547 or
BODIPY-R6G, 5,7-dimethyl-BODIPY (503/512) or BODIPY-FL,
1,3,5,7-tetramethyl-BODIPY (495/503), BODIPY-TMR-X or BODIPY
(564/570)-X, BODIPY-TR-X or BODIPY (589/616)-X, BODIPY (530/550),
BODIPY (564/570), and BODIPY (558/568)), a zanthene dye, a
rhodamine dye (rhodamine green, rhodamine red, tetraethylrhodamine,
5-carboxy rhodamine 6G (R6G), 6-carboxy R6G, tetramethylrhodamine
(TMR), 5-carboxy TMR or 5-TAMRA, 6-carboxy TMR or 6-TAMRA,
rhodamine B, X-rhodamine (ROX), 5-carboxy ROX, 6-carboxy ROX,
lissamine rhodamine B, and Texas Red), a fluorescein dye (FITC,
5-carboxy fluorescein, 6-carboxy fluorescein, fluorescein
diacetate, naphthofluorescein, HEX, TET, 5-carboxy JOE, 6-carboxy
JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514,
erythrosin, eosin), a coumarin dye (7-hydroxycoumarin,
7-dimethylaminocoumarin, 7-methoxycoumarin,
7-amino-4-methylcoumarin-3-acetic acid or (AMCA), and Pacific
Blue), a cyanine (Cy) dye (Cy3, Cy3.5, Cy5, Cy5.5, Cy7), a
phthalocyanine dye, a phycobiliprotein dye, (B-phycoerythrin
(B-PE), R-phycoerythrin (R-PE), and allophycocyanin (APC)), a
pyrene and a sulfonated pyrene, (cascade blue), a squaraine dye, an
Alexa dye (Alexa 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546,
Alexa 568, Alexa 594) and Lucifer yellow.
IV. Excitation Sources
[0050] A central principle of the PME technology is the
discrimination of a mixture of different fluorophores by the time
correlation of "colorblind" fluorescence emission triggered by
serially pulsing different excitation lasers. This approach
significantly contrasts that of the widely used method of
wavelength discrimination of fluorescence emission, where a single
excitation source, typically an argon ion laser (488 nm and 514.5
nm) excites four spectrally resolvable fluorescent dyes. The dye of
this set, which emits at the longest emitting wavelength is usually
the least optimally excited, which is due to poor spectral overlap
between the excitation source and the dye's absorption maximum.
This inefficient excitation has been partially overcome by the use
of fluorescence resonance energy transfer (FRET) dye-primers (Ju et
al., 1995; Metzker et al., 1996) and dye-terminators (Rosenblum et
al., 1997) to increase signal intensities. Obviously, the optimal
method in obtaining the highest emission signal possible would be
matching the excitation source with the absorption maxima for every
fluorophore in DNA sequencing assays.
[0051] The invention may use at least one laser and is flexible to
accommodate as many different lasers as is feasibly possible. There
may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more
lasers, depending on the system.
[0052] A single laser may produce 1, 2, 3, 4, 5, 6, 7, 8 or more
different wavelengths for the excitation of fluorophores with
different absorption maxima. This can be accomplished using the
technique of Stimulated Raman Shifting (SRS). This technique may be
employed for conversion to either shorter or longer wavelength(s).
The Raman effect enables a laser frequency to be modified by
discrete increments, (the Stokes and Anti-Stokes shifts). Frequency
conversion is accomplished by passing laser light through a
suitable crystal or a stainless steel cell containing gas at an
elevated pressure, (i.e. several atmospheres). Conversion
efficiency for the principal Stokes shift to longer wavelength can
be as high as 35%. The nature of the crystal or gas determines the
frequency output, for example, N.sub.2, O.sub.2, H.sub.2, D.sub.2,
and CH.sub.4 give shifts of 2330 cm.sup.-1, 1550 cm.sup.-1, 4155
cm.sup.-1, 2987 cm.sup.-1, and 2917 cm.sup.-1 respectively, while
Ba(NO.sub.3).sub.2 gives a shift of 1047 cm.sup.-1. A preferred
Raman medium for this invention is molecular nitrogen, as 2330
cm.sup.-1 is about the desired spacing between excitation
frequencies.
[0053] Until recently, gas lasers have been widely used for the
excitation of "blue" and "green" fluorophores with absorption
maxima ranging between 488 nm and 543 nm for DNA sequencing
applications. In general, these lasers include the argon ion, the
krypton ion, and the helium-neon (He--Ne) lasers. These lasers are
large in size, highly inefficient and relatively expensive devices.
Moreover, the lifetime of gas laser is approximately 1,000-to-3,000
hours of use, which imposes high maintenance cost for these
instruments. Despite these disadvantages, the argon ion laser has
been widely used in automated DNA sequencing instrumentation for 15
years now (Smith et al., 1986; Probe et al., 1987) and is
frequently described as the excitation source in many capillary
electrophoresis systems, see below.
[0054] On the other hand, semiconductor lasers or laser diodes are
much smaller, lighter, and more rugged than any other laser types
and have been employed in a wide variety of applications such as CD
players, laser printers, and telecommunication systems. These
compact lasers typically produce monochromatic light between 630 nm
and 1100 nm. These extremely compact, but durable lasers can
produce power in the 10-100 mW range and have a useful lifetime of
up to 100,000 hours.
[0055] The neodymium:YAG (Yttrium Aluminum Garnet) laser is the
most common solid-state laser in use today with instruments being
found in a variety of applications such as in industry welding of
heavy metals, in surgical operating devices, in laboratory
spectroscopic equipment, or on unmanned space probes. A solid-state
laser is a source in which the active medium is usually a
transparent crystal containing a transition metal, (typically 1% or
less), such as neodymium, chromium or holmium. Transitions in the
metal ion are responsible for the laser's action. These lasers are
optically pumped by either broadband flash-lamp sources, or one or
more diode laser sources. For blue and green excitation,
solid-state lasers contain a frequency doubling or second harmonic
generating (SHG) crystal such as lithium borate or potassium
titanyl phosphate. For example, the frequency doubled Nd:YAG laser
has a fundamental excitation line of 1064 nm, which is doubled by
an SHG crystal to generate green 532 nm light.
[0056] Until recently, the application of the PME technology has
been unrecognized by the lack of available and reliable solid-state
lasers that produce monochromatic light at wavelengths between 400
nm and 630 nm. This emerging field, however, has recently produced
solid-state lasers that generate monochromatic light at wavelengths
of approximately 400 nm, 473 nm, and 488 nm, which becomes suitable
for DNA sequencing applications. Thus, the development of PME is
uniquely coupled to this emerging field of laser development and
well positioned to incorporate new advances in laser technology,
when available.
V. Inverse Dispersion
[0057] Because of the need for multiple laser beams incident on a
single sample, the laser beams must be steered so that they all
pass through or contact the sample. This can be accomplished by
spatially combining the different laser beams into one overlapping
"white" beam.
[0058] Other groups have developed devices to combine two or more
laser beams. Conemac (U.S. Pat. No. 6,226,126) describes a laser
beam mixer having a beam combining element with a transmissive
portion and a reflective portion. However, this technology requires
that the cross sectional shape of the second, third and so forth
laser beams be distorted. Another limitation is that for each laser
beam added, the combined beam must pass through an additional
optical element, which introduces loss into the system.
[0059] U.S. Pat. No. 5,991,082 discloses a lens system that forms
narrow superimposable focal lines from multiple focal lines. This
system uses a prism with multiple longitudinally arranged facets
bounded by parallel ridge lines and can be used to obtain a high
energy radiation beam for use in pumping an X-ray laser.
[0060] U.S. Pat. No. 6,215,598 discloses an apparatus for
concentrating laser beams, which comprises collimating devices that
converge the laser beams into a laser beam sheet. A digital optics
device shapes and concentrates the laser beam sheet into a narrow
overlapping laser beam.
[0061] In the present invention, an inverse dispersion system can
be used to steer the light from multiple lasers onto a single
sample. Inverse dispersion uses optical dispersion elements such as
a prism assembly or a diffraction grating positioned such that
light from discrete locations is steered to be substantially
colinear and/or substantially coaxial upon exiting the system. The
term "substantially colinear" means that the laser beams or
excitation lines diverge from each other at angles of less than
5.degree.. The term "substantially coaxial" means that the laser
beams or excitation lines diverge from each other at angles of less
than 5.degree..
[0062] The inverse dispersion system may be configured as shown in
FIG. 2 in which five excitation lines from discrete locations are
combined into a single colinear and/or coaxial line.
[0063] High dispersion equilateral prisms constructed from high
grade glass, quartz or silica are used in one preferred embodiment
of the invention. The preferred orientation for the prisms relative
to each other when using high dispersion flint glass prisms is
40.degree.-50.degree., or more preferred 45.degree.. The preferred
orientation for the prisms relative to each other when using quartz
prisms is 25.degree.-55.degree. or more preferred
30.degree.-50.degree.. This angle allows efficient overlap of the
multiple beams by inverse dispersion into a single beam. A two
prism assembly is preferred, however, a single prism or an assembly
with three or more prisms is also contemplated. Similarly, a
diffraction grating such as a ruled or holographic grating can be
used to combine the multiple beams. Multiple excitation lines can
be steered onto a diffraction grating such that the diffraction of
the grating causes the beams to combine.
[0064] In addition to being colinear, the beams may be coaxial,
with all of the laser beams passing through the same columnar space
in the sample. The same sample molecules will be exposed to each of
the laser beams sequentially in turn as the lasers are fired.
[0065] The inverse dispersion approach uses the same principle
first demonstrated by Sir Isaac Newton, but in reverse direction.
In his experiment, collimated white light, from the sun, passed
through an equilateral prism, and the various wavelengths became
separated by angle. The beam of light passes into the prism,
forming a non-zero angle with respect to the normal to the entrance
surface. According to Snell's law, all of the rays will be bent
towards the normal as the light passes into the more optically
dense medium. Due to dispersion of the glass, the shorter
wavelengths deviate more. When the rays exit the prism, all are
bent a second time, but again, the shorter wavelengths bend more.
The result is that the shorter wavelengths now have an angular
separation from the longer wavelengths. Blue light is deviated more
than green, which is deviated more than yellow, and that in turn
more than red.
[0066] This process may be reversed, and that is the principle
utilized for inverse dispersion. If the separated rays are made to
trace paths that are just the reverse of above, then the various
wavelengths are combined into a single beam of "white" light. For
example, light from lasers, light emitting diodes, arc lamps,
incandescent lamps, etc. may be combined (after collimation and
spectral filtering, if appropriate) by this inverse dispersion
method. The shorter wavelength rays or beams enter the prism at the
appropriate larger off-normal angle than the longer wavelength
beams. When the correct angles are determined from Snell's law, the
beams will all combine into a single coaxial beam. If desired, the
beams may be spatially offset to provide colinear beams that, while
parallel to each other, pass through the sample at slightly
different positions. In the former case, the fluorescence from all
of the beams may be imaged onto a single detector. In the latter
case, the beams may be imaged onto four or more separate detectors,
or separate regions of one detector, such as a CCD camera. If the
light sources are pulsed in rapid succession, the combined beam
appears to be white or nearly so. If the pulsing is slow enough for
the eye to follow, the combined beam will exhibit a changing color
pattern originating from the same spatial location.
[0067] The prism is typically used at the minimum deviation angle,
whereby the entering and exiting angles for a given beam are equal,
or as nearly so as practical. The apex bisector will also bisect
the angle formed by the entering and exiting beam. If the prism is
then rotated, then these two angles are no longer equal. This is
advantageous in that it increases both the overall deviation angle
and the amount of dispersion. However, this also causes anamorphic
changes in the beam diameter. Such anamorphic expansion can be
useful if it is desirable to change a round beam cross section to
an oblong one, or an oblong beam shape to a round shape. If the
inverse dispersion combining of beams is to be done with minimal
distortion of the original beam shape, then the minimum deviation
angle is preferred.
[0068] The angular separation of the beams is increased in
proportion to the number of prisms the beam passes through. For
example, the use of two identical prisms doubles the angular
separation. The anamorphic beam changes can be nearly canceled by
using a pair of prisms at non-minimum deviation angles. Use of high
dispersion glass, such as flint glass also increases the angular
separation of the incoming beams. This in turn reduces the distance
needed to achieve spatial separation of the incoming beams, and
provides for a more compact optical apparatus. As an example, the
present apparatus utilizes two F2 flint glass prisms.
[0069] A diffraction grating may also be used as a suitable inverse
dispersion element. For a grating exposed to a collimated beam of
white light, the beam is diffracted in accordance with the grating
equation. If it is a transmission grating, the beam is diffracted
away from a straight line path, called the zero order, with the
longer wavelengths deviating at a larger angle than the shorter
wavelengths. The order of the dispersed wavelengths is the reverse
of that for prisms. If a reflection grating is used, again the
longer wavelength beam is deviated further from the zero order
reflection than a shorter wavelength. As with the prism combiner
above, beams of different wavelengths incident on the grating in
the reverse direction will provide the same sort of inverse
dispersion, leading to a colinearity of the beams. They may be made
coaxial if the beams are incident on the same area on the grating
but at the appropriate angles for each of the wavelengths.
Conventionally ruled gratings are suitable for this purpose,
however holographic gratings generally exhibit less scattered
light. Gratings generally can be obtained that have considerably
higher dispersion than prisms, and hence dispersion angles are
larger and the spacing between the light sources can be reduced.
They are generally less efficient, so that light losses are
greater. Gratings and prisms are both sensitive to the polarization
of the light. Since the fluorescence emission is also sensitive to
the direction of the polarization, proper orientation of the
electric vector of the light should be considered. Polarization
rotation devices may be added to improve the transmission
efficiency.
VI. Collection Devices
[0070] Of the possible choices for detection of fluorescence, the
optimum one will depend upon the level of fluorescence intensity.
For all detectors described herein, a photon striking the detector
is converted into a charge carrier, which is then detected
electronically. Charge carriers, however, are generated by
extrinsic processes unrelated to the fluorescence signal from a
variety of sources: thermal generation, cosmic rays, and natural
radioactivity. All carriers generated both from fluorescence and
from unrelated sources contribute to shot noise, a well-understood
statistical phenomenon. Of the extrinsic sources of excess carrier
noise, thermal generation of carriers is usually the dominant
extrinsic noise source, which can be reduced by cooling the
detector. Fundamentally all that is needed for satisfactory
detection is that the number of charge carriers generated by
fluorescence during an observation time window is much greater than
the square root of the number of all carriers generated during the
same time interval. However, once the carriers leave the detector,
the amplifying electronics will introduce noise as well, and this
electronic noise may dominate. To avoid this situation, devices
have been invented that incorporate their own very low noise
amplifiers. There are two such devices: the photomultiplier tube
(PMT) and the silicon avalanche photodiode (APD). Another approach
to reducing amplifier noise is to collect carriers for a period of
time and then rapidly read the collected charges out. This mode of
operation is used for photodiode array charge-coupled device (CCD)
detectors.
[0071] For light levels with high enough signals that the noise
generated in the external amplifiers is negligible, the simplest
device for fluorescence detection is the silicon PIN detector. This
device consists of a thin hole rich region (p) and an electron rich
region (n) separated by thick carrier deficient region (i). It is
back-biased with a negative voltage applied to the p side and a
positive voltage applied to the n side. Light striking it
penetrates the p region and is absorbed in the i region generating
an electron hole pair with the electron being attracted to the n
side and the hole to the p side creating a current through the
device. The quantum efficiency of this process is very high
(.about.80%). Because it is the simplest, most compact, and
cheapest detector, the silicon PIN is a preferred detector. Other,
more sensitive detectors may also be used for detecting low levels
of light emission for a multi-capillary electrophoresis system and
direct detection assays.
[0072] The silicon PIN detector can be made suitable for the
detection of very low light levels by introducing carrier gain into
it. A photon strikes the detector creating an electron hole pair.
The electron is accelerated by an electric field and creates
additional carriers by ionization. The additional electrons are
accelerated to produce more carriers resulting in an avalanche
process and current gain. Silicon avalanche detectors operate in
two modes analogous to that of the PMT: an analog current
measurement mode and a digital counting mode. When operated in the
analog mode, the current gain (.about.300) is less than that of a
PMT (.about.10.sup.5-10.sup.6), but with exception to the lowest
light levels, the signal is still much larger than the noise in the
external amplification circuit. In addition, the wavelength
response range (300 to 1100 nm) of silicon detectors is much wider
than any individual PMT photocathode, covering the fluorescent
maximum of any dye that might be used for DNA sequencing
instrumentation.
[0073] Alternatively, a silicon APD can be thermoelectrically
cooled and operated in the Geiger counting mode, where individual
fluorescence photons are counted. This provides a high quantum
efficiency (.about.80%) with dark count levels approaching a cooled
PMT (quantum efficiency typically 10%). The thermoelectrically
cooled silicon APD provides a compact form combined with
state-of-the-art sensitivity. While higher sensitivity may not be
required for detecting fluorescent signals from standard sequencing
reactions, silicon APDs in the counting mode can be ideal for
detecting fluorescent signals directly from genomic DNA assays
(i.e., without amplification).
[0074] Detectors with the required characteristics are commercially
available and include the simple silicon PIN detector, the silicon
APD, or a photodiode array (CCD). The simple silicon detector is
the cheapest, the silicon avalanche is the most sensitive, and the
CCD is most useful for multiple capillary systems as it provides
many detectors in a single unit.
VII. Coupled Systems
[0075] The PME technology has sufficient flexibility for coupling
to a variety of formats, for example, the PME system can be coupled
to conventional capillary electrophoresis (CE) or to separation
and/or purification using high-density arrays/biochips. Ideally, a
DNA sequencing system capable of direct detection of fluorescent
assays for genomic DNA should (i) optimally excite all fluorescent
dyes, (ii) be capable of efficiently collecting photons over a
large part of the UV, visible and infrared spectrum, (iii)
continuous monitoring of fluorescent signals in high-throughput
array or high-density formats, (iv) maximize fluorescence emission
signals for detection, (v) be configured to minimize background
scattered light, and be automated using replaceable gel
matrices.
[0076] a. Capillary Electrophoresis
[0077] The PME fluorescence detection system of the present
invention can be coupled to conventional capillary electrophoresis
(CE) as a preferred method for resolving DNA fragments.
[0078] Microcapillary array electrophoresis generally involves the
use of a thin capillary or channel, which may or may not be filled
with a particular separation medium. Electrophoresis of a sample
through the capillary provides a size-based separation profile for
the sample. The use of microcapillary electrophoresis in size
separation of nucleic acids has been reported in, e.g., Woolley and
Mathies (1994). The high surface to volume ratio of these
capillaries allows for the application of higher electric fields
across the capillary without substantial thermal variation across
the capillary, consequently allowing for more rapid separations.
Furthermore, when combined with confocal imaging methods, these
methods provide sensitivity in the range of attomoles, which is
comparable to the sensitivity of radioactive sequencing methods.
Microfabrication of microfluidic devices including microcapillary
electrophoretic devices has been discussed previously (e.g.,
Jacobsen et al., 1994; Effenhauser et al., 1994; Harrison et al.,
1993; Effenhauser et al., 1993; Manz et al., 1992; and U.S. Pat.
No. 5,904,824). Typically, these methods comprise photolithographic
etching of micron scale channels on a silica, silicon or other
crystalline substrate or chip, and can be readily adapted for use
in the present invention. In some embodiments, the capillary arrays
may be fabricated from the same polymeric materials described for
the fabrication of the body of the device, using the injection
molding techniques described herein.
[0079] Tsuda et al., (1990), describes rectangular capillaries, an
alternative to the cylindrical capillary glass tubes. Some
advantages of these systems are their efficient heat dissipation
due to the large height-to-width ratio and, hence, their high
surface-to-volume ratio and their high detection sensitivity for
optical on-column detection modes. These flat separation channels
have the ability to perform two-dimensional separations, with one
force being applied across the separation channel, and with the
sample zones detected by the use of a multi-channel array
detector.
[0080] In many capillary electrophoresis methods, the capillaries,
e.g., fused silica capillaries or channels etched, machined or
molded into planar substrates, are filled with an appropriate
separation/sieving matrix. Typically, a variety of sieving matrices
are known in the art, which may be used in the microcapillary
arrays. Examples of such matrices include, e.g., hydroxyethyl
cellulose, polyacrylamide, agarose and the like. Generally, the
specific gel matrix, running buffers and running conditions are
selected to maximize the separation characteristics of the
particular application, e.g., the size of the nucleic acid
fragments, the required resolution, and the presence of native or
undenatured nucleic acid molecules. For example, running buffers
may include denaturants, chaotropic agents such as urea or the
like, to denature nucleic acids in the sample.
[0081] The use of replaceable gel matrices, which suppress
electroendoosmotic flow and DNA-capillary wall interactions such as
polydimethylacrylamide (Madabhushi, 1998), may be used for
electrophoretic separations in the present invention.
[0082] b. Chromatographic Techniques
[0083] Alternatively, chromatographic techniques may be coupled to
the PME fluorescence detection system of the present invention.
There are many kinds of chromatography, which may be used including
liquid chromatography, HPLC and many specialized techniques, such
as reverse phase HPLC, normal phase HPLC, anion exchange, cation
exchange, denaturing HPLC, size exclusion or gel permeation, and
hydrophobic interaction.
[0084] c. Microfluidic Techniques
[0085] Microfluidic techniques can be used for fluid flow with the
PME system, and includes the use of a platform such as
microcapillaries, designed by ACLARA BioSciences Inc., or the
LabChip.TM. "liquid integrated circuits" made by Caliper
Technologies Inc. Miniaturizing some of the processes involved in
genetic analysis has been achieved using microfluidic devices. For
example, published PCT Application No. WO 94/05414, by Northrup and
White, incorporated herein by reference, reports an integrated
micro-PCR.TM. apparatus for collection and amplification of nucleic
acids from a specimen. U.S. Pat. Nos. 5,304,487 to Wilding et al.,
and 5,296,375 to Kricka et al., discuss devices for collection of
cell containing samples and are incorporated herein by reference.
U.S. Pat. No. 5,856,174 describes an apparatus, which combines the
various processing and analytical operations involved in nucleic
acid analysis and is incorporated herein by reference.
[0086] d. Chip Technologies
[0087] Specifically contemplated by the present inventors for
combining with the PME system are chip-based DNA technologies.
These techniques involve quantitative methods for analyzing large
numbers of genes rapidly and accurately.
[0088] Chip based technologies that can be used in the current
invention include the those described in U.S. Pat. No. 6,153,379
and Shumaker et al. (1996) where a method of analyzing
oligonucleotides is described in which oligonucleotides are
extended with fluorescent dideoxynucleotides, and detected using an
automated fluorescent DNA sequencer. The oligonucleotide length
identifies the known mutation site, and the fluorescence emission
of the ddNTP identifies the mutation. Another method of analyzing
oligonucleotides involves using template DNA annealed to an
oligonucleotide array. The analysis is done using a Phosphor Imager
and alpha-.sup.32P labels. Kurg et al., (2000) describes an
integrated system with DNA chip and template preparation, multiplex
primer extension on the array, fluorescence imaging, and data
analysis. The method includes annealing DNA to immobilized primers,
which promote sites for template-dependent DNA polymerase extension
reactions using four unique fluorescently labeled dideoxy
nucleotides. A mutation is detected by a change in the color code
of the primer sites.
[0089] Motorola BioChip Systems has the I-based SNP systems with
array technology centered on a three-dimensional gel pad format
consisting of flexible content architectures.
[0090] The MassARRAY system, developed by SEQUENOM (U.S. Pat. Nos.
5,547,835, 6,238,871, and 6,235,478) has a platform capable of high
throughput SNP analysis using enzymology, bioinformatics and
miniaturized chip-based disposables with mass spectrometry
detection. The MassARRAY technology can be used to distinguish
genotypes using MALDI-TOF mass spectrometry. DNA fragments
associated with genetic variants are simultaneously separated and
detected, measuring target DNA associated with SNPs and other forms
of genetic variation directly.
[0091] f. Bead Technologies
[0092] The PME system may be coupled with microbeads containing
bound DNA or RNA segments. These beads may be plain or coated with
material such as biotin, terminal amines, or Protein G to
facilitate binding of the biomolecule to the bead. Microbeads may
be used in conjunction with, for example, microfluidic systems or
electrophoretic systems and PME detection. The beads may be porous
or solid and made of polymers such as polystyrene or agarose and
may optionally contain magnetic particles, such as those obtained
from Dynal thus allowing use in magnetic separation techniques. The
beads may also be porous thereby providing increased surface area
for binding. Magnetic beads are used in a manner similar to polymer
beads. However, these beads contain a magnetic source such as
Fe.sub.2O.sub.3 or Fe.sub.3O.sub.2 that can be used for rapid and
simple separation.
VIII. Continuous Fluorescence Monitoring
[0093] To capture minute fluorescent signals for multiple capillary
array or other formats derived from direct assays, the high duty
cycle of continuous monitoring systems has significant advantage
over scanning systems. Moreover, continuous systems have other
benefits that simplify the mechanical operation of the system, such
as no moving mechanical stages that wear down, break down, or
become misaligned. These systems use the laser light power more
efficiently allowing greater operation of fluorescence excitation
under photobleaching conditions. Basically, there are two known
methods for continuous monitoring of fluorescent signals, namely
on-column and post-column detection. Both methods can be used with
the PME technology of the current invention for DNA sequencing
applications using capillary electrophoresis.
[0094] a. On-Column Detection
[0095] The first on-column detection schemes were described using
single capillary systems (Luckey et al., 1990; Swerdlow et al.,
1990; Drossman et al., 1990; Cohen et al., 1990). In 1990, the
Smith group described the first 4-color on-column system using a
multi-line argon-ion laser (488 nm and 514.5 nm) to illuminate a
single capillary. The fluorescence emitted from FAM, JOE, TAMRA,
and ROX dye-labeled sequencing reactions was collected orthogonal
to the excitation source and using a set of beamsplitters, the
emitted fluorescence was directed to a set of 4 PMT detectors
(Luckey et al., 1990). Karger et al. described the first on-column,
spectral dispersion system using a CCD camera (Sweedler et al.,
1991) to detect emission wavelengths approximately in the range of
500 nm to 650 nm derived from the fluorescence of a 4-color
sequencing reaction. Similar to the Smith design, the fluorescence
was collected perpendicular to the excitation path, but the authors
describe the unique feature of 4 target areas that correspond to
the emission properties of FAM, JOE, TAMRA, and ROX dye-labeled
reactions and the binning of pixels within the target areas for
enhanced readout speed and reduced readout noise (Karger et al.,
1991).
[0096] The first capillary array instrument was a modified DuPont
GENESIS 2000 DNA sequencing instrument (Probe et al., 1987), which
the slab gel was replaced with a 12-capillary array (Zagursky et
al., 1990). An argon-ion laser beam (single line 488 nm) was
scanned across the capillaries and fluorescence was detected using
the 510 nm and 540 nm resolved, two PMT detector scheme. The
Mathies group has described a confocal-fluorescence system, which
has potential for improved signal to noise and scanned back and
forth using a motor-driven translation stage across a 24-capillary
array (Huang et al., 1992). Fluorescence was collected using a
180.degree. geometry to the argon ion laser (488 nm) excitation
source by confocal detection. Originally, a two PMT detector system
coupled to a two-dye binary coding method was used with different
mole fraction combinations of FAM and JOE dye reactions to
differentiate the four-termination reaction set (Huang et al.,
1992). Recently, the Mathies group developed a 4-color confocal
scanning detection system that directs the fluorescence emission
through a number of different dichroic beamsplitters to 4 PMT
detectors (Kheterpal et al., 1996). The 4-color confocal scanning
system described here represents the core technology for the
commercially available 96-capillary MegaBACE 1000 instrument
(Molecular Dynamics). More recently, the Riken group has described
a 384-capillary DNA sequencer, which uses an argon ion laser in a
scanning mode and splits the fluorescence emission signal to 4
different band-pass filters coupled to dedicated PMT detectors
(Shibata et al., 2000). Although mechanical scanning systems can
uniformly illuminate each capillary in the array, in general, they
can be problematic due to breakdown and misalignment, low duty
cycle, and potential photobleaching by duty cycle compensation with
higher laser power levels.
[0097] Several side-entry illumination schemes directly through
capillary arrays have been problematic in scaling from a single
capillary to array systems, mainly because of reflection and
refraction of the laser beam at the capillary boundaries. The Yeung
group described a 10-capillary system that used axial beam
illumination and CCD detection, in which individual optical fibers
coupled via an argon ion laser were directly inserted into the ends
of the capillary tubes (Taylor et al., 1993). The intrusion of
optical fibers into separate capillaries, however, affected the
electroendoosmotic flow and increased the possibility for
contamination and clogging (Lu et al., 1995). This group also
described illumination by a line of laser light focused across the
array using a plano-convex cylindrical lens to illuminate a
100-capillary array (Ueno et al., 1994); however, this design
inefficiently used less than 0.5% of the laser power to illuminate
each capillary (Lu et al., 1995). Quesada and Zhang (1996)
described an 8-capillary prototype in which argon ion illumination
and fluorescence detection were achieved by using individual
optical fibers constructed in an orthogonal geometry and imaged
through a spectrograph using a CCD camera. Scaling beyond the
initial individual optical fiber design, however, was reported to
be problematic because of the increased demand on laser power, and
the bulkiness and irregular alignment of the optical junction
connectors to each capillary tube (Quesada et al., 1998).
[0098] To address the loss of laser illumination by refraction, two
independent groups have demonstrated that refracted laser light at
the capillary surface can be focused repeatedly under optimized
optical conditions and produce a waveguiding effect (Quesada et
al., 1998; Anazawa et al., 1996). Quesada et al. (1998)
demonstrated that a bi-directionally illuminated waveguide system
using an argon ion laser showed good illumination across a
12-capillary array with a difference of less than 10% across the
array, and with potential for scaling to a 96-capillary system with
near uniform illumination. Alternatively, index matching of the
capillary array has also been shown to reduce laser light
refraction and scattering across the array (Lu et al., 1995), but
to a smaller degree than waveguide illumination (Quesada et al.,
1998).
[0099] b. Post-Column Detection
[0100] The first post-column detection schemes also described
single capillary systems (Swerdlow et al., 1990; Swerdlow et al.,
1991). The Dovichi group first reported a 4-color post-column
detection system using a sheath flow cuvette (Swerdlow et al.,
1990). These authors demonstrated the sheath flow concept using the
four-spectral channel system based on the work described by the
Smith group (Luckey et al., 1990) and a two-spectral channel system
based on the work described by Prober et al. (1987). Unlike the
beamsplitter design, the four-spectral channels were discriminated
using a rotating wheel containing 4 specific band-pass filters,
which was synchronized to a sector wheel that alternated the
excitation source between an argon-ion laser (488 nm) and a green
He--Ne laser (543.5 nm). The appropriate orientation of the filter
wheel directed the specific emission light wavelengths of FAM, JOE,
TAMRA, and ROX dye-labeled sequencing reactions to a single PMT
detector. The two-spectral channel, two intensity system used a
single argon-ion laser (488 nm) to excite the 4-different
succinyl-fluorescein dye-labeled reactions, which have limited
spectral resolution. The emission fluorescence centered at 510 nm
and 540 nm was uniquely split using a single dichroic mirror and
detected with two PMT detectors. The assignment of the nucleotide
sequence was performed by determining the ratio of
baseline-corrected peak intensities (Prober et al., 1987).
[0101] The post-column sheath-flow approach has the advantage of
eliminating excitation light scattering at the capillary surfaces
and in illuminating all capillary tracks simultaneously. Kambara
and Takahashi described the first multiple sheath-flow capillary
array system using a He--Ne laser (594 nm) and single color Texas
Red (ROX) labeled DNA sequencing reactions (Kambara et al., 1993).
This system was later developed into a 4-color system using the
combined excitation lines from both an argon ion laser (488 nm) and
a YAG laser (532 nm) to simultaneously irradiate FAM, JOE, TAMRA,
and ROX dye-labeled reactions. The fluorescence was dispersed using
an image-splitting prism, passed through 4 different optical
filters, and detected as two-dimensional line images using a cooled
CCD camera (Takahashi et al., 1994). The Hitachi technology
described here represents the core technology for the commercially
available 96-capillary 3700 DNA sequencer instrument (Applied
Biosystems).
[0102] Another application of the sheath flow approach to
post-column detection was described in U.S. Pat. No. 6,139,800
where fluorescent detection of labeled particles is accomplished
for capillary electrophoresis. Multiple wavelength sources excite
the labeled particles and multiple wavelength discriminating
detectors detect the sample emissions.
[0103] Capillary array sheath-flow cuvettes require careful
attention to hydrodynamic focusing, which can be achieved by
uniformly spacing the capillaries in the cuvette holder. Recently,
the Dovichi group has described two sheath flow cuvettes, the
rectangular cuvette that is tapered to force 5-capillaries to
squeeze together (Zhang et al., 1999) and the micro-machined
cuvette with uniformly spaced etched grooves to align 16 individual
capillaries (Crabtree et al., 2000). Of the two designs, the latter
one shows more promise for scaling to a 96-capillary array.
IX. Obtaining High Sensitivity and Low Background Scattered
Light
[0104] In 1990, several groups reported limit of detection values
corresponding to 10.sup.-19 moles for CE systems and were performed
using a 10.sup.-11 M solution of fluorescein flowing continuously
in an open capillary (Swerdlow et al., 1990; Drossman et al.,
1990). These systems, however, were roughly 10-fold less sensitive
than the sheath flow detector system, which has a reported
detection limit of 10.sup.-20 moles (Swerdlow et al., 1990; Kambara
et al., 1993). Coupled to an APD operating in the Geiger counting
mode, this sheath flow system described recently by the Dovichi
group showed a limit of detection of 130.+-.30 fluorescein
molecules (Zhang et al., 1999).
[0105] The number of fluorescence counts generated is the product
of two factors: (i) the number of fluorescence photons generated
and (ii) the overall counting efficiency. The number of
fluorescence photons generated is given by
N p = .sigma. NP Ahv QYt ##EQU00001##
where N is the number of dye molecules being excited, P is the
laser power (J/s), .sigma. is the absorption cross-section
(cm.sup.2), A is the area of the laser beam, h.nu. is the energy of
an excitation photon (J), QY is the quantum yield for fluorescence,
and t is the observation time (s). The overall counting efficiency
is given by
Eff = SA QE 4 .pi. ##EQU00002##
where SA is the solid angle of fluorescent light collection (in
steradians) and QE is the quantum efficiency of the detector.
Assuming that the cross-section is 3.8.times.10.sup.-16 cm.sup.2
(.epsilon.=100,000 liters/(mol-cm)), the wavelength of the
excitation is 600 nm, the QY is unity, the numerical aperture is
unity, and the quantum efficiency is 0.8, then
N p = 73 NPt A ##EQU00003##
[0106] The PME system of the current invention is useful as a DNA
sequencing device for analyzing SNPs directly from genomic DNA
without cloning or PCR amplification. Estimating 10.sup.6 white
blood cells per cm.sup.3 of blood, one calculates 73,000 counts in
a second could be expected for a single SNP probing of 1 ml of
blood without any concentration of solution with a laser power of 1
mW and an area of 1 cm.sup.2. Concentration would reduce A without
reducing N. Thus, there is an easily detectable signal without
electrophoresis or heroic measures, granted that the sequencing
assays are free of unincorporated dye. Note that focusing the laser
reduces N, but simultaneously reduces A by the same factor so that
the signal does not depend upon focusing as long as the numerical
aperture can be maintained (defocusing limit) or the dye is not
destroyed by two photon absorption effects (tight focusing
limit).
[0107] The situation is somewhat different when determining a
number of SNPs simultaneously. Then electrophoresis becomes
necessary in order to separate the various fragments. Typically in
Sanger sequencing, a 10-to-30 .mu.L sample is introduced into the
3700 DNA sequencing instrument, but only about 10 mL is actually
introduced into the capillary. This is a loss in N of about a
factor of 1000-to-3000 reducing N to .about.100-to-300. However, if
using the sheath flow approach (Anazawa et al., 1996; Zhang et al.,
1999), the dye-tagged fragments emerging from a 50 .mu.m ID
capillary will occupy a cylinder about 2 mm long and perhaps 100
.mu.m in diameter. Its cross-sectional area A will be
2.times.10.sup.-3 cm.sup.2 and the number of counts in a 1 s
counting interval will be
N p = 73 300 0.001 1 0.002 = 11 , 000 ##EQU00004##
[0108] This calculation is consistent with the report by Zhang et
al. (1999) that 130 fluorescein molecules emerging from a 50 .mu.m
capillary could be detected in 0.2 sec counting time. It will be
possible to reduce the wastage factor of 3000 cited above to
perhaps 100 by devising low volume methodologies to use a 1 .mu.L
sample.
[0109] Thus, a capillary electrophoresis system that utilizes
multiple, compact solid-state lasers and laser diodes coupled to
highly efficient detection devices, which employ continuous
illumination and sheath flow detection features is a preferred
embodiment of the current invention. These integrated technologies
are well suited for the application of the PME technology and have
sufficient feasibility for direct detection assays.
X. Single Nucleotide Polymorphisms (SNPs)
[0110] Spontaneous mutations that arise during the course of
evolution in the genomes of organisms are often not immediately
transmitted throughout all of the members of the species, thereby
creating polymorphic alleles that co-exist in the species
populations. Often polymorphisms are the cause of genetic diseases.
Several classes of polymorphisms have been identified. For example,
variable nucleotide tandem repeats (VNTRs) are polymorphic and
arise from spontaneous tandem duplications of di- or trinucleotide
repeated motifs of nucleotides. If such variations alter the
lengths of DNA fragments generated by restriction endonuclease
cleavage, the variations are referred to as restriction fragment
length polymorphisms (RFLPs). RFLPs are been widely used in human
and animal genetic analyses and forensic and paternity testing.
[0111] Another class of polymorphisms is generated by the
replacement of a single nucleotide. Such single nucleotide
polymorphisms (SNPs) rarely result in changes in a restriction
endonuclease site. SNPs are the most common genetic variations and
occur once every 300-to-1000 bases, and several SNP mutations have
been found that affect a single nucleotide in a protein-encoding
gene in a manner sufficient to actually cause a genetic disease.
SNP diseases are exemplified by hemophilia, sickle-cell anemia,
hereditary hemochromatosis, late-onset Alzheimer disease, etc.
[0112] SNPs can be the result of deletions, point mutations and
insertions and in general any single base alteration, whatever the
cause, can result in a SNP. The greater frequency of SNPs means
that they can be more readily identified than the other classes of
polymorphisms. The greater uniformity of their distribution permits
the identification of SNPs "nearer" to a particular trait of
interest. The combined effect of these two attributes makes SNPs
extremely valuable. For example, if a particular trait reflects a
mutation at a particular locus, then any polymorphism that is
linked to the particular locus can be used to predict the
probability that an individual will be exhibit that trait.
[0113] Several methods have been developed which can be combined
with PME technology to screen polymorphisms and some non-limiting
examples are listed below. Such methods include the direct or
indirect sequencing of the site, the use of restriction enzymes
where the respective alleles of the site create or destroy a
restriction site, the use of allele-specific hybridization probes,
the use of antibodies that are specific for the proteins encoded by
the different alleles of the polymorphism, or any other biochemical
interpretation.
[0114] a. DNA Sequencing
[0115] Traditionally, DNA sequencing has been accomplished by the
"dideoxy-mediated chain termination method," also known as the
"Sanger Method" (Sanger, F., et al., 1975), which involves the
chain termination of DNA synthesis by the incorporation of
2',3'-dideoxynucleotides (ddNTPs) using DNA polymerase. The
reaction also includes the natural 2'-deoxynucleotides (dNTPs),
which extend the DNA chain by DNA synthesis. Thus, balanced
appropriately, competition between chain extension and chain
termination results in the generation of a set of nested DNA
fragments, which are uniformly distributed over thousands of bases
and differ in size as base pair increments. Electrophoresis is used
to resolve the nested set of DNA fragments by their respective
size. The fragments are then detected by the previous attachment of
four different fluorophores to the four bases of DNA (i.e., A, C,
G, and T), which fluoresce at their respective emission wavelengths
after excitation at their respective excitation wavelengths using
PME technology. The DNA sequencer may be based on an
electrophoresis system with the throughput capacity of a single
column, 4, 8, 16, 48, 96 or 384-capillary instrument or may
integrate with other separation platforms, including high-density
chip arrays.
[0116] Similar methods which can be used with PME technology
include the "chemical degradation method," also known as the
"Maxam-Gilbert method" (Maxam, A. M., et al., 1977). Sequencing in
combination with genomic sequence-specific amplification
technologies, such as the polymerase chain reaction may be utilized
to facilitate the recovery of the desired genes (Mullis, K. et al.,
1986; European Patent Appln. 50,424; European Patent Appln. 84,796,
European Patent Application 258,017, European Patent Appln.
237,362; European Patent Appln. 201,184; U.S. Pat. No. 4,683,202;
U.S. Pat. No. 4,582,788; and U.S. Pat. No. 4,683,194).
[0117] b. Primer Extensions Methods for SNP Detection
[0118] A preferred assay for the detection of multiple SNPs is the
single nucleotide primer extension method, which has also been
called single nucleotide incorporation assay and primer-guided
nucleotide incorporation assay. These methods rely on the specific
hybridization of an identically complementary oligonucleotide
sequence to the genetic region or target of interest, which has
been amplified by the polymerase chain reaction (PCR) or cloned
using standard molecular biology techniques (Sambrook et al.,
1989). However, unlike the Sanger reaction, the primer extension
method is assayed using either single unlabeled or labeled dNTPs,
3'-modified-dNTPs, base-modified-dNTPs, or alpha-thio-dNTPs or a
mixture of ddNTPs, which all can chain terminate DNA synthesis
under appropriate conditions following the incorporation of a
single nucleotide (U.S. Pat. Nos. 4,656,127; 5,846,710; 5,888,819;
6,004,744; 6,013,431; 6,153,379, herein incorporated by
references). Here, the word usage of 2'-deoxynucleoside
triphosphate, 2'-deoxyribonucleoside triphosphate, dNTP,
2'-deoxynucleotide, 2'-deoxyribonucleotide, nucleotide or natural
nucleotide are used as synonymous terms and used interchangeably in
the current patent document.
[0119] 1. Using Single Unlabeled dNTPs
[0120] A method, called pyrosequencing, uses singly added unlabeled
dNTPs and is based on a repetitive cyclic method of start-stop DNA
synthesis of single nucleotide addition. Pyrosequencing is a
mini-sequencing technique and relies on a multi-enzyme cascade to
generate light by luciferase as the mode of detection (Nyren et
al., 1993; Ronaghi et al., 1998). PCR amplified DNA fragments,
which contain a 5'-biotin group are immobilized on
streptavidin-coated magnetic beads. An incorporated unlabeled
nucleotide event is monitored by the release of inorganic
pyrophosphate and the subsequent release of light following the
primer extension step. Because pyrosequencing is a cyclic DNA
sequencing strategy, the placement of the oligonucleotide
immediately adjacent to the 5'-position is not always required, and
the primer can be placed within the sequencing read-length of the
method, usually 20 bases. Major disadvantages with the
pyrosequencing technique are the method has low sensitivity, the
high cost of the reagents, particularly the enzymes, and sequence
difficulties with homopolymer repeats (i.e., AAAAA) and high "GC"
rich regions.
[0121] 2. Using Single Labeled dNTPs
[0122] Unlike the pyrosequencing method, which can extend the
primer beyond a single nucleotide position, other investigators
have reported single nucleotide incorporation assays using single
nucleotides labeled with radioactive, non-radioactive, or
fluorescent tags (Sokolov, 1990; Syvanen et al., 1990; Kuppuswamy
et al., 1991; Prezant and Ghodsian, 1992). In these strategies, the
placement of the oligonucleotide is immediately adjacent to the
5'-position of the single nucleotide mutation site under
investigation. Sokolov showed the specific incorporation and
correct identification of single nucleotide sequences of a known
sequence in the cystic fibrosis gene using alpha .sup.32P-dCTP and
alpha .sup.32P-dGTP (Sokolov, 1990). In another report, a similar
approach was described for the detection of the .DELTA.508 mutation
in the cystic fibrosis gene and point mutations in exon 8 of the
factor IX gene (Hemophilia B) (Kuppuswamy et al., 1991). Following
PCR amplification of specific target regions, specific
oligonucleotides, which hybridized immediately adjacent to the
5'-position of the mutation under investigation, were extended by
one nucleotide using single alpha .sup.32P-dNTPs. Moreover, a dual
labeling strategy for SNP detection was reported for exon 4 of the
apolipoprotein E gene using different combinations of
.sup.3H-labeled dTTP, alpha .sup.32P-labeled dCTP, or
digoxigenin-11-dUTP. Following immobilization of PCR-amplified
fragments on avidin-coated polystyrene beads, single nucleotide
extension assays were performed and incorporated nucleotides were
detected using a liquid scintillation counter at different window
settings for .sup.3H and .sup.32P radioactivity or colorimetrically
using an alkaline phosphatase assay (Syvanen et al., 1990). A
similar method, called Trapped-Oligonucleotide Nucleotide
Incorporation (TONI) using a biotinylated primer immobilized on
streptavidin magnetic beads and singly added alpha .sup.32P-labeled
dNTPs was described for genetic screening of mitochondrial
polymorphisms and different hemoglobin genotypes (Prezant and
Ghodsian, 1992).
[0123] 3. Using Single 3'-Modified dNTPs
[0124] Metzker et al. proposed the base addition sequencing
strategy (BASS), which is a mini-sequencing technique and involves
stepwise single nucleotide sequencing by repetitive cycles of
incorporation of 3'-O-modified nucleotides, detection of the
incorporated nucleotide, and deprotection of the 3'-O-modified
nucleotide to generate the 3'-OH substrate and allow for the next
cycle of DNA synthesis (Metzker et al., 1994). Eight different
3'-O-modified dNTPs were synthesized and tested for incorporation
activity by a variety of DNA polymerases. 3'-O-(2-Nitrobenzyl)-dATP
is a UV sensitive nucleotide and was shown to be incorporated by
several thermostable DNA polymerases. Base specific termination and
efficient photolytic removal of the 3'-protecting group was
demonstrated. Following deprotection, DNA synthesis was reinitiated
by the incorporation of natural nucleotides into DNA. The
identification of this labile terminator and the demonstration of a
one-cycle stop-start DNA synthesis identified the initial steps in
the development of a novel sequencing strategy. The major challenge
for SNP detection using BASS, however, is the continued synthesis
and identification of novel 3'-modified nucleotides that give the
desired properties of termination with removable protecting
groups.
[0125] 4. Using Single Base-Modified 3'-dNTPs
[0126] Kornher and Livak (1989) described another method by
incorporating mobility shifting modified-dNTPs (i.e., the
attachment of a biotin group or a fluorescein group to the base)
into a PCR amplified DNA sample. The SNP is identified by
denaturing gel electrophoresis by observing a "slower" migrating
band, which corresponds to the incorporated modified nucleotide
into the DNA fragment.
[0127] 5. Using Single Alpha-Thio-dNTPs
[0128] Other methods that can be employed to determine the identity
of a nucleotide present at a polymorphic site utilize modified
alpha-thio-dNTPs, which are resistant to exonuclease cleavage (U.S.
Pat. No. 4,656,127). An oligonucleotide, of identical sequence to a
complementary target region, immediately flanks the 5'-position of
the single nucleotide mutation site under investigation. If the
polymorphic site on the DNA contains a nucleotide that is
complementary to the particular exonuclease-resistant nucleotide
derivative present, then that derivative will be incorporated by a
polymerase and extend the oligonucleotide by one base. Such
incorporation makes the primer resistant to exonuclease cleavage
and thereby permits its detection. As the identity of the
exonuclease-resistant nucleotide derivative is known one can
determine the specific nucleotide present in the polymorphic site
of the DNA.
[0129] 6. Using Single Labeled 2',3'-Dideoxynucleotides
[0130] Several groups have reported methods for single nucleotide
incorporation assays using labeled 2',3'-dideoxynucleotides to
specifically assay given SNPs of interest, which are detected by
autoradiography, colorimetrically, or fluorescently (Lee and
Anvret, 1991; Livak and Hainer, 1994; Nikiforov et al., 1994;
Shumaker et al., 1996). All of these methods rely on PCR to amplify
genomic DNA from patient material and careful design of
oligonucleotide sequences to target specific known mutations in
different genes. The separation of dideoxynucleotide incorporated
DNA fragments can be achieved electrophoretically (Lee and Anvret,
1991; Livak and Hainer, 1994; Nikiforov et al., 1994; Shumaker et
al., 1996) or by using high-density array chip formats (Shumaker et
al., 1996).
[0131] 7. By Direct Detection from Genomic DNA
[0132] One aspect of this invention is to develop a DNA sequencing
device for analyzing SNPs directly from genomic DNA without cloning
or PCR amplification. The present invention circumvents the
problems associated with the previously described methods for SNP
detection, which rely on a prior PCR or cloning step. These steps
potentially add errors in sample handling, introduction of
exogenous contamination, significant costs in reagents and labor
and seriously hamper the introduction of high-throughput SNP
detection into a clinical or medical setting. Because of its
simplicity, the PME technology has the capability of greatly
increasing the multiplexing of numerous SNPs simultaneously, which
is significantly limited in other previously described systems. For
example, 4-, 8-, 12- and 16-different fluorophores identified
herein can be coupled to appropriate ribonucleotides,
2'-deoxynucleotides, 2',3'-dideoxynucleotides,
2'3'-unsaturated-dNTPs and/or other modified nucleotides.
[0133] Moreover, the assay can be multiplexed, and coupled to a
high-throughput electrophoresis system, and in this configuration,
it has the capability of analyzing 2,000-to-4,000 independent SNPs
in approximately 30-to-60 minutes. Multiplexing is accomplished by
varying the length of the specific primers by increments of 2-to-3
bases, and by increasing the number of fluorophores detected in the
SNP assay. Twenty to 100, or more specifically 30-to-50 primers,
all differing in length, could be assayed in a single nucleotide
primer extension assay, which could be resolved by electrophoresis
and detected by PME in a single capillary. Since the longest primer
sequence will generally not exceed 100 bases in length, fast
separation times are expected. It is noteworthy that SNP specific
primers can also be arrayed in a high-density chip format, thus
eliminating the need for electrophoresis. The scalability of the
DNA sequencer is multiplied by 96-capillaries.
[0134] c. Massively Parallel Signature Sequencing (MPSS)
Strategy
[0135] Brenner et al. (2000) recently presented data on their
massively parallel signature sequencing (MPSS) strategy, which is
another cyclic process involving type II restriction
digestion/ligation/hybridization to sequence over 269,000
signatures of 16-20 bases in length (Brenner et al., 2000). The
main disadvantage of MPSS is low efficiency where only 25% of the
starting DNA templates yield signatures after application of the
base-calling algorithms.
[0136] d. Ligase Chain Reaction (LCR)
[0137] LCR can also amplify short DNA regions of interest by
iterative cycles of denaturation and annealing/ligation steps
(Barany, 1991). LCR utilizes four primers, two adjacent ones that
specifically hybridize to one strand of target DNA and a
complementary set of adjacent primers that hybridize to the
opposite strand. LCR primers must contain a 5'-end phosphate group
such that thermostable ligase (Barany, 1991) can join the 3'-end
hydroxyl group of the upstream primer to the 5'-end phosphate group
of the downstream primer. Successful ligations of adjacent primers
can subsequently act as the LCR template resulting in an
exponential amplification of the target region. LCR is well suited
for the detection of SNPs since a single-nucleotide mismatch at the
3'-end of the upstream primer will not ligate and amplify, thus
discriminating it from the correct base. Any or all of the LCR
primers can be labeled with different fluorescent dyes for
unambiguous discrimination of specific SNPs. Although LCR is
generally not quantitative, linear amplifications using one set of
adjacent primers, called the Ligase Detection Reaction, can be
quantitative. Coupled to PCR, linear ligation assays can also be
used as a mutation detection system for the identification of SNPs
using both wild-type-specific and mutant-specific primers in
separate reactions.
[0138] e. Oligonucleotide Ligation Assay (OLA)
[0139] The Oligonucleotide Ligation Assay was first reported to
detect SNPs from both cloned and clinical materials using a 5'-end
biotin group attached to the upstream primer and a nonisotopic
label attached to the downstream primer (Landegren et al., 1988).
Allele-specific hybridizations and ligations can be separated by
immobilization to a streptavidin-coated solid support and directly
imaged under appropriate conditions without the need for gel
electrophoretic analysis. Subsequently, Nickerson et al. have
described an automated PCR/OLA method for the diagnosis of several
common genetic diseases. Following PCR amplification, the upstream
5'-end biotinylated primer and digoxigenin labeled downstream
primer are ligated together under appropriate and specific
annealing conditions, captured on streptavidin coated microtiter
plates and detected colorimetrically by an alkaline phosphatase
assay (Nickerson et al., 1990)
[0140] f. Ligase/Polymerase-Mediated Genetic Bit Analysis
[0141] U.S. Pat. No. 5,952,174 describes a method that also
involves two primers capable of hybridizing to abutting sequences
of a target molecule. The hybridized product is formed on a solid
support to which the target is immobilized. Here the hybridization
occurs such that the primers are separated from one another by a
space of a single nucleotide. Incubating this hybridized product in
the presence of a polymerase, a ligase, and a nucleoside
triphosphate mixture containing at least one deoxynucleoside
triphosphate allows the ligation of any pair of abutting hybridized
oligonucleotides. Addition of a ligase results in two events
required to generate a signal, that is extension and ligation. This
provides a higher specificity and lower "noise" than methods using
either extension or ligation alone and unlike the polymerase-based
assays, this method enhances the specificity of the polymerase step
by combining it with a second hybridization and a ligation step for
a signal to be attached to the solid phase.
XI. Data Acquisition and Analysis
[0142] The PME system, including switching the lasers and
collecting the data are under computer control in a unified
hardware/software framework. Cross-platform versatility is
achieved, for example, by using PCI-bus data acquisition and
controller cards and LabView.TM. software from National
Instruments. The graphically oriented acquisition and analysis
environment provided by LabView has led to its widespread adoption
in laboratory use. Software programs have been developed to perform
several operations for the PME sequencing prototypes including: (i)
generating a trigger signal for the TTL clock chip to govern the
basic 4 sub-cycles (serial pulsing of lasers 1 through 4) of each
cycle, (ii) controlling the blocking of scattered light, (iii)
acquisition of the time-integrated signals from the photodetector,
and (iv) controlling various operations for automated capillary
electrophoresis methods. Scattered light is controlled by use of a
liquid crystal tunable filter under electronic command (e.g., the
VariSpec from CRI, Inc.) to provide a different edge block for each
of the four lasers. When using a PMT or avalanche photodiode,
sampling should be taken several times per sub-cycle for purposes
of time-integration. For the full-scale multichannel CCD operation,
only one read per sub-cycle is necessary. These different modes of
operation are easily handled in software. As mentioned above, a
primary goal of this invention is direct detection, which would
eliminate the need for PCR amplification. This requires high direct
sensitivity such as can be obtained with a spectroscopic-grade CCD
camera with very low readout noise (e.g., a few accumulated photons
per readout).
[0143] In addition, software programs that perform a number of data
analysis steps, including spectral matrix correction, baseline
correction, electrophoretic mobility corrections, base-calling of
the single nucleotide, quantitation of peak heights for
heterozygote analyses, allele association by electrophoretic
position and order of different fluorescently labeled gene targeted
primers are developed. Excitation by PME produces some level of
cross-talk from the non-matched laser pulses other than from the
best matched laser. As discussed previously, laser-dye combinations
that minimize non-matched laser cross-talk can be easily
identified, so that time correlated excitation of the correct
fluorophore can be identified and made with high confidence. In
general, however, it will be necessary to accommodate heterozygous
base pairs, particularly for SNP analyses in which more than one
fluorophore is excited at a time. Under non-saturating conditions,
this leads to linear relations between the number N.sub.i of
photons detected due to illumination by laser i and the number
n.sub.j of molecules of dye j,
N.sub.i=.SIGMA..sub.j.alpha..sub.i,jn.sub.j
The matrix .alpha. implicitly contains factors including the molar
extinction coefficients of the different dyes at frequency i, their
quantum yields, the efficiencies of the laser and the detection
system, and attenuation effects. From a practical point of view,
the relative magnitudes of the elements .alpha..sub.i,j are
calibrated experimentally. The matching of the dye maxima to the
laser colors makes the matrix diagonally dominant, allowing it to
be inverted without numerical difficulties. The inversion of
.alpha. removes the residual cross-talk between the dyes. Thus it
should be possible to directly obtain the relative numbers of dye
molecules with maximum contrast from the four-color experiments.
Corrections to this may come from scattered light. While a simple
baseline correction is easily accommodated, light fluctuations will
add some noise to the experiments. Several full cycles of the four
lasers will pass during each elution component, allowing reduction
of the noise by signal averaging. At the same time, the
quantification of the noise provides a real-time diagnostic for
estimating confidence levels on the signal measurements. Base-calls
should in any case proceed with high confidence since the precise
handling of the cross-talk will ordinarily yield one dye population
that is much higher than the others. In those cases where
heterozygotes are present, it is straightforward to distinguish
these mixed-populations since they will yield two dyes with higher
(and approximately equal) populations.
[0144] As used herein, the term "timing program" is meant to
include either software or hardware configured to signal a laser
firing sequence. The timing program will also contain information
from the laser firing sequence, which can be correlated with the
fluorescence emission signal.
[0145] As used herein, the term "excitation line" means a laser
beam or output from another excitation source having a spectral
wavelength or its corresponding frequency.
[0146] As used herein, the term "substantially all" means at least
90%. For example, "substantially all of the fluorescence signal" is
at least 90% of the signal.
[0147] As used herein, the term "substantially corresponds" means
that the difference between the two is less than 5%. For
differences in wavelengths in the visible spectrum, this
corresponds to differences of 20-33 nm, or more preferably 10-20
nm, or even more preferably 5-10 nm, or most preferably, when the
absorption maxima of a dye "substantially corresponds" to the
excitation wavelength of an excitation line, the two wavelengths
are less than 5 nm.
[0148] As used herein, the term "optically matched" means that the
wavelength maxima are within one nm of each other.
[0149] The term "substantially colinear" means that the laser beams
or excitation lines diverge from each other at angles of less than
5.degree..
[0150] The term "substantially coaxial" means that the laser beams
or excitation lines diverge from each other at angles of less than
5.degree..
[0151] The term "phased shifted," means that the phase relationship
between two alternating quantities of the same frequency is
changed. For example, consider two trains of repeating pulses such
as pulse train (1) laser 1 on followed by laser 1 off for three
times as long and pulse train (2) laser 2 on followed by laser 2
off for three times as long. One would say that the sequence of
equal time periods of laser 1 on, laser 1 off, laser 2 on, laser 2
off corresponds to the sum of pulse train (1) and pulse train (2)
with the phase of pulse train (2) delayed by a phase shift of
180.degree. or a half cycle. Note that for 180.degree., delayed or
advanced phase shifts are equivalent.
[0152] As used herein, the term an "on-time window" is defined as
the window of time corresponding to when the excitation line is
incident on the sample and includes the window of time
corresponding to the time after the excitation line has ceased
firing and before a second excitation line is incident on the
sample.
[0153] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising", the words "a" or "an" may mean one or
more than one. As used herein "another" may mean at least a second
or more.
XII. Examples
[0154] The following example is included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow, represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Optical System
[0155] To test the concept of the PME system, a simple breadboard
device was built to test the feasibility of discriminating
different fluorescent signals from a mixture of two BODIPY
fluorophores (Metzker et al., 1996). The optical path for combining
the pulsed 532 nm and 635 nm lines is depicted schematically in
FIG. 1 as solid lines. The laser light emitted from the green 532
nm solid state, diode-pumped, frequency-doubled Nd:YAG laser
(Intelite, Minden, NY) and the red 635 nm SPMT diode laser module
with external potentiometer (Blue Sky Research, San Jose, Calif.)
were each directed using two commercial grade aluminum steering
mirrors (Edmund Industrial Optics, Barrington, N.J.) to a dual
prism assembly. The prisms were coated with a single layer HEBBAR
antireflection material, which reduced polarization at the prism
surfaces by increasing total transmittance. The high dispersion
equilateral prisms were constructed from F2, grade "A" fine
annealed flint glass and were positioned at a forty-five degree
angle relative to one another to allow efficient overlap of the two
beams by inverse dispersion into a single beam, FIG. 2. The
flexibility of this design allows as many as eight excitation lines
originating from discrete point sources (five lasers are shown in
FIG. 1 for illustration) to be combined efficiently by the inverse
dispersion strategy.
[0156] The combined laser beams were directed into the cuvette
assembly box, which consists of a hollow aluminum lightproof box.
The box was modified by affixing two "floating" adjustable iris
fixtures (Edmund Industrial Optics) to minimize the amount of stray
light entering the box. A 10 cm cylindrical optically correct glass
cuvette (NSG Precision Cells, Inc, Farmingdale, N.Y.) was installed
and mounted using two black delrin holders. A 500K multi-alkali PMT
detector, which has good sensitivity in the range of 280 nm to 850
nm, was coupled to the cuvette assembly box. The fluorescence was
detected directly from the cuvette using a collection lens in an
orthogonal geometry to the propagation direction of the excitation
laser beams.
Example 2
Pulse Generation System
[0157] There are a number of methods to serially pulse multiple
lasers, including mechanical chopping and TTL control. One strategy
was to serially pulse the 532 nm solid-state laser and the 635 nm
diode laser by TTL control using 74174-clock chip, (FIG. 3). The
advantages of the clock chip TTL circuit are its simplicity and
flexibility as it is designed to pulse of up to eight discrete
sources. As an alternative, a TTL bucket brigade circuit was
constructed because of its simplistic design in pulsing 4-lasers
using 2 dual J/K flip/flop chips. The TTL Clock Chip essentially
provides a means for distributing the timing pulses from a master
clock on the computer to the appropriate lasers. As each clock
pulse is received, the chip output sequentially shifts one step
from Q0 to Q1 . . . and finally to Q7 on the 8th clock pulse. Each
laser is turned on in turn for one and only one clock pulse. The
total cycle time may be easily varied by several orders of
magnitude, from tens of seconds to milliseconds simply by changing
the master clock frequency, and this has been successfully tested.
The duration of the laser pulses are always identical to each
other, and the off time between the pulses remain in the same exact
proportion to each other, so the overall cycle time may be changed
with a single parameter.
Example 3
Results and Discussion from the 2 Color PME Study
[0158] A preliminary experiment was performed to determine the
feasibility of the PME approach to discriminate each fluorophore
from a mixture of fluorescence dyes. To test the concept of
"colorblind" detection, this experiment was performed without the
aid of fluorescence band pass filters, laser line blocking filters,
gratings, prisms, or any other dispersing elements to aid in
distinguishing one dye's emission from the other. Moreover, the raw
output from the photomultiplier was sent directly to an
oscilloscope, without signal averaging or any other type of
processing enhancement. Each laser was alternately pulsed for 1.2
msec and was configured with the red laser connected to Q0 and the
green laser connected to Q3, FIG. 3. Altering the green laser to Q2
gave the correct firing sequence, which verified the proper
configuration of the TTL circuit (data not shown). The remaining Q
inputs were idle and resulted in dark spacing between laser pulses
of 2.4 msec (red-to-green) and of 4.8 msec (green-to-red). The red
and green lasers provided 5.5 mW and 4 mW of power, respectively.
The difference in power settings was purposeful to partially offset
the higher detector sensitivity of the green fluorescence over the
red fluorescence. Photographs were taken by a digital camera in
real time, and the fluorescent signals were recorded in a downward
(negative) direction, as the PMT multiplies electrons.
[0159] The two dyes examined were BODIPY (523/547) and BODIPY
(630/650), which have narrow absorption/emission half-bandwidths
(Metzker et al., 1996), and therefore are ideal for the color-blind
PME detection scheme. The dyes were analyzed at a concentration of
approximately 10.sup.-5 M in ethanol. This moderately high
concentration was chosen to assure that the signals were derived
entirely from the dye solutions, and not from other sources, such
as stray light, thus providing an accurate measure of the contrast
ratio. The emission from each dye fell in toto onto the
red-sensitive photomultiplier, which is a key feature of the
color-blind methodology.
[0160] In FIG. 4A, the oscilloscope trace was obtained from an
equal mixture of BODIPY dyes in the cuvette. The two channels from
the oscilloscope photographs were set to record the trigger signal,
which turns on the red laser (upper trace) and the fluorescence
signal from the PMT detector (lower trace). The green laser pulse
was subjected to a partial modulation, which gives the fluorescence
output a "two-finger" appearance, making it easily distinguishable
from the smooth red laser fluorescence. The small variation
observed in the red laser pulse-to-pulse fluorescence intensity was
due to 120 Hz leakage from an inadequately rectified AC power
supply, and can be corrected by insertion of a capacitor .pi.
filter in the power supply in the experimental section. As shown,
the timing of the sequential firing of the red laser and then the
green laser resulted in significant fluorescence signals when both
BODIPY dyes were present in solution.
[0161] FIG. 4B shows the total fluorescence signal from the serial
pulsing of the green and red lasers with only the BODIPY 630/650
(red dye) present in the cuvette. Whereas the large fluorescence
signal is time correlated with the firing of the red laser, the
green laser only imparts a small "cross-talk" signal to the red
dye. This cross-talk was measured to be approximately 4% of the red
laser signal, which is attributed to the highly efficient coupling
of a laser precisely matched to the red dye absorption peak and the
narrower absorption spectral properties of BODIPY dyes. Both
aspects give the desired low excitation efficiency of the
off-resonant green laser. This result clearly illustrates good
contrast between laser excitations for the same dye using the PME
approach. Since the ratio of red to green excitation efficiency can
be determined, a cross-talk matrix can be computed and
mathematically applied to yield a much higher contrast ratio.
[0162] FIG. 4C shows the total fluorescence signal from the serial
pulsing of the red and green lasers with only the BODIPY 523/547
(green dye) present in the cuvette. The only fluorescence signal
observed is time correlated with the firing of the green laser,
which gives the "two-finger" signature. Unlike that of the BODIPY
630/650, the cross-talk signal observed from the red laser is
negligible on this scale, and further signal amplification revealed
it to be considerably less than 1% of that from the green laser.
This observation is expected because longer wavelength excitation
sources should not impose photon absorption on shorter wavelength
dyes (i.e., the red laser does not excite the green dye). This
feature illustrates an important and key advantage of reduced
cross-talk of fluorophores using the PME strategy. Consider a four
dye system, which is made-up of blue, green, yellow, and red dyes.
The blue dye should not exhibit cross-talk from the sequential
firing of the green, yellow, and red lasers. The green dye, on the
other hand, will exhibit cross-talk from the only blue laser, but
not the yellow or red lasers. The yellow dye will exhibit
cross-talk from the blue and green lasers, but not the red laser
and so forth. In other words, the observed cross-talk on the "blue"
portion of the spectrum relative to the absorption/excitation
maxima of a given dye is negated, which is significantly different
from emission cross-talk of blue green, yellow, and red dyes
excited using a single excitation source.
[0163] An excellent contrast ratio for the discrimination of two
different BODIPY dyes using the PME technology by detecting all of
the fluorescence emission in a true color-blind fashion is
demonstrated. The experiment was designed using no spectral
filtering elements of any kind, and the signal was taken directly
from the oscilloscope in real time without signal averaging or
other processing. These data show that the choice of experimental
conditions provided significant fluorescence signal for which
scattered laser light signals are negligible. Therefore, the 25:1
contrast ratio observed for this unaveraged raw signal obtained
with laser pulses on the msec timescale provides a genuine
comparison of the time correlated fluorescence detection
technique.
Prophetic Example 4
Development of a 1-Capillary 4-Color PME Prototype
[0164] a. Develop 4-Color: Identification of the Optimal 4
Laser-Dye Combination
[0165] Six different solid-state lasers and/or laser diode modules
have been identified with excitation wavelengths that match the
absorption maxima of a number of commercially available
fluorophores, most of which have been used for DNA sequencing.
Other lasers and fluorophores can also be identified by comparing
and matching the excitation maxima of the fluorophore with the
emission wavelength of the laser. Candidate dyes should show good
quantum yields and have narrow absorption spectra. The non-limiting
list of dyes listed herein below initially meet these requirements,
although other commercially available fluorophores and lasers may
be tested as well. For experimentation purposes, each dye is
coupled to the universal sequencing primer (5'-TTGTAAAACGACGGCCAGT
(Metzker et al., 1996) (Sequence ID No. 1) as representative of
termination products for the SNP assay.
TABLE-US-00001 TABLE 1 Laser Fluorophore Blue 399 nm solid state,
indium 7-dimethylaminocoumarin (409/473), gallium nitride laser
cascade blue (396/410), and 7- hydroxycoumarin (386/448). Blue 473
nm or 488 nm solid 5-carboxyfluorescein (494/518), 1,3,5,7- state,
diode-pumped, frequency- tetramethyl-BODIPY (495/503), Oregon
doubled Nd: YAG laser green 488 (496/524), and the 5,7-
dimethyl-BODIPY (503/512).* Green 532 nm solid state, diode- BODIPY
(523/547) (536/554 when pumped, frequency-doubled Nd: coupled to a
primer) YAG laser Yellow 594 nm He--Ne BODIPY 589/617 and BODIPY
581/591 (592/603 when coupled to a primer) Red 635 nm SPMT diode
laser BODIPY 630/650 (643/651 when coupled module with external to
a primer) potentiometer Red 670 nm SPMT diode laser The BODIPY
(650/665) dye (661/667 module with external when coupled to a
primer) and cyanine potentiometer 5 dye
[0166] Although some dyes listed in Table 1 have a listed
absorption maxima on the blue side of the excitation source, these
dyes will be considered for use since the attachment of dyes to DNA
usually results in a red shift in the absorption/emission spectra.
The absorption/emission values for the dyes given in parenthesis
correspond to the absorption and emission wavelengths when coupled
to a universal sequencing primer.
[0167] A systematic evaluation of each laser-dye combination will
be compared for good excitation characteristics and between
laser-dye pairs to identify an optimal set of 4 laser-dyes for DNA
sequencing applications. Each laser will be set-up, similar to the
green and red laser experiment and pulsed using the TTL clock chip
for excitation and cross-talk experiments as described in Example
3. It is anticipated that numerous new solid-state lasers and laser
diodes and new fluorescent dyes will continue to be developed and
commercialized with unique emission wavelengths ranging below 400
nm to beyond 1100 nm that can be used in the current invention. Due
to the modular configuration of the PME system, the testing of
additional laser-dye pairs is straightforward.
[0168] b. Construction of the 1-Capillary Breadboard Prototype
[0169] A 1-capillary electrophoresis unit will be set-up on a
breadboard platform, and the electrophoresis will be driven
initially using a 30 kV power supply. A Plexiglas box equipped with
a safety interlock will be constructed to enclose the samples and
the running buffers. Initially, electrophoresis will be performed
under ambient conditions due to the nature of the short primer
extension products; however, a temperature controlled heating
jacket to improve electrophoretic resolution will be constructed if
necessary. The separation format will use fused silica capillaries
(150 .mu.M OD, 50 .mu.M ID, and 50 cm in length), POP-6 solution as
the separation matrix, and TBE as the running buffer. A 1-capillary
sheath flow cuvette will be constructed using the rectangular,
tapered design described by Zhang et al. (1999) and sheath flow
will be driven by syringe pump at a flow rate of approximately 0.3
mL per hour.
[0170] c. Sensitivity Experiments for Direct Detection
[0171] Although limited sensitivity information can be obtained
from the 2-color PME system (FIG. 1), more data will be obtained
from conducting sensitivity experiments directly using the
1-capillary instrument. Subsequent to the identification of the 4
laser-dye set, but overlapping with the construction of the
1-capillary instrument, the PME laser system will be coupled to the
electrophoresis device. For limit of detection assays, both the PMT
and the silicon APD (current and counting modes) will be
investigated over a wide range of fluorophore-labeled universal
primer concentrations. Sensitivity assays will be conducted using
free zone and POP-6-based capillary electrophoresis. A unique
feature of the PME system is that limit of detection experiments
will be performed for the 4 laser-dye sets identified previously.
It should be noted that limit of detection experiments are only
informative regarding sensitivity when the test dye is optimally
excited and producing maximum fluorescence signal. Sensitivity
experiments are not possible or practical using all four
fluorophores with the standard spectrally resolved DNA sequencing
systems because the longer wavelength dyes are inefficiently
excited. Therefore, the limit of detection experiments typically
published in the literature are performed using the dye most
closely matched to the laser source (out of the set of four), which
is usually fluorescein and the argon ion laser (Swerdlow et al.,
1990; Drossman et al., 1990; Swerdlow et al., 1990; Zhang et al.,
1999). Here, limit of detection experiments will be performed using
all four PME fluorophores because the lasers are closely matched
for optimal excitation and therefore will produce a more robust
picture for sensitivity with respect to the entire sequencing
chemistry.
[0172] d. Development of Rapid Sample Preparation Methods for
Direct Fluorescent Assays from Genomic DNA
[0173] The PME instrument should be able to detect as few as
10.sup.4-to-10.sup.5 fluorescent molecules. Given that 1 mL of
whole blood contains approximately 10.sup.6 white blood cells,
direct detection (without the need for amplification of the sample)
of multiple SNPs is possible. Initially, SNP assays will be
performed using standard PCR techniques and diluted appropriately
to simulate direct genomic DNA levels. This approach will allow the
performance of limit of detection experiments without dependence or
delay for the development of optimized sample preparation methods
and direct genomic SNP assays.
[0174] Sample preparation methods will be developed from whole
blood to be fast, simple, and amenable to direct single nucleotide
primer extension assays. Typically, most methods involve the
fractionation of whole blood into serum, red blood cells, and white
blood cells, of which the latter is used for analysis. Sample
preparation experiments can rely on commercially available kits and
published protocols for evaluation and optimization.
[0175] As discussed hereinabove, sequencing assays are typically
performed in .mu.L quantities, but loaded onto commercial capillary
electrophoresis instruments in nL quantities. To minimize wasting
direct assay samples, reaction volume assays will be optimized in a
target volume of 1 .mu.L. Since electrokinetic injection will be
implemented as the injection method for each prototype, injection
biases will most likely occur depended on sample purity (Huang et
al., 1988). Therefore, several solid-phase and affinity-based
purification schemes will be investigated for producing highly
purified fluorescently labeled SNP assays, which are devoid of
contaminants, such as unincorporated fluorescent terminators,
salts, and other electro-competing macromolecules.
[0176] In the event that the sensitivity limit of the PME
technology is several orders of magnitude higher than anticipated
(i.e., 10.sup.6-to-10.sup.7 fluorescent molecules), direct
detection from whole blood can still be achieved. This can be done
by increasing the amount of blood analyzed from 1-to-10 mL, and/or
performing a linear amplification of the primer extension assay by
temperature cycling, typically used in Sanger sequencing
reactions.
Prophetic Example 5
Construction of a Portable 8-Capillary PME DNA Sequencer
[0177] a. Construction of 8-APD Detector for a Portable System
[0178] A PME DNA sequencer that is portable will be useful for any
applications where there are space limitations or where it is
important to be able to move the sequencer. The technology to
modify the PME DNA sequencer such that it is portable is currently
available. For the portable DNA sequencer, the optical system
developed for the breadboard will be adapted to become more compact
and robust. The highly efficient dual prism combiner will be
initially adapted to the portable sequencer. Constructed with
microbench components, the optics train will be incorporated into a
4-rail structure, which was developed for the very rigid needs of
laser cavity mirror supports. The commercially available miniature
4-rail system will also be used to support the sheath flow cuvette
and collection optics. As previously discussed, commercial diode
laser modules are remarkably compact, typically 1'' or 2'' long,
and are generally available with optical fiber coupled outputs.
Fiber splitter/combiners have been developed for laser based
communications, and contingent on this rapidly emerging technology,
it may be feasible to combine the beams and deliver the 4-laser
sources to the sheath flow cuvette in a single optical fiber. For
this design, only a conventional achromatic lens will be needed to
project the collimated alternating multicolor beam through the
sheath flow cuvette.
[0179] A wide field f/1 (NA .about.0.5) lens will be used to
collect the fluorescent light from the capillary plumes and project
it onto 8 APDs. A microscope objective is often used for this
purpose, but may suffer vignetting and consequent loss of
fluorescence signal from the outermost capillary plumes. The lens
mount will be equipped with opposing adjustment screws that lock
the lens into place after optimization. The light path will be
shielded with baffles to reduce scattered laser light reaching the
detectors. An optional liquid crystal device will be used as an
edge filter to block scattered laser light, as needed; this device
has a rejection ratio of about 4 orders of magnitude. Unlike the
familiar rotating filter wheel, the liquid crystal device has no
mechanical moving parts. The liquid crystal filter has a response
time of several milliseconds, so that it will be cycled under
computer control along with the 4 lasers, blocking scattered light
from each one in turn, while passing essentially all of the
fluorescent light to the color blind APD detectors.
[0180] Although it is preferable to imaged fluorescent light
directly onto the APDs, the collection lens, which typically
magnifies the image 20.times. may not be enough to resolve 8 images
for detection. One solution is to use a small mirror or prism
affixed to each APD housing, which can deflect the beam at right
angles. This design can be mounted in a staggered array and thereby
reduce this congestion. Individual gradient refractive index (GRIN)
lenses and optical fiber couplings to each APD will also be
considered in the portable configuration. However, the insertion
loss for a properly coated beam steering prism is about 2%, and it
is most unlikely that the fiber coupling will perform as well. This
fiber coupling problem is much more significant for the projected
fluorescent image, which behaves as an extended source, than it is
for a laser beam. Finally, the same rigid 4-rail structure
mentioned above will be used to support the detection system.
[0181] The laser and TTL circuit power supplies typically have a
footprint of a few square inches, and the power supplies for the
APDs and the 10 kV electrophoresis modules are slightly larger, but
still only a few inches long. These various electronic components
will be easily positioned beneath and around the capillaries, which
will travel around the perimeter of the system, thus avoiding sharp
bends.
[0182] b. PME of Residual Signal
[0183] Residual fluorescence may be detected immediately after very
short excitation laser pulses have irradiated the sample. With such
an approach, the lasers will be off during the period that the
fluorescent signal is collected. This may be particularly important
for the development of sequencing on a chip, because the chip
almost inevitably will generate large amounts of scattered
light.
[0184] Specifically the intent is to sequentially fire pico-second
laser pulses at a fluorescently labeled DNA sample and then "look"
for a fluorescent response on the nanosecond time-scale,
immediately after the laser pulse ends. This novel approach is a
logical extension to the central principle of operation intrinsic
to the core PME technology. This innovative experimental strategy
is referred to as "Looking In The Dark" or "PME-LITD".
[0185] The primary advantage of the Looking In The Dark strategy is
the complete elimination of scattered light, which at low levels of
fluorescence is likely to be a main source of noise in the PME
instrument. This technique, if successful, could have enormous
implications for improving the signal-to-noise ratio and
potentially improve the overall sensitivity of the instrument.
[0186] The following example details a simple sequence of events to
illustrate the PME-LITD concept: [0187] 1. The first laser in
sequence is pulsed for 50 pico-seconds. [0188] 2. A 500 pico-second
time delay is applied after the laser has been switched off. Note
that during the delay period no fluorescence is sampled by the
detector. [0189] 3. A fast photon counter is used to look for any
fluorescent response from the labeled DNA during the ensuing 50
nano-second gated window. [0190] 4. Steps 1 through 3 are repeated
in sequence for each laser in the subcycle. [0191] 5. The
pico-second pulsed excitation and nano-second gated detection
windows cycle continuously.
[0192] For a four color system, the above steps would generate a
subcycle time that is 202.2 nanoseconds. This implies that over an
eight second time window, (approximate time for a labeled DNA band
to pass through an ABI 3700 cuvette), around 40 million complete
subcycles would be completed. The data collected will then be
appropriately averaged and further processed to yield high quality
analyzed data.
[0193] To conduct these types of experiments, lasers that are
capable of generating very short pulses of sub-nanosecond duration
will be required. Pico-second laser sources are commercially
available from a number of companies including Coherent Laser
Group, Newport, and PicoQuant. For example, Coherent has a diode
pumped mode-locked laser with fundamental wavelengths at either
1047 nm, 1053 nm or 1064 nm, which generates pulses as short as 2
ps. The second harmonics can easily be generated from picosecond
sources, hence the following wavelengths would be available: 532
nm, 526.5 nm, and 523.5 nm. Newport also manufactures a "NanoLaser"
that generates sub-nanosecond green, (532 nm) light, at an average
power of more than 6 mW.
[0194] In addition, an instrument that is capable of counting
photons on a sub-nanosecond time scale will also be needed; such
devices are available from a variety of manufacturers. For example,
"FAST ComTec" produces a single photon counting instrument, which
has resolution on the time-scale of 500 pico-seconds. Becker &
Hickl GmbH also manufactures a four channel correlated single
photon counting device that has resolution down to 813
femto-seconds. Furthermore, it should be noted that when coupled
into an appropriately configured electronic circuit the recovery
time of a silicon avalanche photo-diode detector, (following
illumination by scattered light from a excitation laser pulse), is
of the order of 500 pico-seconds. This rapid recovery time will
permit the effective observation of fluorescence from a dye with a
fluorescent lifetime of several nanoseconds in the complete absence
of any laser excitation, i.e. "in the dark".
[0195] Four wavelengths can be generated from a single laser, as
opposed to using four synchronized and mode-locked lasers. This can
be done using Stimulated Raman Shifting, (SRS).
[0196] An experiment to test the PME-LITD strategy comprises a
simple two-color system. Specifically, a mode-locked Nd:YAG laser
generating 50 pico-second pulses will be coupled to a Raman cell
filled with molecular nitrogen. The superimposed multi-wavelength
output from the Raman cell will be then dispersed and ultimately
recombined using a four-prism assembly. In the middle of the
four-prism assembly, (i.e. where the various excitation lines are
separated and traveling approximately in parallel), a pair of
electro-optic modulators will be used to chop the colored
pulses--selecting alternate pulses from each beam. The recombined
beams will then be directed into a cuvette assembly--similar to the
prototype described in FIG. 1. Finally, the time-resolved
fluorescence will be detected using a fast photon counter that
looks for photons in a window that spans the range from 0.5 ns-50.5
ns after the cessation of each laser pulse.
[0197] c. Construction of a 96-Capillary PME Suitcase DNA
Sequencer
[0198] A portable 96-capillary PME DNA sequencer is envisioned as
an aspect of the current invention. In one embodiment, the
four-laser illumination system described in the 8-capillary sheath
flow cuvette system will be used for the 96 capillary system. All
four alternating excitation lines will be coaxial and well
collimated to facilitate the illumination of the 96 fluorescent
plumes. Although the compact multi-laser source will remain
unchanged, it will not be practical to scale the detection system
from 8 APDs to 96 discrete detectors. A CCD camera, however, will
be more suited to perform this operation. A fast lens such as f/1,
with good imaging quality will be installed for efficient light
collection. A second lens will be used to re-image the light onto
the CCD. The computer-controlled liquid crystal filter may be
interposed between the two lenses to block scattered laser light,
if needed. Baffles will be used to minimize stray light, but there
are no restricting apertures that reduce the wide cone angle of
collection, or dispersing elements that further attenuate the
signal.
[0199] Essentially all of the fluorescent light from one sheath
flow plume will fall on one particular group of pixels, and these
pixels are binned together so they are read out as a single unit,
which will reduce readout noise. Binning of all of the fluorescence
from a capillary into effectively one giant pixel provides a single
robust signal from each capillary plume even when the amount of
fluorescing dye is quite small.
[0200] The CCD camera is quite compact, and even with adding
thermoelectric cooling to reduce background noise, fitting the CCD
detector into a compact device will not be problematic. A portable
computer will read out the CCD contents at the end of each laser
pulse. For a standard video rate of 30 Hz, the entire cycle
frequency of 4 lasers will be 7.5 Hz (5 Hz with the incorporation
of a liquid crystal laser blocker), and this will allow for the
data from dozens of readout cycles to be signal averaged per one
elution event. Following the construction of the CCD suitcase
system, detailed limit of detection experiments will be performed
to compare it to the performance of the 8-capillary APD suitcase
prototype.
[0201] All of the methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the methods and in the steps or in the sequence of steps
of the method described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
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Sequence CWU 1
1
1119DNAArtificial SequenceSynthetic primer 1ttgtaaaacg acggccagt
19
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