U.S. patent application number 12/577078 was filed with the patent office on 2010-08-05 for methods and systems for molecular fingerprinting.
This patent application is currently assigned to California Institute of Technology. Invention is credited to Hou-Pu Chou, Stephen R. Quake.
Application Number | 20100196892 12/577078 |
Document ID | / |
Family ID | 27393324 |
Filed Date | 2010-08-05 |
United States Patent
Application |
20100196892 |
Kind Code |
A1 |
Quake; Stephen R. ; et
al. |
August 5, 2010 |
Methods and Systems for Molecular Fingerprinting
Abstract
This invention relates in general to a method for molecular
fingerprinting. The method can be used for forensic identification
(e.g. DNA fingerprinting, especially by VNTR), bacterial typing,
and human/animal pathogen diagnosis. More particularly, molecules
such as polynucleotides (e.g. DNA) can be assessed or sorted by
size in a microfabricated device that analyzes the polynucleotides
according to restriction fragment length polymorphism. In a
microfabricated device according to the invention, DNA fragments or
other molecules can be rapidly and accurately typed using
relatively small samples, by measuring for example the signal of an
optically-detectable (e.g., fluorescent) reporter associated with
the polynucleotide fragments.
Inventors: |
Quake; Stephen R.;
(Stanford, CA) ; Chou; Hou-Pu; (Foster City,
CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
California Institute of
Technology
Pasadena
CA
|
Family ID: |
27393324 |
Appl. No.: |
12/577078 |
Filed: |
October 9, 2009 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11582838 |
Oct 17, 2006 |
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12577078 |
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10917665 |
Aug 13, 2004 |
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11582838 |
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09826373 |
Apr 4, 2001 |
6833242 |
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10917665 |
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09325667 |
May 21, 1999 |
6540895 |
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09826373 |
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08932774 |
Sep 23, 1997 |
6221654 |
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09325667 |
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60194422 |
Apr 4, 2000 |
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
B01L 2200/0647 20130101;
B01L 2300/0816 20130101; C12Q 1/6816 20130101; B01L 2400/0415
20130101; B01L 2400/0633 20130101; C12Q 1/683 20130101; C12Q 1/6816
20130101; B01L 3/502738 20130101; B01L 2400/0622 20130101; B01L
2400/0484 20130101; G01N 2015/149 20130101; B01L 2300/0864
20130101; B01L 2400/0421 20130101; B01L 2200/0652 20130101; B01L
3/502761 20130101; B01L 2400/0418 20130101; B01L 2300/0654
20130101; B01L 2400/0406 20130101; B01L 3/502707 20130101; B01L
2400/0487 20130101; C12Q 2565/629 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A molecular fingerprinting method comprising the steps of: (a)
identifying a target polynucleotide, (b) selecting at least one
fragment of the target polynucleotide, wherein the fragment is a
fixed distance from a restriction site, to generate a set of one or
more polynucleotide fragments, and (c) designating some or all of
the set of fragments as molecular fingerprint corresponding to the
target polynucleotide.
2-3. (canceled)
4. A method for identifying a polynucleotide sample comprising the
steps of: (a) identifying a target polynucleotide; (b) selecting at
least one fragment of the target polynucleotide, wherein the
fragment is a fixed distance from a restriction site, to generate a
set of one or more polynucleotide fragments; (c) designating some
or all of the set of fragments as a fingerprint corresponding to
the target polynucleotide; (d) synthesizing one or more
oligonucleotide probes to complement the set of polynucleotide
fragments; (e) combining the probes, a polynucleotide sample,
nucleotide triphosphates, and polymerase to synthesize at least one
polynucleotide strand; (f) cutting the strands with restriction
enzymes to yield a set of sample fragments of fixed length; and (g)
comparing the set of sample fragments to the fingerprint.
5-15. (canceled)
16. A method for detecting a particular nucleic acid in a sample,
which particular nucleic acid has at least one restriction site,
and which method comprises: (a) contacting the sample with a primer
that hybridizes to the particular nucleic acid a predetermined
distance from the restriction site, a polymerase and a plurality of
nucleotides, so that a complementary nucleic acid is synthesized
from the primer at least to the restriction site; (b) contacting
the complementary nucleic acid with a restriction enzyme under
conditions capable of cutting the complementary nucleic acid at the
restriction site; and (c) detecting a nucleic acid fragment having
a particular length equal to the fixed distance, wherein the
presence of the nucleic acid fragment in the sample indicates that
the particular nucleic acid is present in the sample.
17-35. (canceled)
36. A method for detecting a particular nucleic acid in a sample,
which particular nucleic acid has at least one restriction site,
and which method comprises: (a) contacting the sample with a
restriction enzyme under conditions capable of cutting the
particular nucleic acid at the restriction site; (b) contacting the
sample with a primer that hybridizes to the nucleic acid a
predetermined distance from the cut at the restriction site, a
polymerase and a plurality of nucleotides, so that a complementary
nucleic acid fragment is synthesized; and (c) detecting the
complementary to nucleic acid fragment, wherein the presence of the
complementary nucleic acid fragment in the sample indicates that
the particular nucleic acid is present in the sample.
37-55. (canceled)
Description
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to copending U.S. provisional patent application Ser.
No. 60/194,422 filed on Apr. 4, 2000. The present application is
also a continuation-in-part of copending U.S. patent application
Ser. Nos. 08/932,774 and 09/325,667, filed on Sep. 23, 1997 and May
21, 1999, respectively. Each of these prior applications is hereby
incorporated by reference in its entirety.
1. FIELD OF THE INVENTION
[0002] This invention relates in general to a method for molecular
fingerprinting. The method can be used for forensic identification
(e.g. DNA fingerprinting, especially by VNTR), bacterial typing,
and human/animal pathogen diagnosis. More particularly, molecules
such as polynucleotides (e.g. DNA) can be assessed or sorted by
size in a micro fabricated device that analyzes the polynucleotides
according to restriction fragment length polymorphism. In a micro
fabricated device according to the invention, DNA fragments or
other molecules can be rapidly and accurately typed using
relatively small samples, by measuring for example the signal of an
optically-detectable (e.g., fluorescent) reporter associated with
the polynucleotide fragments.
[0003] More generally, the invention relates to a method of
analyzing or sorting molecules such as polynucleotides (e.g., DNA)
by size or some other characteristic. In particular, the invention
relates to a method of analyzing and/or sorting individual
polynucleotide molecules in a microfabricated device by measuring
the signal of an optically-detectable (e.g., fluorescent,
ultraviolet, radioactive or color change) reporter associated with
the molecules. These methods and devices can also be adapted to
analyze or sort cells or particles.
[0004] The devices and methods of the invention are advantageous,
particularly in comparison with conventional gel electrophoresis
techniques. For example, the invention provides less costly and
more rapid equipment, can use smaller molecular samples, is less
labor-intensive and is more readily automated. The invention is
also advantageously flexible. Additional functions can be
incorporated into the design as desired, such as in-line digestion,
separation, etc.
2. BACKGROUND OF THE INVENTION
[0005] When DNA is broken into fragments using restriction enzymes,
each of which cuts the DNA in a known way, the resulting DNA
fragments or polypeptides of different sizes produce a unique
pattern or profile which can be used to uniquely identify the
source of the DNA molecules. In the invention, a reporter or other
measurable signal varies as a function of molecule size, and in
this way profiles based on size can be efficiently generated and
compared, particularly on a small scale and in an automated or
semi-automated fashion.
[0006] Methods enabling the matching of unidentified tissue samples
to specific individuals have wide application in many fields. For
DNA fingerprinting, commonly used methods include RFLP analysis
(53, 54), variable nucleotide tandem repeats (55), and
microsatellites (56). With the possible exception of monozygotic
twins, each individual in the human population has a unique genetic
composition which can be used to specifically identify each
individual. This phenomenon has allowed law enforcement officials
to use DNA sequence variation to determine, for example, whether a
forensic sample was derived from any given individual. The fields
of forensic and medical serology, paternity testing, and tissue and
sample origin have seen increasing use of such techniques,
including the forensic and diagnostic use of DNA sequence
variation, e.g., statistical evaluations based on satellite
sequences and variable number of tandem repeats (VNTRS) or
amplified fragment length polymorphisms (AMP-FITS). These methods
are being used in crime laboratories, courts, hospitals and
research and testing labs. Inclusion probabilities stated by the
laboratories performing the analyses in such cases often exceed
1:1,000,000. That is, only one individual in one million is
predicted, on a statistical basis, to have a given DNA
"fingerprint" obtained by analyzing a pattern of DNA fragments
generated according to these techniques.
[0007] The first implementation of DNA typing in forensics was
Jeffreys' use of a multilocus DNA probe "fingerprint" that
identified a suspect in a murder case in England. (55) In the
United States, DNA profiling has been established using a battery
of unlinked highly polymorphic single locus VNTR probes. (57) The
use of these batteries of probes permits the development of a
composite DNA profile for an individual. These profiles can be
compared to databases, for example using the principles of
Hardy-Weinberg to determine the probability of a match between a
suspect and an unknown forensic sample.
[0008] Although these methods have markedly improved the power of
the forensic and medical scientists to distinguish between
individuals, they suffer from a number of shortcomings including a
lack of sensitivity, the absence of internal controls, expense,
time intensity, relatively large sample size, an inability to
perform precise allele (gene pair) identification, and problems
with identifying degraded DNA samples.
[0009] For example, the most frequently used method for forensic
identification is the "Southern" hybridization technique, which has
been widely used in forensic identification and medical diagnosis.
Also called a "Southern blot," this technique treats an extracted
molecule (a DNA sample) with a restriction endonuclease, an enzyme
that cuts a polynucleotide chain wherever a specific and relatively
short sequence of nucleic acids in the chain occurs. Examples of
well known restriction enzymes used in this way are the
endocucleases HaeIII, EcoRI, HpaI and HindIII. In DNA
fingerprinting, restriction sites are typically used to isolate
VNTRs (variable number of tandem repeats), which are regions in
which a short sequence of DNA has been repeated a number of times.
The number of repeating units within these regions vary between
individuals, and when cut with a restriction endonuclease result in
multiple fragments of different size called] RFLPs (restriction
fragment length polymorphisms). These fragments can be used as a
"fingerprint" because they vary in number and size from one
individual to another.
[0010] The resulting nucleotide fragments (i.e. the RFLPs) are
separated by size via gel electrophoresis, in which different sized
charged molecules are separated by their different rates of
movement through a stationary gel under the influence of an
electric current. Following electrophoresis, the separated
nucleotides are denatured and transferred to the surface of a nylon
membrane by blotting; the so-called "Southern Blot". The Southern
Blot is then incubated in a solution containing a radioactive
single locus probe under conditions of temperature and salt
concentration that favor hybridization. (A single locus probe is
also called a "primer.") The locations of radioactive probe
hybridization on the Southern Blot are detected and recorded via
X-ray film or some other detection technique, thus providing a
"profile" of the nucleotide. (Hybridization is used to pull out
VNTR fragments, i.e. to separate them from irrelevant fragments.)
In this approach, sample DNA is digested, and the resulting
fragments are separated by size using gel electrophoresis. The
separated fragments are transferred to a membrane by blotting, and
are subjected to primer hybridization. (58)
[0011] This technique is time-consuming, labor intensive, and the
gel may have a limited resolving power, making it potentially
difficult to interpret the results. Another disadvantage is that
these techniques generally require the use of a polymerase chain
reaction (PCR) to multiply the polynucleotide in the sample. That
is, the conventional tests are not very sensitive, and require
relatively large DNA samples which often are not available. In such
cases the sample concentration is increased to a meaningful
detectable level by PCR. While this addresses some problems of
sensitivity and sample degradation, PCR has been open to challenge
because of possible sample contamination, and consequent
undesirable amplification of contaminants leading to unreliable
results. PCR approaches are also difficult to multiplex. For
example, the probes and primers must be chosen with care, and
generally only one set can be used. The sample may be consumed by
one round of PCR, and different sets of probes or primers may
require different reaction conditions, such as temperature. A
simpler, more powerful technique is needed, which can accommodate
small samples, does not rely on PCR, and which makes use of the
most recent advances in DNA technology.
[0012] As described herein, the invention addresses these problems.
In preferred embodiments a DNA sample is digested, primers are used
to extend specifically desired DNA regions (e.g. VNTRs), without
successive rounds of PCR, and highly sensitive or specific reporter
molecules, such as fluorescently-labeled single nucleotides, are
used to efficiently determine the length of the resulting DNA. A
micro fabricated or microfluidic device may be used to implement
these techniques, for example to separate and optically detect
labeled fragments.
[0013] The identification and separation of nucleic acid fragments
by size, such as in sequencing of DNA or RNA, is a widely used
technique in many fields, including molecular biology,
biotechnology, and medical diagnostics. The most frequently used
method for such separation is gel electrophoresis, in which
different sized charged molecules are separated by their different
rates of movement through a stationary gel under the influence of
an electric current. Gel electrophoresis presents several
disadvantages, however. The process can be time consuming, and
resolution is typically about 10%. Efficiency and resolution
decrease as the size of fragments increases; molecules larger than
40,000 base pairs are difficult to process, and those larger than
10 million base pairs cannot be distinguished.
[0014] Methods have been proposed for determination of the size of
nucleic acid molecules based on the level of fluorescence emitted
from molecules treated with a fluorescent dye. See Keller, et al.,
1995 (31); Goodwin, et al., 1993 (28); Castro, et. al., 1993 (27);
and Quake, et al., 1999 (59). Castro (27) describes the detection
of individual molecules in samples containing either uniformly
sized (48 Kbp) DNA molecules or a predetermined 1:1 ratio of
molecules of two different sizes (48 Kbp and 24 Kbp). A resolution
of approximately 12-15% was achieved between these two sizes. There
is no discussion of sorting or isolating the differently sized
molecules.
[0015] In order to provide a small diameter sample stream, Castro
(27) uses a "sheath flow" technique wherein a sheath fluid
hydrodynamically focuses the sample stream from 100 .mu.m to 20
.mu.m. This method requires that the radiation exciting the dye
molecules, and the emitted fluorescence, must traverse the sheath
fluid, leading to poor light collection efficiency and resolution
problems caused by lack of uniformity. Specifically, this method
results in a relatively poor signal-to-noise ratio of the collected
fluorescence, leading to inaccuracies in the sizing of the DNA
molecules.
[0016] Goodwin (28) mentions the sorting of fluorescently stained
DNA molecules by flow cytometry. This method, however, employs,
costly and cumbersome equipment, and requires atomization of the
nucleic acid solution into droplets, with the requirement that each
droplet contains at most one analyte molecule. Furthermore, the
flow velocities required for successful sorting of DNA fragments
were determined to be considerably slower than used in conventional
flow cytometry, so the method would require adaptations to
conventional equipment. Sorting a usable amount (e.g., 100 ng) of
DNA using such equipment would take weeks; if not months, for a
single run, and would generate inordinately large volumes of DNA
solution requiring additional concentration and/or precipitation
steps.
[0017] Quake (59) relates to a single molecule sizing
microfabricated device (SMS) for sorting polynucleotides or
particles by size, charge or other identifying characteristics, for
example, characteristics that can be optically detected. The
invention includes a fluorescence activated sorter (FAS), and
methods for analyzing and sorting polynucleotides by measuring a
signal produced by an optically-detectable (e.g., fluorescent,
ultraviolet or color change) reporter associated with the
molecules. These methods and microfabricated devices allow for high
sensitivity, no cross-contamination, and lower cost than
conventional gel techniques. In one embodiment of the invention, it
has been discovered that devices of this kind can be advantageously
designed for use in molecular fingerprinting applications, such as
DNA fingerprinting.
[0018] It is thus desirable to provide a method of rapidly
analyzing and sorting differently sized nucleic acid molecules with
high resolution, using simple and inexpensive equipment. In a
microfabricated system, a short optical path length is desirable to
reduce distortion and improve signal-to-noise of detected
radiation. Ideally, sorting of fragments can be carried out using
any size-based criteria.
3. SUMMARY OF THE INVENTION
[0019] The invention provides a molecular fingerprinting method and
system, including for example microfabricated devices for sorting
reporter-labeled polynucleotides or polynucleotide molecules by
size.
[0020] An object of the present invention is a method for DNA
fingerprinting using synthetic repeat polymorphisms.
[0021] An additional object of the present invention is a method
for identifying the source of DNA in a forensic or medical
sample.
[0022] A further object of the present invention is to provide an
automated DNA profiling assay. This case be used, for example for
DNA mapping, e.g. of BAC or YAC libraries.
[0023] An additional object of the present invention is to provide
a kit for detecting synthetic repeat polymorphisms.
[0024] In accomplishing these and other objectives, the invention
provides a method for molecular fingerprinting using a synthetic
version of restriction fragment length polymorphism. The method
includes choosing at random a short (20-50 bp) sequence of the
polynucleotide that is a fixed distance away from a restriction
site. This can be repeated any number of times for enhanced
statistical discrimination, with different locations in the
polynucleotide and different distances to a restriction site. Thus,
a unique set of fragments can be generated, resulting in a
fingerprint that can be obtained without relying on naturally
occurring repeat sequences or restriction sites.
[0025] The method also provides for identification of a fingerprint
in a sample. To identify a fingerprinted polynucleotide in a
sample, an oligonucleotide (i.e. a short polynucleotide probe) is
synthesized to complement the randomly chosen sequences. The probes
are mixed with the sample along with nucleotide triphosphates and
polymerase. The nucleotides can be fluorescently labeled. Through
this technique a set of fluorescent strands of polynucleotide will
be synthesized. Each complementary strand is cut with restriction
enzymes to yield a polynucleotide of a fixed length. The
polynucleotides can then be sized, either by gel electrophoresis or
in a single molecule sizing device (SMS). One oligonucleotide probe
derived from a references sample can be used, resulting in one
complementary strand in a test sample containing matching
sequences. If multiple oligonucleotides are designed, the reaction
can be multiplexed and the different length fragments can be
resolved into a multiple fragment fingerprint that can be compared
to the standard or reference fingerprint. Preferably, a digestion
is performed before enzyme/primer extension to prevent non-specific
binding of primers. A six-base cutter (digestion enzyme) is
particularly preferred to cut the sample into fragments of tens of
thousands of base pairs. Alternatively, digestion after extension
to fix the length can be performed.
[0026] A number of variations and modifications to this technique
will be apparent to the practitioner of ordinary skill. For
example, instead of using labeled nucleotides, complementary
polynucleotides can be post-stained with an intercalating dye.
Another variation is to use affinity purification to pull down the
fragment of interest, i.e., using biotinylated oligonucleotides and
streptavidin coated magnetic beads.
[0027] In a preferred embodiment, a micro fabricated device is used
for detecting or sorting the nucleotide fragments in a fingerprint
based on size. The SMS device is fast, allowing analysis in as
little as 10 minutes, and requires only femtograms of material,
thus, the SMS device provides relatively high sensitivity without
the need for PCR.
[0028] Microfabricated Device. The device includes a chip having a
substrate with at least one microfabricated analysis unit. Each
analysis unit includes a main channel, having at one end a sample
inlet, having along its length a detection region, and having,
adjacent and downstream of the detection region, an outlet or a
branch point discrimination region leading to a plurality of branch
channels originating at the discrimination region and in
communication with the main channel. The analysis unit also
provides a stream of solution, preferably continuous, containing
the molecules and passing through the detection region, such that
on average only one molecule occupies the detection region at any
given time. The level of reporter from each molecule is measured as
it passes within the detection region. If desired, the molecule is
directed to a selected branch channel based on the level of
reporter.
[0029] In a preferred embodiment, the substrate is planar, and
contains a microfluidic chip made from a silicone elastomer
impression of an etched silicon wafer according replica methods in
soft-lithography (11). In one embodiment, the channels meet to form
a "T" (T junction). A Y-shaped junction, and other shapes and
geometries may also be used. A detection region is typically
upstream from the branch point. Molecules or cells are diverted
into one or another outlet channel based on a predetermined
characteristic that is evaluated as each cell passes through the
detection region. The channels are preferably sealed to contain the
flow, for example by fixing a transparent coverslip, such as glass,
over the chip, to cover the channels while permitting optical
examination of one or more channels or regions, particularly the
detection region. In a preferred embodiment the coverslip is pyrex,
anodically bonded to the chip.
[0030] Other devices such as electrophoresis chips may also be
used. Exemplary devices are described in U.S. Pat. Nos. 6,042,709;
5,965,001; 5,948,227; 5,880,690; and 6,007,690.
[0031] Channel Dimensions. The channels in a molecular analysis
device are preferably between about 1 .mu.m and about 20 .mu.m in
width and between about 1 .mu.m and about 20 .mu.m in depth, and
the detection region has a volume of between about 1 fl and about 1
pl. In a cell analysis device the channels are preferably between
about 1 and 500 microns in width and between about 1 and 500
microns in depth, and the detection region has a volume of between
about 1 fl and 100 nl. In preferred embodiments, the device
includes a transparent (e.g., glass) cover slip bonded to the
substrate and covering the channels to form the roof of the
channels. The channels may be of any dimensions suitable to
accommodate the largest dimension of the molecules to be
analyzed.
[0032] Manifolds. A device which contains a plurality of analysis
units may further include a plurality of manifolds, the number of
such manifolds typically being equal to the number of branch
channels in one analysis unit, to facilitate collection of
molecules from corresponding branch channels of the different
analysis units.
[0033] Flow of Molecules. In one embodiment, the molecules are
directed or sorted by electroosmotic force. A pair of electrodes
apply an electric field or gradient across the discrimination
region that is effective to move the flow of molecules through the
device. In a sorting embodiment the electrodes can be switched to
direct a particular molecule into a selected branch channel based
on the amount of reporter signal detected from that molecule. In
another embodiment, a flow of molecules is maintained through the
device via a pump or pressure differential, and a valve structure
can be used at the branch point effective to permit each molecule
to enter only one selected branch channel. Alternatively, a valve
can be placed in one or more channels downstream of the branch
point to allow or curtail flow through each channel. In a related,
pressure can be adjusted at the outlet of each branch channel
effective to allow or curtail flow through the channel.
[0034] Optical Detection. Preferably the molecules are optically
detectable when passing through the detection region. For example
the molecules may be labeled with a reporter, for example a
fluorescent reporter. The optically detectable signal can be
measured, and generally is proportional to or is a function of a
characteristic of the molecules, such as size or molecular weight.
A fluorescent reporter, generating a quantitative optical signal
can be used. Fluorescent reporters are known, and can be associated
with molecules such as polynucleotides using known techniques.
[0035] In a preferred molecular fingerprinting embodiment, the
reporter label is a fluorescently-labeled single nucleotides, such
as fluorescein-dNTP, rhodamine-dNTP, Cy3-dNTP, Cy5-dNTP, where dNTP
represents dATP, dTTP, dUTP or dCTP. The reporter can also be
chemically-modified single nucleotides, such as biotin-dNTP.
Alternatively, chemicals can be used that will react with an
attached functional group such as biotin.
[0036] Sorting Molecules. In another aspect, the invention includes
a method of isolating polynucleotides having a selected size. The
method includes: a) flowing a continuous stream of solution
containing reporter-labeled polynucleotides through a channel
comprising a detection region having a selected volume, where the
concentration of the molecules in the solution is such that the
molecules pass through the detection region one-by-one, c)
determining the size of each molecule as it passes through the
detection region by measuring the level of the reporter, d) in the
continuous stream of solution, diverting (i) molecules having the
selected size into a first branch channel, and (ii) molecules not
having the selected size into a second branch channel.
Polynucleotides diverted into any channel can be collected as
desired.
[0037] Flow Control. In preferred embodiments, the concentration of
polynucleotides in the solution is between about 10 fM and about 1
nM and the detection region volume is between about 1 fl and about
1 pl. The molecules can be diverted, for example, by transient
application of an electric field effective to bias (i) a molecule
having the selected size (e.g., between about 100 bp and about 10
mb) to enter one branch channel, and (ii) a molecule not having the
selected size to enter another branch channel. Alternatively,
molecules can be directed into a selected channel, based on size,
by temporarily blocking the flow in other channels, such that the
continuous stream of solution carries the molecule having the
selected size into the selected channel. Pumps and valves may also
be used to divert flow, and carry molecules into one or another
channels, and mechanical switches may also be used. These methods
can also be used in combination, and likewise molecules can be
diverted based on whether they have a selected property or size, or
do not have that property or size, or exceed or do not exceed a
selected threshold measurement.
[0038] Synchronization. In each embodiment where molecules are
measured and then diverted, as opposed to being measured only, the
molecules are detected and measured one-by-one within the detection
region, and are diverted one-by-one into the appropriate channels,
by coordinating or synchronizing the diversion of flow with the
detection step and with the flow entering the detection, as
described for example in more detail below. In certain embodiments
the flow rate may be adjusted, for example delayed, to maintain
efficient detection and switching, and as described below the flow
may in some cases be temporarily reversed to improve accuracy.
[0039] Sizing Molecules. In yet another aspect, the invention
includes a method of sizing polynucleotides in solution. This
method includes: a) flowing a continuous stream of solution
containing reporter-labeled polynucleotides through a
microfabricated channel comprising a detection region having a
selected volume, where the concentration of the molecules in the
solution is such that most molecules pass through the detection
region one by one, and b) determining the size of each molecule as
it passes through the detection region by measuring the level of
the reporter.
[0040] Multiparameter Embodiments. In addition to analyzing or
sorting fluorescent and non-fluorescent nucleotide fragments, the
SMS can also provide multiparameter analysis. For example, sizing
or sorting can be done according to a window or threshold value,
meaning that molecules (e.g. polynucleotides) are selected based on
the presence of a signal above or below a certain value or
threshold. There can also be several points of analysis on the same
chip for multiple time course measurements.
[0041] Thus, the invention provides for the rapid and accurate
determination of the "profile" of a polynucleotide in high
resolution using minimal amounts of material in these simple and
inexpensive microfabricated devices. The methods and devices of the
invention can replace or be used in combination with conventional
gel based approaches.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1 shows a nucleic acid sorting device in accordance
with one embodiment of the invention.
[0043] FIG. 2 shows a partial perspective view of a nucleic acid
sorting device, showing a sample solution reservoir and sample
inlet.
[0044] FIG. 3A shows one embodiment of a detection region used in a
nucleic acid sorting device, having an integrated photodiode
detector.
[0045] FIG. 3B shows another embodiment of a detection region,
having an integrated photodiode detector, and providing a larger
detection volume (than the embodiment of FIG. 3A).
[0046] FIGS. 4A-4B show one embodiment of a valve within a branch
channel of a nucleic acid sorting device, and steps in fabrication
of the valve.
[0047] FIG. 5A shows one embodiment of a discrimination region used
in a nucleic acid sorting device, having electrodes disposed within
the channels for electrophoretic discrimination.
[0048] FIG. 5B shows another embodiment of a discrimination region
used in a nucleic acid sorting device, having electrodes disposed
for electroosmotic discrimination.
[0049] FIGS. 5C and 5D show two further embodiments of a
discrimination region, having valves disposed for pressure
electrophoretic separation, where the valves are within the branch
point, as shown in FIG. 5C, or within the branch channels, as shown
in FIG. 5D.
[0050] FIG. 6 shows a device with analysis units containing a
cascade of detection and discrimination regions suitable for
successive rounds of polynucleotide or cell sorting.
[0051] FIGS. 7A-7D show initial steps in photolithographic
microfabrication of a nucleic acid sorting device from a silicon
wafer, using photolithography and several stages of etching.
[0052] FIG. 8 shows a schematic representation of a process for
obtaining a silicone elastomer impression of a silicon mold to
provide a microfabricated chip according to the invention.
[0053] FIG. 9 shows a schematic representation of an apparatus of
the invention, in which a silicone elastomer chip is mounted on an
inverted microscope for optical detection of a laser-stimulated
reporter. Electrodes are used to direct cells in response to the
microscope detection.
[0054] FIG. 10 is a photograph of an apparatus of the invention,
showing a chip with an inlet channel and reservoir, a detection
region, a branch point, and two outlet channels with
reservoirs.
[0055] FIGS. 11A and 11B show a sorting scheme according to the
invention, in diagrammatic form.
[0056] FIGS. 12A and 12B show a reversible sorting scheme according
to the invention.
[0057] FIG. 13 shows the results a comparison between a fingerprint
for T7 phage and a known T7 sample, using the method and a
microfabricated device of the invention.
[0058] FIG. 14 shows the results a comparison between a fingerprint
for T7 phage and a known lambda phage sample, using the method and
a microfabricated device of the invention.
[0059] FIG. 15 shows a comparison between a T7 phage sample and a
lambda phage sample against a T7 fingerprint, using a threshold
detection algorithm in a microfabricated device of the
invention.
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. Definitions
[0060] The terms used in this specification generally have their
ordinary meanings in the art, within the context of the invention,
and in the specific context where each term is used. Certain terms
are discussed below, or elsewhere in the specification, to provide
additional guidance to the practitioner in describing the devices
and methods of the invention and how to make and use them. For
convenience, certain terms are highlighted, for example using
italics and/or quotation masks. The use of highlighting has no
influence on the scope and meaning of a term; the scope and meaning
of a term is the same, in the same context, whether or not it is
highlighted. It will be appreciated that the same thing can be said
in more than one way. Consequently, alternative language and
synonyms may be used for any one or more of the terms discussed
herein, nor is any special significance to be placed upon whether
or not a term is elaborated or discussed herein. Synonyms for
certain terms are provided. A recital of one or more synonyms does
not exclude the use of other synonyms. The use of examples anywhere
in this specification, including examples of any terms discussed
herein, is illustrative only, and in no way limits the scope and
meaning of the invention or of any exemplified term. Likewise, the
invention is not limited to the preferred embodiments.
[0061] General Definitions. As used herein, the term "isolated"
means that the referenced material is removed from the environment
in which it is normally found. Thus, an isolated biological
material can be free of cellular components, i.e., components of
the cells in which the material is found or produced. In the case
of nucleic acid molecules, an isolated nucleic acid includes a PCR
product, an isolated mRNA, a cDNA, or a restriction fragment. In
another embodiment, an isolated nucleic acid is preferably excised
from the chromosome in which it may be found, and more preferably
is no longer joined to non-regulatory, non-coding regions, or to
other genes, located upstream or downstream of the gene contained
by the isolated nucleic acid molecule when found in the chromosome.
In yet another embodiment, the isolated nucleic acid lacks one or
more introns. Isolated nucleic acid molecules include sequences
inserted into plasmids, cosmids, artificial chromosomes, and the
like. Thus, in a specific embodiment, a recombinant nucleic acid is
an isolated nucleic acid. An isolated protein may be associated
with other proteins or nucleic acids, or both, with which it
associates in the cell, or with cellular membranes if it is a
membrane-associated protein. An isolated organelle, cell, or tissue
is removed from the anatomical site in which it is found in an
organism. An isolated material may be, but need not be,
purified.
[0062] The term "purified" as used herein refers to material that
has been isolated under conditions that reduce or eliminate the
presence of unrelated materials, i.e., contaminants, including
native materials from which the material is obtained. For example,
a purified protein is preferably substantially free of other
proteins or nucleic acids with which it is associated in a cell; a
purified nucleic acid molecule is preferably substantially free of
proteins or other unrelated nucleic acid molecules with which it
can be found within a cell. As used herein, the term "substantially
free" is used operationally, in the context of analytical testing
of the material. Preferably, purified material substantially free
of contaminants is at least 50% pure; more preferably, at least 90%
pure, and more preferably still at least 99% pure. Purity can be
evaluated by chromatography, gel electrophoresis, immunoassay,
composition analysis, biological assay, and other methods known in
the art.
[0063] Methods for purification are well-known in the art. For
example, nucleic acids can be purified by precipitation,
chromatography (including preparative solid phase chromatography,
oligonucleotide hybridization, and triple helix chromatography),
ultracentrifugation, and other means. Polypeptides and proteins can
be purified by various methods including, without limitation,
preparative disc-gel electrophoresis, isoelectric focusing, HPLC,
reversed-phase HPLC, gel filtration, ion exchange and partition
chromatography, precipitation and salting-out chromatography,
extraction, and countercurrent distribution. For some purposes, it
is preferable to produce the polypeptide in a recombinant system in
which the protein contains an additional sequence tag that
facilitates purification, such as, but not limited to, a
polyhistidine sequence, or a sequence that specifically binds to an
antibody, such as FLAG and GST. The polypeptide can then be
purified from a crude lysate of the host cell by chromatography on
an appropriate solid-phase matrix. Alternatively, antibodies
produced against the protein or against peptides derived therefrom
can be used as purification reagents. Cells can be purified by
various techniques, including centrifugation, matrix separation
(e.g., nylon wool separation), panning and other immunoselection
techniques, depletion (e.g., complement depletion of contaminating
cells), and cell sorting (e.g., fluorescence activated cell sorting
which is also referred to as FACS). Other purification methods are
possible. A purified material may contain less than about 50%,
preferably less than about 75%, and most preferably less than about
90%, of the cellular components with which it was originally
associated. The "substantially pure" indicates the highest degree
of purity which can be achieved using conventional purification
techniques known in the art.
[0064] A "sample" as used herein refers to a biological material
which can be tested, e.g., for the presence of CK-2 polypeptides or
CK-2 nucleic acids, e.g., to identify cells that specifically
express the CK-2 gene and its gene product. Such samples can be
obtained from any source, including tissue, blood and blood cells,
including circulating hematopoietic stem cells (for possible
detection of protein or nucleic acids), plural effusions,
cerebrospinal fluid (CSF), ascites fluid, and cell culture. In
preferred embodiments samples are obtained from bone marrow.
[0065] Non-human animals include, without limitation, laboratory
animals such as mice, rats, rabbits, hamsters, guinea pigs, etc.;
domestic animals such as dogs and cats; and, farm animals such as
sheep, goats, pigs, horses, and cows.
[0066] In preferred embodiments, the terms "about" and
"approximately" shall generally mean an acceptable degree of error
for the quantity measured given the nature or precision of the
measurements. Typical, exemplary degrees of error are within 20
percent (%), preferably within 10%, and more preferably within 5%
of a given value or range of values. Alternatively, and
particularly in biological systems, the terms "about" and
"approximately" may mean values that are within an order of
magnitude, preferably within 5-fold and more preferably within
2-fold of a given value. Numerical quantities given herein are
approximate unless stated otherwise, meaning that the term "about"
or "approximately" can be inferred when not expressly stated.
[0067] The term "molecule" means any distinct or distinguishable
structural unit of matter comprising one or more atoms, and
includes, for example, polypeptides and polynucleotides.
[0068] As used herein, the term "cell" means any cell or cells
(e.g., biological cells) as well as viruses or any other particle
having a microscopic size; e.g., a size that similar to (such as
having the same order of magnitude as) the size of a biological
cell. The terms cell therefore encompasses both prokaryotic and
eukaryotic cells; including bacteria, fungi, plant and animal
cells. Cells are typically spherical, but can also be elongated,
flattened, deformable and asymmetrical, i.e., non-spherical. The
size or diameter of a cell typically ranges from about 0.1 to 120
microns, and typically is from about 1 to 50 microns. A cell may be
living or dead. Since the microfabricated device of the invention
is directed to sorting materials having a size similar to a
biological cell (e.g. about 0.1 to 120 microns) any material having
a size similar to a biological cell can be characterized and sorted
using the microfabricated device of the invention. Thus, the term
cell shall further include microscopic beads (such as
chromatographic and fluorescent beads), liposomes, emulsions, or
any other encapsulating biomaterials and porous materials.
Non-limiting examples include latex, glass, or paramagnetic beads;
and vesicles such as emulsions and liposomes, and other porous
materials such as silica beads. Beads ranging in size from 0.1
micron to 1 mm can also be used, for example in sorting a library
of compounds produced by combinatorial chemistry. As used herein, a
cell may be charged or uncharged. For example, charged beads may be
used to facilitate flow or detection, or as a reporter. Biological
cells, living or dead, may be charged for example by using a
surfactant, such as SDS (sodium dodecyl sulfate).
[0069] A "reporter" is any molecule, or a portion thereof, that is
detectable, or measurable, for example, by optical detection. In
addition, the reporter associates with a molecule or cell or with a
particular marker or characteristic of the molecule or cell, or is
itself detectable, to permit identification of the molecule or
cell, or the presence or absence of a characteristic of the
molecule or cell. In the case of molecules such as polynucleotides
such characteristics include size, molecular weight, the presence
or absence of particular constituents or moieties (such as
particular nucleotide sequences or restrictions sites). The term
"label" can be used interchangeably with "reporter". The reporter
is typically a dye, fluorescent, ultraviolet, or chemiluminescent
agent, chromophore, or radio-label, any of which may be detected
with or without some kind of stimulatory event, e.g., fluoresce
with or without a reagent. Typical reporters for molecular
fingerprinting include without limitation fluorescently-labeled
single nucleotides such as fluorescein-dNTP, rhodamine-dNTP,
Cy3-dNTP, Cy5-dNTP, where dNTP represents dATP, dTTP, dUTP or dCTP.
The reporter can also be chemically-modified single nucleotides,
such as biotin-dNTP. Alternatively, chemicals can be used that
react with an attached functional group such as biotin.
[0070] A "marker" is a characteristic of a molecule or cell that is
detectable or is made detectable by a reporter, or which may be
coexpressed with a reporter. For molecules, a marker can be
particular constituents or moieties, such as restrictions sites or
particular nucleic acid sequences in the case of polynucleotides.
The marker may be directly or indirectly associated with the
reporter or can itself be a reporter. Thus, a marker is generally a
distinguishing feature of a molecule, and a reporter is generally
an agent which directly or indirectly identifies or permits
measurement of a marker. These terms may, however, be used
interchangeably.
[0071] The term "flow" means any movement of liquid or solid
through a device or in a method of the invention, and encompasses
without limitation any fluid stream, and any material moving with,
within or against the stream, whether or not the material is
carried by the stream. For example, the movement of molecules or
cells through a device or in a method of the invention, e.g.
through channels of a microfluidic chip of the invention, comprises
a flow. This is so, according to the invention, whether or not the
molecules or cells are carried by a stream of fluid also comprising
a flow, or whether the molecules or cells are caused to move by
some other direct or indirect force or motivation, and whether or
not the nature of any motivating force is known or understood. The
application of any force may be used to provide a flow, including
without limitation, pressure, capillary action, electro-osmosis,
electrophoresis, dielectrophoresis, optical tweezers, and
combinations thereof, without regard for any particular theory or
mechanism of action, so long as molecules or cells are directed for
detection, measurement or sorting according to the invention.
[0072] An "inlet region" is an area of a microfabricated chip that
receives molecules or cells for detection measurement or sorting.
The inlet region may contain an inlet channel, a well or reservoir,
an opening, and other features which facilitate the entry of
molecules or cells into the device. A chip may contain more than
one inlet region if desired. The inlet region is in fluid
communication with the main channel and is upstream therefrom.
[0073] An "outlet region" is an area of a microfabricated chip that
collects or dispenses molecules or cells after detection,
measurement or sorting. An outlet region is downstream from a
discrimination region, and may contain branch channels or outlet
channels. A chip may contain more than one outlet region if
desired.
[0074] An "analysis unit" is a microfabricated substrate, e.g., a
microfabricated chip, having at least one inlet region, at least
one main channel, at least one detection region and at least one
outlet region. Sorting embodiments of the analysis unit include a
discrimination region and/or a branch point, e.g. downstream of the
detection region, that forms at least two branch channels and two
outlet regions. A device of the invention may comprise a plurality
of analysis units.
[0075] A "main channel" is a channel of the chip of the invention
which permits the flow of molecules or cells past a detection
region for detection (identification), measurement, or sorting. In
a chip designed for sorting, the main channel also comprises a
discrimination region. The detection and discrimination regions can
be placed or fabricated into the main channel. The main channel is
typically in fluid communication with an inlet channel or inlet
region, which permit the flow of molecules or cells into the main
channel. The main channel is also typically in fluid communication
with an outlet region and optionally with branch channels, each of
which may have an outlet channel or waste channel. These channels
permit the flow of cells out of the main channel.
[0076] A "detection region" is a location within the chip,
typically within the main channel where molecules or cells to be
identified, measured or sorted are examined on the basis of a
predetermined characteristic. In a preferred embodiment, molecules
or cells are examined one at a time, and the characteristic is
detected or measured optically, for example, by testing for the
presence or amount of a reporter. For example, the detection region
is in communication with one or more microscopes, diodes, light
stimulating devices, (e.g., lasers), photomultiplier tubes, and
processors (e.g., computers and software), and combinations
thereof, which cooperate to detect a signal representative of a
characteristic, marker, or reporter, and to determine and direct
the measurement or the sorting action at the discrimination region.
In sorting embodiments the detection region is in fluid
communication with a discrimination region and is at, proximate to,
or upstream of the discrimination region.
[0077] A "discrimination region" or "branch point" is a junction of
a channel where the flow of molecules or cells can change direction
to enter one or more other channels, e.g., a branch channel,
depending on a signal received in connection with an examination in
the detection region. Typically, a discrimination region is
monitored and/or under the control of a detection region, and
therefore a discrimination region may "correspond" to such
detection region. The discrimination region is in communication
with and is influenced by one or more sorting techniques or flow
control systems, e.g., electric, electro-osmotic, (micro-) valve,
etc. A flow control system can employ a variety of sorting
techniques to change or direct the flow of molecules or cells into
a predetermined branch channel.
[0078] A "branch channel" is a channel which is in communication
with a discrimination region and a main channel. Typically, a
branch channel receives molecules or cells depending on the
molecule or cell characteristic of interest as detected by the
detection region and sorted at the discrimination region. A branch
channel may be in communication with other channels to permit
additional sorting. Alternatively, a branch channel may also have
an outlet region and/or terminate with a well or reservoir to allow
collection or disposal of the molecules or cells.
[0079] The term "forward sorting" or flow describes a one-direction
flow of molecules or cells, typically from an inlet region
(upstream) to an outlet region (downstream), and preferably without
a change in direction, e.g., opposing the "forward" flow.
Preferably, molecules or cells travel forward in a linear fashion,
i.e., in single file. A preferred "forward" sorting algorithm
consists of running molecules or cells from the input channel to
the waste channel, until a molecule or cell is identified to have
an optically detectable signal (e.g. fluorescence) that is above a
pre-set threshold, at which point voltages are temporarily changed
to electroosmotically divert the molecule or to the collection
channel.
[0080] The term "reversible sorting" or flow describes a movement
or flow that can change, i.e., reverse direction, for example, from
a forward direction to an opposing backwards direction. Stated
another way, reversible sorting permits a change in the direction
of flow from a downstream to an upstream direction. This may be
useful for more accurate sorting, for example, by allowing for
confirmation of a sorting decision, selection of particular branch
channel, or to correct an improperly selected channel.
[0081] Different algorithms for sorting in the microfluidic device
can be implemented by different programs, for example under the
control of a personal computer. As an example, consider a
pressure-switched scheme instead of electro-osmotic flow.
Electro-osmotic switching is virtually instantaneous and throughput
is limited by the highest voltage that can be applied to the sorter
(which also affects the run time through ion depletion effects). A
pressure switched-scheme does not require high voltages and is more
robust for longer runs. However, mechanical compliance in the
system is likely to cause the fluid switching speed to become
rate-limiting with the "forward" sorting program. Since the fluid
is at low Reynolds number and is completely reversible, when trying
to separate rare molecules or cells one can implement a sorting
algorithm that is not limited by the intrinsic switching speed of
the device. The molecules or cells flow at the highest possible
static (non-switching) speed from the input to the waste. When an
interesting molecule or cell is detected, the flow is stopped. By
the time the flow stops, the molecule or cell may be past the
junction and part way down the waste channel. The system is then
run backwards at a slow (switchable) speed from waste to input, and
the molecule or cell is switched to the collection channel when it
passes through the detection region. At that point, the molecule or
cell is "saved" and the device can be run at high speed in the
forward direction again. Similarly, an device of the invention that
is used for analysis, without sorting, can be run in reverse to
re-read or verify the detection or analysis made for one or more
molecules or cells in the detection region. This "reversible"
analysis or sorting method is not possible with standard gel
electrophoresis technologies (for molecules) nor with conventional
FACS machines (for cells). Reversible algorithms are particularly
useful for collecting rare molecules or cells or making multiple
time course measurements of a molecule or single cell.
[0082] Molecular Biology Definitions. In accordance with the
present invention, there may be employed conventional molecular
biology, microbiology and recombinant DNA techniques within the
skill of the art. Such techniques are explained fully in the
literature. See, for example, Sambrook, Fitsch & Maniatis,
Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (referred
to herein as "Sambrook et al., 1989"); DNA Cloning: A Practical
Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide
Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D.
Hames & S. J. Higgins, eds. 1984); Animal Cell Culture (R. I.
Freshney, ed. 1986); Immobilized Cells and Enzymes (IRL Press,
1986); B. E. Perbal, A Practical Guide to Molecular Cloning (1984);
F. M. Ausubel et al. (eds.), Current Protocols in Molecular
Biology, John Wiley & Sons, Inc. (1994).
[0083] The term "polymer" means any substance or compound that is
composed of two or more building blocks (`mers`) that are
repetitively linked together. For example, a "dimer" is a compound
in which two building blocks have been joined together; a "trimer"
is a compound in which three building blocks have been joined
together; etc.
[0084] The term "polynucleotide" or "nucleic acid molecule" as used
herein refers to a polymeric molecule having a backbone that
supports bases capable of hydrogen bonding to typical
polynucleotides, wherein the polymer backbone presents the bases in
a manner to permit such hydrogen bonding in a specific fashion
between the polymeric molecule and a typical polynucleotide (e.g.,
single-stranded DNA). Such bases are typically inosine, adenosine,
guanosine, cytosine, uracil and thymidine. Polymeric molecules
include "double stranded" and "single stranded" DNA and RNA, as
well as backbone modifications thereof (for example,
methylphosphonate linkages).
[0085] Thus, a "polynucleotide" or "nucleic acid" sequence is a
series of nucleotide bases (also called "nucleotides"), generally
in DNA and RNA, and means any chain of two or more nucleotides. A
nucleotide sequence frequently carries genetic information,
including the information used by cellular machinery to make
proteins and enzymes. The terms include genomic DNA, cDNA, RNA, any
synthetic and genetically manipulated polynucleotide, and both
sense and antisense polynucleotides. This includes single- and
double-stranded molecules; i.e., DNA-DNA, DNA-RNA, and RNA-RNA
hybrids as well as "protein nucleic acids" (PNA) formed by
conjugating bases to an amino acid backbone. This also includes
nucleic acids containing modified bases, for example, thio-uracil,
thio-guanine and fluoro-uracil.
[0086] The polynucleotides herein may be flanked by natural
regulatory sequences, or may be associated with heterologous
sequences, including promoters, enhancers, response elements,
signal sequences, polyadenylation sequences, introns, 5'- and
3'-non-coding regions and the like. The nucleic acids may also be
modified by many means known in the art. Non-limiting examples of
such modifications include methylation, "caps", substitution of one
or more of the naturally occurring nucleotides with an analog, and
internucleotide modifications such as, for example, those with
uncharged linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoroamidates, carbamates, etc.) and with charged linkages
(e.g., phosphorothioates, phosphorodithioates, etc.).
Polynucleotides may contain one or more additional covalently
linked moieties, such as proteins (e.g., nucleases, toxins,
antibodies, signal peptides, poly-L-lysine, etc.), intercalators
(e.g., acridine, psoralen, etc.), chelators (e.g., metals,
radioactive metals, iron, oxidative metals, etc.) and alkylators to
name a few. The polynucleotides may be derivatized by formation of
a methyl or ethyl phosphotriester or an alkyl phosphoramidite
linkage. Furthermore, the polynucleotides herein may also be
modified with a label capable of providing a detectable signal,
either directly or indirectly. Exemplary labels include
radioisotopes, fluorescent molecules, biotin and the like. Other
non-limiting examples of modification which may be made are
provided, below, in the description of the present invention.
[0087] As used herein, the term "oligonucleotide" refers to a
nucleic acid, generally of at least 10, preferably at least 15, and
more preferably at least 20 nucleotides, preferably no more than
100 nucleotides, that is hybridizable to a genomic DNA molecule, a
cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or
other nucleic acid of interest. Oligonucleotides can be labeled,
e.g., with .sup.32P-nucleotides or nucleotides to which a label,
such as biotin or a fluorescent dye (for example, Cy3 or Cy5) has
been covalently conjugated. In one embodiment, a labeled
oligonucleotide can be used as a probe to detect the presence of a
nucleic acid. In another embodiment, oligonucleotides (one or both
of which may be labeled) can be used as PCR primers, either for
cloning full length or a fragment of a particular nucleic acid
(e.g., a particular gene or a particular gene sequence), or to
detect the presence of particular nucleic acids (e.g., of a
particular gene or a particular gene sequence). Generally,
oligonucleotides are prepared synthetically, preferably on a
nucleic acid synthesizer. Accordingly, oligonucleotides can be
prepared with non-naturally occurring phosphoester analog bonds,
such as thioester bonds, etc.
[0088] Specific non-limiting examples of synthetic nucleotides
(including polynucleotides and oligonucleotides) envisioned for
this invention include, in addition to the nucleic acid moieties
described above, oligonucleotides that contain phosphorothioates,
phosphotriesters, methyl phosphonates, short chain alkyl, or
cycloalkyl intersugar linkages or short chain heteroatomic or
heterocyclic intersugar linkages. Most preferred are those with
CH.sub.2--NH--O--CH.sub.2, CH.sub.2--N(CH.sub.3)--O--CH.sub.2,
CH.sub.2--O--N(CH.sub.3)--CH.sub.2,
CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2, and
O--N(CH.sub.3)--CH.sub.2--CH.sub.2 backbones (where phosphodiester
is O--PO.sub.7--O--CH.sub.2). U.S. Pat. No. 5,677,437 describes
heteroaromatic olignucleoside linkages. Nitrogen linkers or groups
containing nitrogen can also be used to prepare oligonucleotide
mimics (U.S. Pat. Nos. 5,792,844 and 5,783,682). U.S. Pat. No.
5,637,684 describes phosphoramidate and phosphorothioamidate
oligomeric compounds. Also envisioned are oligonucleotides having
morpholino backbone structures (U.S. Pat. No. 5,034,506). In other
embodiments, such as the peptide-nucleic acid (PNA) backbone, the
phosphodiester backbone of the oligonucleotide may be replaced with
a polyamide backbone, the bases being bound directly or indirectly
to the aza nitrogen atoms of the polyamide backbone (Nielsen et
al., Science 254:1497, 1991). Other synthetic oligonucleotides may
contain substituted sugar moieties comprising one of the following
at the 2' position: OH, SH, SCH.sub.3, F, OCN,
O(CH.sub.2).sub.nNH.sub.2 or O(CH.sub.2).sub.aCH.sub.3 where n is
from 1 to about 10; C.sub.1 to C.sub.10 lower alkyl, substituted
lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF.sub.3; OCF.sub.3;
O--; S--, or N-alkyl; O--, S--, or N-alkenyl; SOCH.sub.3;
SO.sub.2CH.sub.3; ONO.sub.2; NO.sub.2; N.sub.3; NH.sub.2;
heterocycloalkyl; heterocycloalkaryl; aminoalkylamino;
polyalkylamino; substituted silyl; a fluorescein moiety; an RNA
cleaving group; a reporter group; an intercalator; a group for
improving the pharmacokinetic properties of an oligonucleotide; or
a group for improving the pharmacodynamic properties of an
oligonucleotide, and other substituents having similar properties.
Oligonucleotides may also have sugar mimetics such as cyclobutyls
or other carbocyclics in place of the pentofuranosyl group.
Nucleotide units having nucleosides other than adenosine, cytidine,
guanosine, thymidine and uridine, such as inosine, may be used in
an oligonucleotide molecule.
[0089] A "polypeptide" is a chain of chemical building blocks
called amino acids that are linked together by chemical bonds
called "peptide bonds". The term "protein" refers to polypeptides
that contain the amino acid residues encoded by a gene or by a
nucleic acid molecule (e.g., an mRNA or a cDNA) transcribed from
that gene either directly or indirectly. Optionally, a protein may
lack certain amino acid residues that are encoded by a gene or by
an mRNA. For example, a gene or mRNA molecule may encode a sequence
of amino acid residues on the N-terminus of a protein (i.e., a
signal sequence) that is cleaved from; and therefore may not be
part of, the final protein. A protein or polypeptide, including an
enzyme, may be a "native" or "wild-type", meaning that it occurs in
nature; or it may be a "mutant", "variant" or "modified", meaning
that it has been made, altered, derived, or is in some way
different or changed from a native protein or from another
mutant.
[0090] "Amplification" of a polynucleotide, as used herein, denotes
the use of polymerase chain reaction (PCR) to increase the
concentration of a particular DNA sequence within a mixture of DNA
sequences. For a description of PCR see Saiki et al., Science 1988,
239:487.
[0091] "Chemical sequencing" of DNA denotes methods such as that of
Maxam and Gilbert (Maxam-Gilbert sequencing; see Maxam &
Gilbert, Proc. Natl. Acad. Sci. U.S.A. 1977, 74:560), in which DNA
is cleaved using individual base-specific reactions.
[0092] "Enzymatic sequencing" of DNA denotes methods such as that
of Sanger (Sanger et al, Proc. Natl. Acad. Sci. U.S.A. 1977,
74:5463) and variations thereof well known in the art, in a
single-stranded DNA is copied and randomly terminated using DNA
polymerase.
[0093] A "gene" is a sequence of nucleotides which code for a
functional "gene product". Generally, a gene product is a
functional protein. However, a gene product can also be another
type of molecule in a cell, such as an RNA (e.g., a tRNA or a
rRNA). For the purposes of the present invention, a gene product
also refers to an mRNA sequence which may be found in a cell. For
example, measuring gene expression levels according to the
invention may correspond to measuring mRNA levels. A gene may also
comprise regulatory (i.e., non-coding) sequences as well as coding
sequences. Exemplary regulatory sequences include promoter
sequences, which determine, for example, the conditions under which
the gene is expressed. The transcribed region of the gene may also
include untranslated regions including introns, a 5'-untranslated
region (5'-UTR) and a 3'-untranslated region (3'-UTR).
[0094] A "coding sequence" or a sequence "encoding" an expression
product, such as a RNA, polypeptide, protein or enzyme, is a
nucleotide sequence that, when expressed, results in the production
of that RNA, polypeptide, protein or enzyme; i.e., the nucleotide
sequence "encodes" that RNA or it encodes the amino acid sequence
for that polypeptide, protein or enzyme.
[0095] A "promoter sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating-transcription of a
downstream (3' direction) coding sequence. For purposes of defining
the present invention, the promoter sequence is bounded at its 3'
terminus by the transcription initiation site and extends upstream
(5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background. Within the promoter sequence will be found a
transcription initiation site (conveniently found, for example, by
mapping with nuclease S1), as well as protein binding domains
(consensus sequences) responsible for the binding of RNA
polymerase.
[0096] A coding sequence is "under the control of" or is
"operatively associated with" transcriptional and translational
control sequences in a cell when RNA polymerase transcribes the
coding sequence into RNA, which is then trans-RNA spliced (if it
contains introns) and, if the sequence encodes a protein, is
translated into that protein.
[0097] The term "express" and "expression" means allowing or
causing the information in a gene or DNA sequence to become
manifest, for example producing RNA (such as rRNA or mRNA) or a
protein by activating the cellular functions involved in
transcription and translation of a corresponding gene or DNA
sequence. A DNA sequence is expressed by a cell to form an
"expression product" such as an RNA (e.g., a mRNA or a rRNA) or a
protein. The expression product itself, e.g., the resulting RNA or
protein, may also said to be "expressed" by the cell.
[0098] The term "transfection" means the introduction of a foreign
nucleic acid into a cell. The term "transformation" means the
introduction of a "foreign" (i.e., extrinsic or extracellular)
gene, DNA or RNA sequence into a host cell so that the host cell
will express the introduced gene or sequence to produce a desired
substance, in this invention typically an RNA coded by the
introduced gene or sequence, but also a protein or an enzyme coded
by the introduced gene or sequence. The introduced gene or sequence
may also be called a "cloned" or "foreign" gene or sequence, may
include regulatory or control sequences (e.g., start, stop,
promoter, signal, secretion or other sequences used by a cell's
genetic machinery). The gene or sequence may include nonfunctional
sequences or sequences with no known function. A host cell that
receives and expresses introduced DNA or RNA has been "transformed"
and is a "transformant" or a "clone". The DNA or RNA introduced to
a host cell can come from any source, including cells of the same
genus or species as the host cell or cells of a different genus or
species.
[0099] The terms "vector", "cloning vector" and "expression vector"
mean the vehicle by which a DNA or RNA sequence (e.g., a foreign
gene) can be introduced into a host cell so as to transform the
host and promote expression (e.g., transcription and translation)
of the introduced sequence. Vectors may include plasmids, phages,
viruses, etc. and are discussed in greater detail below.
[0100] A "cassette" refers to a DNA coding sequence or segment of
DNA that codes for an expression product that can be inserted into
a vector at defined restriction sites. The cassette restriction
sites are designed to ensure insertion of the cassette in the
proper reading frame. Generally, foreign DNA is inserted at one or
more restriction sites of the vector DNA, and then is carried by
the vector into a host cell along with the transmissible vector
DNA. A segment or sequence of DNA having inserted or added DNA,
such as an expression vector, can also be called a "DNA construct."
A common type of vector is a "plasmid", which generally is a
self-contained molecule of double-stranded DNA, usually of
bacterial origin, that can readily accept additional (foreign) DNA
and which can readily introduced into a suitable host cell. A large
number of vectors, including plasmid and fungal vectors, have been
described for replication and/or expression in a variety of
eukaryotic and prokaryotic hosts. The term "host cell" means any
cell of any organism that is selected, modified, transformed, grown
or used or manipulated in any way for the production of a substance
by the cell. For example, a host cell may be one that is
manipulated to express a particular gene, a DNA or RNA sequence, a
protein or an enzyme. Host cells can further be used for screening
or other assays that are described infra. Host cells may be
cultured in vitro or one or more cells in a non-human animal (e.g.,
a transgenic animal or a transiently transfected animal).
[0101] The term "expression system" means a host cell and
compatible vector under suitable conditions, e.g. for the
expression of a protein coded for by foreign DNA carried by the
vector and introduced to the host cell. Common expression systems
include E. coli host cells and plasmid vectors, insect host cells
such as Sf9, Hi5 or S2 cells and Baculovirus vectors, Drosophila
cells (Schneider cells) and expression systems, fish cells and
expression systems (including, for example, RTH-149 cells from
rainbow trout, which are available from the American Type Culture
Collection and have been assigned the accession no. CRL-1710) and
mammalian host cells and vectors.
[0102] The term "heterologous" refers to a combination of elements
not naturally occurring. For example, the present invention
includes chimeric RNA molecules that comprise an rRNA sequence and
a heterologous RNA sequence which is not part of the rRNA sequence.
In this context, the heterologous RNA sequence refers to an RNA
sequence that is not naturally located within the ribosomal RNA
sequence. Alternatively, the heterologous RNA sequence may be
naturally located within the ribosomal RNA sequence, but is found
at a location in the rRNA sequence where it does not naturally
occur. As another example, heterologous DNA refers to DNA that is
not naturally located in the cell, or in a chromosomal site of the
cell. Preferably, heterologous DNA includes a gene foreign to the
cell. A heterologous expression regulatory element is a regulatory
element operatively associated with a different gene that the one
it is operatively associated with in nature.
[0103] The terms "mutant" and "mutation" mean any detectable change
in genetic material, e.g., DNA, or any process, mechanism or result
of such a change. This includes gene mutations, in which the
structure (e.g., DNA sequence) of a gene is altered, any gene or
DNA arising from any mutation process, and any expression product
(e.g., RNA, protein or enzyme) expressed by a modified gene or DNA
sequence. The term "variant" may also be used to indicate a
modified or altered gene, DNA sequence, RNA, enzyme, cell, etc.;
i.e., any kind of mutant. For example, the present invention
relates to altered or "chimeric" RNA molecules that comprise an
rRNA sequence that is altered by inserting a heterologous RNA
sequence that is not naturally part of that sequence or is not
naturally located at the position of that rRNA sequence. Such
chimeric RNA sequences, as well as DNA and genes that encode them,
are also referred to herein as "mutant" sequences.
[0104] The term "homologous", in all its grammatical forms and
spelling variations, refers to the relationship between two
proteins that possess a "common evolutionary origin", including
proteins from superfamilies (e.g., the immunoglobulin superfamily)
in the same species of organism, as well as homologous proteins
from different species of organism (for example, myosin light chain
polypeptide, etc.; see, Reeck et al., Cell 1987, 50:667). Such
proteins (and their encoding nucleic acids) have sequence homology,
as reflected by their sequence similarity, whether in terms of
percent identity or by the presence of specific residues or motifs
and conserved positions.
[0105] The term "sequence similarity", in all its grammatical
forms, refers to the degree of identity or correspondence between
nucleic acid or amino acid sequences that may or may not share a
common evolutionary origin (see, Reeck et al., supra). However, in
common usage and in the instant application, the term "homologous",
when modified with an adverb such as "highly", may refer to
sequence similarity and may or may not relate to a common
evolutionary origin.
[0106] In specific embodiments, two nucleic acid sequences are
"substantially homologous" or "substantially similar" when at least
about 80%, and more preferably at least about 90% or at least about
95% of the nucleotides match over a defined length of the nucleic
acid sequences, as determined by a sequence comparison algorithm
known such as BLAST, FASTA, DNA Strider, CLUSTAL, etc. An example
of such a sequence is an allelic or species variant of the specific
genes of the present invention. Sequences that are substantially
homologous may also be identified by hybridization, e.g., in a
Southern hybridization experiment under, e.g., stringent conditions
as defined for that particular system.
[0107] Similarly, in particular embodiments of the invention, two
amino acid sequences are "substantially homologous" or
"substantially similar" when greater than 80% of the amino acid
residues are identical, or when greater than about 90% of the amino
acid residues are similar (i.e., are functionally identical).
Preferably the similar or homologous polypeptide sequences are
identified by alignment using, for example, the GCG (Genetics
Computer Group, Program Manual for the GCG Package, Version 7,
Madison Wis.) pileup program, or using any of the programs and
algorithms described above (e.g., BLAST, FASTA, CLUSTAL, etc.).
[0108] A nucleic acid molecule is "hybridizable" to another nucleic
acid molecule, such as a cDNA, genomic DNA, or RNA, when a single
stranded form of the nucleic acid molecule can anneal to the other
nucleic acid molecule under the appropriate conditions of
temperature and solution ionic strength (see Sambrook et al.,
supra). The conditions of temperature and ionic strength determine
the "stringency" of the hybridization. For preliminary screening
for homologous nucleic acids, low stringency hybridization
conditions, corresponding to a T.sub.m (melting temperature) of
55.degree. C., can be used, e.g., 5.times.SSC, 0.1% SDS, 0.25%
milk, and no formamide; or 30% formamide, 5.times.SSC, 0.5% SDS).
Moderate stringency hybridization conditions correspond to a higher
T.sub.m, e.g., 40% formamide, with 5.times. or 6.times.SSC. High
stringency hybridization conditions correspond to the highest
T.sub.m, e.g., 50% formamide, 5.times. or 6.times.SSC. SSC is a
0.15M NaCl, 0.015M Na-citrate. Hybridization requires that the two
nucleic acids contain complementary sequences, although depending
on the stringency of the hybridization, mismatches between bases
are possible. The appropriate stringency for hybridizing nucleic
acids depends on the length of the nucleic acids and the degree of
complementation, variables well known in the art. The greater the
degree of similarity or homology between two nucleotide sequences,
the greater the value of T.sub.m for hybrids of nucleic acids
having those sequences. The relative stability (corresponding to
higher T.sub.m) of nucleic acid hybridizations decreases in the
following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater
than 100 nucleotides in length, equations for calculating T.sub.m
have been derived (see Sambrook et al., supra, 9.50-9.51). For
hybridization with shorter nucleic acids, i.e., oligonucleotides,
the position of mismatches becomes more important, and the length
of the oligonucleotide determines its specificity (see Sambrook et
al., supra, 11.7-11.8). A minimum length for a hybridizable nucleic
acid is at least about 10 nucleotides; preferably at least about 15
nucleotides; and more preferably the length is at least about 20
nucleotides.
[0109] In a specific embodiment; the term "standard hybridization
conditions" refers to a T.sub.m of 55.degree. C., and utilizes
conditions as set forth above. In a preferred embodiment, the
T.sub.m is 60.degree. C.; in amore preferred embodiment, the
T.sub.m is 65.degree. C. In a specific embodiment, "high
stringency" refers to hybridization and/or washing conditions at
68.degree. C. in 0.2.times.SSC, at 42.degree. C. in 50% formamide,
4.times.SSC, or under conditions that afford levels of
hybridization equivalent to those observed under either of these
two conditions.
[0110] Suitable hybridization conditions for oligonucleotides
(e.g., for oligonucleotide probes or primers) are typically
somewhat different than for full-length nucleic acids (e.g.,
full-length cDNA), because of the oligonucleotides lower melting
temperature. Because the melting temperature of oligonucleotides
will depend on the length of the oligonucleotide sequences
involved, suitable hybridization temperatures will vary depending
upon the oligonucleotide molecules used. Exemplary temperatures may
be 37.degree. C. (for 14-base oligonucleotides), 48.degree. C. (for
17-base oligonucleotides), 55.degree. C. (for 20-base
oligonucleotides) and 60.degree. C. (for 23-base oligonucleotides).
Exemplary suitable hybridization conditions for oligonucleotides
include washing in 6.times.SSC/0.05% sodium pyrophosphate, or other
conditions that afford equivalent levels of hybridization.
5.2. Overview of the Invention
[0111] The invention provides a method and system for molecular
fingerprinting, and devices for molecular fingerprinting, including
microfabricated microfluidic devices for evaluating or sorting
molecules according to size. More particularly, polynucleotides
such as DNA samples can be fragmented, for example using
endonucleases, to produce a set of fragments that vary in size. The
size distribution of these fragments (e.g. the number of fragments
of each size over a range of sizes) may uniquely identify the
source of the sample. Some or all of the fragments can be selected
to serve as a "fingerprint" of the sample. Further, fragments
comprising the fingerprint can be labeled, for example with a
reporter molecule such as fluorescent marker, so that the they can
be more readily detected, measured or sorted.
[0112] These measurements can be detected by any suitable means,
preferably optical, and can be stored for example in a computer as
a representation of the fragments comprising the fingerprint
Depending on the strategy for producing the fragments which
comprise a fingerprint, oligonucleotide probes of known composition
and length may be used to "tag" or label the fragments. For
example, probes having sequences that are complementary to each of
the fragments can be made by combining the fragments with labeled
nucleotide bases in the presence of a polymerase, which is an
enzyme that assembles a single strand of complementary
polynucleotide using another strand (i.e. a fingerprint fragment)
as a template. The nucleotide bases used to make these probes may
be radioactive, or can be labeled with a fluorescent marker, or
with some other readily detectable reporter. The resulting probes
can be used to record a fingerprint of the sample, by detecting and
measuring the lever of reporter as an indication of size, or by
sorting the probes according to size.
[0113] Labeled or unlabeled probes can also be used to "fish out"
matching polynucleotides from a test sample containing unknown DNA
or polynucleotides. Under appropriate hybridizing conditions,
probes will bind to matching fragments in a sample. This can
provide a way to test for a match, for example when the probes
comprising a fingerprint hybridize to complementary fragments in
the sample.
[0114] According to one aspect of the invention polynucleotides can
be fingerprinted using a synthetic version of restriction fragment
length polymorphism. The method includes choosing at random a short
(20-50 bp) sequence of the polynucleotide that is a fixed distance
away from a restriction site. This can be repeated any number of
times for enhanced statistical discrimination, with different
locations in the polynucleotide and different distances to a
restriction site. Samples are digested with a specific restriction
enzyme, such as Bg II, EcoR I, Hind III or Xho I. A final mixture
of DNA fragments of thousands of base pairs is preferred. In this
way a unique fingerprint can be synthesized without relying on
naturally occurring repeat sequences or restriction sites.
[0115] The method also provides for identification of the
fingerprinted nucleotide in a sample. To identify the fingerprinted
polynucleotide in a sample, an oligonucleotide probe is synthesized
to complement each randomly chosen sequence. Each probe is mixed
with the test sample along with nucleotide triphosphates and
polymerase. The nucleotides can be fluorescently labeled. Through
this technique a fluorescent strand of polynucleotide
(complementary to each probe) will be synthesized. Each strand is
then cut with restriction enzymes to yield a polynucleotide of a
fixed length. The polynucleotide can then be sized, either by gel
electrophoresis or in a single molecule sizing device (SMS) (59).
If multiple oligonucleotides are designed, the reaction can be
multiplexed and the different length fragments can then be resolved
into a fingerprint that can be compared to a standard
fingerprint.
[0116] In one aspect of the invention, polynucleotides, e.g., DNA,
can be detected, sized or sorted dynamically in a continuous flow
stream of microscopic dimensions based for example on molecular
weight, using a microfabricated polynucleotide sorting device. The
polynucleotides, suspended in a suitable carrier fluid (e.g.,
ddH.sub.2O or TE), are introduced into an inlet end of a narrow
channel in the sorting device. The molecular weight of each
molecule is calculated from the intensity of signal from an
optically-detectable reporter incorporated into or associated with
the polynucleotide molecule as the molecule passes through a
"detection window" or "detection region" in the device.
[0117] In a sorter embodiment, molecules having a molecular weight
falling within a selected range are diverted into a selected output
or "branch" channel of the device. The sorted polynucleotide
molecules may be collected from the output channels and used in
subsequent manipulations.
[0118] According to another aspect of the invention, a device such
as described above, but not necessarily including components for
sorting the molecules, can be used to measure or quantify the size
range of polynucleotides in a sample, and store or feed this
information into a processor or computer for subsequent analysis or
display, e.g., as a size distribution histogram. Such a device
enables the generation of the type of polynucleotide fragment
length data now commonly obtained from analytical gels, such as
agarose or polyacrylamide gels, or from Southern blot results, in a
fraction of the time required for preparation and analysis of gels,
and using a substantially-smaller amount of sample.
5.3. Microfluidic Chip Architecture and Methods
[0119] A molecular or cell analyzer or sorter according to the
invention comprises at least one analysis unit having an inlet
region in communication with a main channel, a detection region
within or coincident with a portion of the main channel, and a
detector associated with the detection region. Sorter embodiments
also have a discrimination region or branch point in communication
with the main channel and with branch channels, and a flow control
responsive to the detector. The branch channels may each lead to an
outlet region and to a well or reservoir. The inlet region may also
communicate with a well or reservoir. As each molecule or cell
passes into the detection region, it is examined for a
predetermined characteristic (i.e. using the detector), and a
corresponding signal is produced, for example indicating that "yes"
the characteristic is present, or "no" it is not. The signal may
correspond to a characteristic qualitatively or quantitatively.
That is, the amount of the signal can be measured and can
correspond to the degree to which a characteristic is present. For
example, the strength of the signal may indicate the size of the
molecule, or the potency or amount of an enzyme expressed by a
cell. In response to the signal, data can be collected and/or a
flow control can be activated to divert a molecule or cell into one
branch channel or another. Thus, molecules or cells within the
discrimination region can be sorted into an appropriate branch
channel according to a signal produced by the corresponding
examination at the detection region. Optical detection of molecule
or cell characteristics is preferred, for example directly or by
use of a reporter associated with a characteristic chosen for
sorting. However, other detection techniques may also be
employed.
[0120] A variety of channels for sample flow and mixing can be
microfabricated on a single chip and can be positioned at any
location on the chip as the detection and discrimination or sorting
points, e.g., for kinetic studies (12, 14). A plurality of analysis
units of the invention may be combined in one device.
Microfabrication applied according to the invention eliminates the
dead time occurring in conventional gel electrophoresis or flow
cytometric kinetic studies, and achieves a better time-resolution.
Furthermore, linear arrays of channels on a single chip, i.e., a
multiplex system, can simultaneously detect and sort a sample by
using an array of photomultiplier tubes (PMT) for parallel analysis
of different channels (15). This arrangement can be used to improve
throughput or for successive sample enrichment, and can be adapted
to provide a very high throughput to the microfluidic devices that
exceeds the capacity permitted by conventional flow sorters.
Moreover, microfabrication permits other technologies to be
integrated or combined on a single chip, such as PCR (21), moving
molecules or cells using optical tweezer/trapping (16-18),
transformation of cells by electroporation (19), .mu.TAS (22), and
DNA hybridization (6). Detectors and/or light filters that are used
to detect molecule or cell characteristics or their reporters can
also be fabricated directly on the chip.
[0121] A device of the invention can be microfabricated with a
sample solution reservoir or well at the inlet region, which is
typically in fluid communication with an inlet channel. A reservoir
may facilitate introduction of molecules or cells into the device
and into the sample inlet channel of each analysis unit. An inlet
region may have an opening, such as in the floor of the micro
fabricated chip, to permit entry of the sample into the device. The
inlet region may also contain a connector adapted to receive a
suitable piece of tubing, such as liquid chromatography or HPLC
tubing, through which a sample may be supplied. Such an arrangement
facilitates introducing the sample solution under positive pressure
in order to achieve a desired flow rate through the channels.
Outlet channels and wells can be similarly provided.
[0122] Substrate and Flow Channels. A typical analysis unit of the
invention comprises an inlet region that is part of and feeds or
communicates with a main channel, which in turn communicates with
an outlet (for analysis only) or with two (or more) branch channels
at a junction or branch point, forming for example a T-shape or a
Y-shape for sorting. Other shapes and channel geometries may be
used as desired. The region at or surrounding the junction can also
be referred to as a discrimination region, however, precise
boundaries for the discrimination region are not required. A
detection region is identified within or coincident with a portion
of the main channel downstream of the inlet region, and at or
upstream of the discrimination region or branch point. Precise
boundaries for the detection region are not required, but are
preferred. The discrimination region may be located immediately
downstream of the detection region, or it may be separated by a
suitable distance consistent with the size of the molecules, the
channel dimensions, and the detection system. It will be
appreciated that the channels can have any suitable shape or
cross-section, such as tubular or grooved, and can be arranged in
any suitable manner, so long as a flow can be directed from one
channel into at least one of two or more branch channels.
[0123] The channels of the invention are microfabricated, for
example by etching a silicon chip using conventional
photolithography techniques, or using a micromachining technology
called "soft lithography", developed in the late 1990's (11). These
and other microfabrication methods may be used to provide
inexpensive miniaturized devices, and in the case of soft
lithography, can provide robust devices having beneficial
properties such as improved flexibility, stability, and mechanical
strength. When optical detection is employed, the invention also
provides minimal light scatter from molecule or cell suspension and
chamber material. Devices according to the invention are relatively
inexpensive and easy to set up. They can also be disposable, which
greatly relieves many of the concerns of gel electrophoresis (for
molecules) and for sterilization and permanent adsorption of
particles unto the flow chambers and channels of conventional FACS
machines (for cells). Using these kinds of techniques,
microfabricated fluidic devices can replace the conventional gel
electrophoresis and fluidic flow chambers of the prior art.
[0124] A microfabricated device of the invention is preferably
fabricated from a silicon microchip or silicon elastomer. The
dimensions of the chip are those of typical microchips, ranging
between about 0.5 cm to about 5 cm per side and about 1 micron to
about 1 cm in thickness. The device contains at least one analysis
unit containing a main channel having detection and discrimination
regions. Preferably a device also contains at least one inlet
region (which may contain an inlet channel) and two or more outlet
regions (which have fluid communication with a branch channel in
each region). It shall be appreciated that the "regions" and
"channels" are in fluid communication with each other, and
therefore they may overlap, i.e., there may be no clear boundary
where a region or channel begins or ends. A microfabricated device
can be covered with a material having transparent properties, e.g.,
a glass coverslip to permit detection of a reporter for example by
an optical device, such as an optical microscope.
[0125] The dimensions of the channels and in particular of the
detection region are influenced by the size of the molecules or
cells under study. For polynucleotides, which are large by
molecular standards, a typical length or diameter is about 3.4
angstroms per base pair. Thus, a DNA 49 kpbs long, such as Lambda
phage DNA, is about 17 microns long when fully extended. A typical
range of sizes for polynucleotides of the invention is from about 1
to about 200 kpbs, or about 0.3 to about 70 microns. Detection
regions used for detecting molecules have a cross-sectional area
large enough to allow a desired molecule to pass through without
being substantially slowed down relative to the flow of the
solution carrying it. To avoid "bottlenecks" and/or turbulence, and
promote single-file flow, the channel dimensions, particularly in
the detection region, should generally be at least about twice,
preferably at least about five times as large per side or in
diameter as the diameter of the largest molecule that will be
passing through it.
[0126] For molecules such as DNA, the channels in a device are
typically between about 1 to about 100 microns (.mu.m) in diameter,
with preferred channel dimensions ranging from about 2 to about 5
microns in width and between about 2 and about 4 or 5 microns in
depth. Similarly, the volume of the detection region in a molecular
analysis or sorting device may be from about 1 femtoliter (fl) to
about 1 nanoliter (nl). Typically, the detection region will have a
volume between about 10 to about 5000 fl, preferably about 40 or 50
fl to about 1000 or 2000 fl, most preferably on the order of about
200 fl.
[0127] To prevent material from adhering to the sides of the
channels, the channels (and coverslip, if used) may have a coating
which minimizes adhesion. Such a coating may be intrinsic to the
material from which the device is manufactured, or it may be
applied after the structural aspects of the channels have been
microfabricated. "TEFLON" is an example of a coating that has
suitable surface properties.
[0128] A silicon substrate containing the microfabricated flow
channels and other components is preferably covered and sealed,
most preferably with a transparent cover, e.g., thin glass or
quartz, although other clear or opaque cover materials may be used.
When external radiation sources or detectors are employed, the
detection region is covered with a clear cover material to allow
optical access to the molecules or cells. For example, anodic
bonding to a "PYREX" cover slip can be accomplished by washing both
components in an aqueous H.sub.2SO.sub.4/H.sub.2O.sub.2 bath,
rinsing in water, and then, for example, heating to about 350
degrees C. while applying a voltage of 450V.
[0129] Switching and Flow Control. Electro-osmotic and
pressure-driven flow are examples of methods or systems for flow
control, that is, manipulating the flow of molecules cells,
particles or reagents in one or more directions and/or into one or
more channels of a microfluidic device of the invention (8, 12, 13,
23). Other methods may also be used, for example, electrophoresis
and dielectrophoresis. In certain embodiments of the invention, the
flow moves in one "forward" direction, e.g. from the inlet region
through the main and branch channels to an outlet region. In other
embodiments the direction of flow is reversible. Application of
these techniques according to the invention provides more rapid and
accurate devices and methods for sorting, for example, because the
sorting occurs at or in a discrimination region that can be placed
at or immediately after a detection region. This provides a shorter
distance for molecules or cells to travel, they can move more
rapidly and with less turbulence, and can more readily be moved,
examined, and sorted in single file, i.e., one at a time. In a
reversible embodiment, potential sorting errors can be avoided, for
example by reversing and slowing the flow to re-read or resort a
molecule or cell (or a plurality thereof) before irretrievably
committing the molecule or cell to the outlet or to a particular
branch channel.
[0130] Without being bound by any theory, electro-osmosis is
believed to produce motion in a stream containing ions, e.g. a
liquid such as a buffer, by application of a voltage differential
or charge gradient between two or more electrodes. Neutral
(uncharged) molecules or cells can be carried by the stream.
Electro-osmosis is particularly suitable for rapidly changing the
course, direction or speed of flow. Electrophoresis is believed to
produce movement of charged objects in a fluid toward one or more
electrodes of opposite charge, and away from one on or more
electrodes of like charge. Because of its charged nature (2 charges
for each base pair) DNA can be conveniently moved by
electrophoresis in a buffer of appropriate pH.
[0131] Dielectrophoresis is believed to produce movement of
dielectric objects, which have no net charge, but have regions that
are positively or negatively charged in relation to each other.
Alternating, non-homogeneous electric fields in the presence of
particles, such as molecules, cells or beads, cause them to become
electrically polarized and thus to experience dielectrophoretic
forces. Depending on the dielectric polarizability of the particles
and the suspending medium, dielectric particles will move either
toward the regions of high field strength or low field strength.
For example, the polarizability of living cells depends on their
composition morphology, and phenotype and is highly dependent on
the frequency of the applied electrical field. Thus, cells of
different types and in different physiological states generally
possess distinctly different dielectric properties, which may
provide a basis for cell separation, e.g., by differential
dielectrophoretic forces. According to formulas provided in Fiedler
et al. (13), individual manipulation of single particles requires
field differences (inhomogeneties) with dimensions close to the
particles.
[0132] Manipulation is also dependent on permittivity (a dielectric
property) of the particles with the suspending medium. Thus,
polymer particles and living cells show negative dielectrophoresis
at high-field frequencies in water. For example, dielectrophoretic
forces experienced by a latex sphere in a 0.5 MV/m field (10V for a
20 micron electrode gap) in water are predicted to be about 0.2
piconewtons (pN) for a 3.4 micron latex sphere to 15 pN for a 15
micron latex sphere (13). These values are mostly greater than the
hydrodynamic forces experienced by the sphere in a stream (about
0.3 pN for a 3.4 micron sphere and 1.5 pN for a 15 micron sphere).
Therefore, manipulation of individual cells or particles can be
accomplished in a streaming fluid, such as in a cell sorter device,
using dielectrophoresis. Using conventional semiconductor
technologies, electrodes can be microfabricated onto a substrate to
control the force fields in a microfabricated sorting device of the
invention. Dielectrophoresis is particularly suitable for moving
objects that are electrical conductors. The use of AC current is
preferred, to prevent permanent alignment of ions. Megahertz
frequencies are suitable to provide a net alignment, attractive
force, and motion over relatively long distances. E.g. Benecke
(49).
[0133] Optical tweezers can also be used in the invention to trap
and move objects, e.g. molecules or cells, with focused beams of
light such as lasers. Flow can also be obtained and controlled by
providing a pressure differential or gradient between one or more
channels of a device or in a method of the invention.
[0134] Molecules or cells can be moved by direct mechanical
switching, e.g. with on-off valves, or by squeezing the channels.
Pressure control may also be used, for example by raising or
lowering an output well to change the pressure inside the channels
on the chip. See e.g. the devices and methods described in pending.
U.S. application Ser. No. 08/932,774 filed Sep. 25, 1997; No.
60/108,894 filed Nov. 17, 1998; No. 60/086,394 filed May 22, 1998;
and No. 09/325,667 filed May 21, 1999 (molecular analysis systems).
These methods and devices can further be used in combination with
the methods and devices described in pending U.S. application Ser.
Nos. 60/141,503 (filed Jun. 28, 1999), 609/147,199 (filed Aug. 3,
1999) and 60/186,856 (filed Mar. 3, 2000). Each of these references
is hereby incorporated by reference in its entirety.
[0135] Detection and Discrimination for Sorting. The detector can
be any device or method for interrogating a molecule or cell as it
passes through the detection region. Typically, molecules or cells
are to be analyzed or sorted according to a predetermined
characteristic that is directly or indirectly detectable, and the
detector is selected or adapted to detect that characteristic. A
preferred detector is an optical detector, such as a microscope,
which may be coupled with a computer and/or other image processing
or enhancement devices to process images or information produced by
the microscope using known techniques. For example, molecules can
be sorted by size or molecular weight. Cells can be sorted for
whether they contain or produce a particular protein, by using an
optical detector to examine each cell for an optical indication of
the presence or amount of that protein. The protein may itself be
detectable, for example by a characteristic fluorescence, or it may
be labeled or associated with a reporter that produces a detectable
signal when the desired protein is present, or is present in at
least a threshold amount. There is no limit to the kind or number
of molecule or cell characteristics that can be identified or
measured using the techniques of the invention, which include
without limitation surface characteristics of the cell and
intracellular characteristics, provided only that the
characteristic or characteristics of interest for sorting can be
sufficiently identified and detected or measured to distinguish
cells having the desired characteristic(s) from those which do not.
For example, any label or reporter as described herein can be used
as the basis for sorting molecules or cells, i.e. detecting them to
be collected.
[0136] In preferred embodiments, the molecules or cells are
analyzed and/or separated based on the intensity of a signal from
an optically-detectable reporter bound to or associated with them
as they pass through a detection window or "detection region" in
the device. Molecules or cells having an amount or level of the
reporter at a selected threshold or within a selected range are
diverted into a predetermined outlet or branch channel of the
device. The reporter signal is collected by a microscope and
measured by a photomultiplier tube (PMT). A computer digitizes the
PMT signal and controls the flow via valve action or
electro-osmotic potentials. Alternatively, the signal can be
recorded or quantified, as a measure of the reporter and/or its
corresponding characteristic or marker, e.g. for purposes of
evaluation without necessarily proceeding to sort the molecules or
cells.
[0137] In one embodiment, the chip is mounted on an inverted
optical microscope. Fluorescence produced by a reporter is excited
using a laser beam focused on molecules (e.g. DNA) or cells passing
through a detection region. Fluorescent reporters include, e.g.,
rhodamine, fluorescein, Texas red, Cy 3, Cy 5, phycobiliprotein,
green fluorescent protein (GFP), YOY-1, and picogreen. In molecular
fingerprinting applications, the reporter labels are preferably a
fluorescently-labeled single nucleotides, such as fluorescein-dNTP,
rhodamine-dNTP, Cy3-dNTP, Cy5-dNTP, where dNTP represents dATP,
dTTP, dUTP or dCTP. The reporter can also be chemically-modified
single nucleotides, such as biotin-dNTP. Thus, in one aspect of the
invention, the device can determine the size or molecular weight of
molecules such as polynucleotide fragments passing through the
detection region, or the presence or degree of some other
characteristic indicated by a reporter. If desired, the molecules
can be sorted based on this analysis.
[0138] To detect a reporter or determine whether a molecule has a
desired characteristic, the detection region may include an
apparatus for stimulating a reporter for that characteristic to
emit measurable light energy, e.g., a light source such as a laser,
laser diode, high-intensity lamp, (e.g., mercury lamp), and the
like. In embodiments where a lamp is used, the channels are
preferably shielded from light in all regions except the detection
region. In embodiments where a laser is used, the laser can be set
to scan across a set of detection regions from different analysis
units. In addition, laser diodes may be microfabricated into the
same chip that contains the analysis units. Alternatively, laser
diodes may be incorporated into a second chip (i.e., a laser diode
chip) that is placed adjacent to the microfabricated sorter chip
such that the laser light from the diodes shines on the detection
region(s).
[0139] In preferred embodiments, an integrated semiconductor laser
and/or an integrated photodiode detector are included on the
silicon wafer in the vicinity of the detection region. This design
provides the advantages of compactness and a shorter optical path
for exciting and/or emitted radiation, thus minimizing
distortion.
[0140] Sorting Schemes. According to the invention, molecules or
cells are sorted dynamically in a flow stream of microscopic
dimensions, based on the detection or measurement of a
characteristic, marker or reporter that is associated with the
molecules or cells. The stream is typically but not necessarily
continuous, and may be stopped and started, reversed, or changed in
speed. Prior to sorting, a liquid that does not contain sample
molecules or cells can be introduced at an inlet region of the chip
(e.g., from an inlet well or channel) and is directed through the
device by capillary action, to hydrate and prepare the device for
sorting. If desired, the pressure can be adjusted or equalized for
example by adding buffer to an outlet region. The liquid typically
is an aqueous buffer solution, such as ultrapure water (e.g., 18
mega ohm resistivity, obtained for example by column
chromatography), ultrapure water, 10 mM Tris HCL and 1 mM EDTA
(TE), phosphate buffer saline (PBS), and acetate buffer. Any liquid
or buffer that is physiologically compatible with the population of
molecules or cells to be sorted can be used.
[0141] A sample solution containing a mixture or population of
molecules or cells in a suitable carrier fluid (such as a liquid or
buffer described above) is supplied to the inlet region. The
capillary force causes the sample to enter the device. The force
and direction of flow can be controlled by any desired method for
controlling flow, for example, by a pressure differential, by valve
action, or by electro-osmotic flow, e.g., produced by electrodes at
inlet and outlet channels. This permits the movement of the
molecules or cells into one or more desired branch channels or
outlet regions.
[0142] A "forward" sorting algorithm, according to the invention,
includes embodiments where molecules or cells from an inlet channel
flow through the device to a predetermined branch or outlet channel
(which can be called a "waste channel"), until the level of
measurable reporter is above a pre-set threshold. At that time, the
flow is diverted to deliver the molecule or cell to another
channel. For example, in an electro-osmotic embodiment, where
switching is virtually instantaneous and throughput is limited by
the highest voltage, the voltages are temporarily changed to divert
the chosen molecule or cell to another predetermined outlet channel
(which can be called a "collection channel"). Sorting, including
synchronizing detection of a reporter and diversion of the flow,
can be controlled by various methods including computer or
microprocessor control. Different algorithms for sorting in the
microfluidic device can be implemented by different computer
programs, such as programs used in conventional FACS devices for
sorting cells. For example, a programmable card can be used to
control switching, such as a Lab PC 1200 Card, available from
National Instruments, Austin, Tex. Algorithms as sorting procedures
can be programmed using C++, LABVIEW, or any suitable software. The
method is advantageous, for example, because conventional gel
electrophoresis methods are generally not automated or under
computer control.
[0143] A "reversible" sorting algorithm can be used in place of a
"forward" mode, for example in embodiments where switching speed
may be limited. For example, a pressure-switched scheme can be used
instead of electro-osmotic flow and does not require high voltages
and may be more robust for longer runs. However, mechanical
constraints may cause the fluid switching speed to become
rate-limiting. In a pressure-switched scheme the flow is stopped
when a molecule or cell of interest is detected. By the time the
flow stops; the molecule or cell may be past the branch point and
be part-way down the waste channel. In this situation, a reversible
embodiment can be used. The system can be run backwards at a slower
(switchable) speed (e.g., from waste to inlet), and the molecule or
cell is then switched to a different channel. At that point, a
potentially mis-sorted molecule or cell is "saved", and the device
can again be run at high speed in the forward direction. This
"reversible" sorting method is not possible with standard FACS
machines or in gel electrophoresis technologies. FACS machines
mostly sort aerosol droplets which cannot be reversed back to the
chamber, in order to be redirected. The aerosol droplet sorter are
virtually irreversible. In gel electrophoresis, molecules such as
polynucleotides are drawn through a gel by an electric current and
migrate at different rates proportional to their molecular weights.
Individual molecules can not be reversed through the gel, and
indeed, altering the rate or direction of migration would prevent
meaningful use of the technique. Reversible sorting is particularly
useful for identifying rare molecules or cells (e.g., in molecular
evolution and cancer cytological identification), or molecules or
cells that are few in number, which may be misdirected due to a
margin of error inherent to any fluidic device. The reversible
nature of the device of the invention permits a reduction in this
possible error.
[0144] A "reversible" sorting method permits multiple time course
measurements of a single molecule or cell. This allows for
observations or measurements of the same molecule or cell at
different times, because the flow reverses the molecule or cell
back into the detection window before directing it to a downstream
channel. Measurements can be compared or confirmed, and changes in
molecule or cell properties over time can be examined, for example
in kinetic studies.
[0145] When trying to separate molecules or cells in a sample at a
very low ratio to the total number of molecules or cells, a sorting
algorithm can be implemented that is not limited by the intrinsic
switching speed of the device. Consequently, the molecules or cells
flow at the highest possible static (non-switching) speed from the
inlet channel to the waste channel. Unwanted molecules or cells can
be directed into the waste channel at the highest speed possible,
and when a desired molecule or cell is detected, the flow can be
slowed down and then reversed, to direct it back into the detection
region, from where it can be redirected (i.e. to accomplish
efficient switching). Hence the molecules or cells can flow at the
highest possible static speed.
[0146] Preferably, the fluid carrying the molecules or cells has a
relatively low Reynolds Number, for example 10.sup.-2. The Reynolds
Number represents an inverse relationship between the density and
velocity of a fluid and its viscosity in a channel of given length.
More viscous, less-dense, slower moving fluids over a shorter
distance will have a lower Reynolds Number, and are easier to
divert, stop, start, or reverse without turbulence. Because of the
small sizes and slow velocities, microfabricated fluid systems are
often in a low Reynolds number regime (<<1). In this regime,
inertial effects, which cause turbulence and secondary flows, are
negligible; viscous effects dominate the dynamics. These conditions
are advantageous for sorting, and are provided by microfabricated
devices of the invention. Accordingly the microfabricated devices
of the invention are preferably if not exclusively operated at a
low or very low Reynold's number. Exemplary sorting schemes are
shown diagrammatically in FIGS. 11A and B and FIGS. 12A and B.
6. EXAMPLES
[0147] The present invention is also described by means of
particular examples. However, the use of such examples anywhere in
the specification is illustrative only and in no way limits the
scope and meaning of the invention or of any exemplified term.
Likewise, the invention is not limited to any particular preferred
embodiments described herein. Indeed, many modifications and
variations of the invention will be apparent to those skilled in
the art upon reading this specification and can be made without
departing from its spirit and scope. The invention is therefore to
be limited only by the terms of the appended claims along with the
full scope of equivalents to which the claims are entitled.
6.1. A Microfabricated Polynucleotide Sorting Device
[0148] FIG. 1 shows an embodiment of a microfabricated
polynucleotide sorting device 20 in accordance with the invention.
The device is preferably fabricated from a silicon microchip 22.
The dimensions of the chip are those of typical microchips, ranging
between about 0.5 cm to about 5 cm per side and about 0.1 mm to
about 1 cm in thickness. The device contains a solution inlet 24,
two or more solution outlets, such as outlets 26 and 28, and at
least one analysis unit, such as the unit at 30.
[0149] Each analysis unit includes a main channel 32 having at one
end a sample inlet 34, and downstream of the sample inlet, a
detection region 36, and downstream of the detection region 36 a
discrimination region 38. A plurality of branch channels, such as
channels 40 and 42, are in fluid communication with and branch out
from the discrimination region. The dimensions of the main and
branch channels are typically between about 1 .mu.m and 10 .mu.m
per side, but may vary at various points to facilitate analysis,
sorting and/or collection of molecules.
[0150] In embodiments such as shown in FIG. 1, where the device
contains a plurality of analysis units, the device may further
contain collection manifolds, such as manifolds 44 and 46, to
facilitate collection of sample from corresponding branch channels
of different analysis units for routing to the appropriate solution
outlet. The manifolds are preferably microfabricated into different
levels of the device, as indicated by the dotted line representing
manifold 46. Similarly, such embodiments may include a sample
solution reservoir, such as reservoir 48, to facilitate
introduction of sample into the sample inlet of each analysis
unit.
[0151] Also included with the device is a processor, such as
processor 50. The processor can be integrated into the same chip as
contains the analysis unit(s), or it can be separate, e.g., an
independent microchip connected to the analysis unit-containing
chip via electronic leads, such as leads 52 (connected to the
detection region(s) and 54 (connected to the discrimination
region(s)).
[0152] As mentioned above, the device may be microfabricated with a
sample solution reservoir to facilitate introduction of a
polynucleotide solution into the device and into the sample inlet
of each analysis unit. With reference to FIG. 2, the reservoir is
microfabricated into the silicon substrate of the chip 62, and is
covered, along with the channels (such as main channel 64) of the
analysis units, with a glass coverslip 66. The device solution
inlet comprises an opening 68 in the floor of the microchip. The
inlet may further contain a connector 70 adapted to receive a
suitable piece of tubing, such as liquid chromatography or HPLC
tubing, through which the sample may be supplied. Such an
arrangement facilitates introducing the sample solution under
positive pressure, to achieve a desired flow rate through the
channels as described below.
[0153] Downstream of the sample inlet of the main channel of each
analysis unit is the detection region, designed to detect the level
of an optically-detectable reporter associated with polynucleotides
present in the region. Exemplary embodiments of detection regions
in devices of the invention are shown in FIGS. 3A and 3B.
6.2. Photodiode Detectors
[0154] With reference to FIG. 3A, each detection region is formed
of a portion of the main channel of an analysis unit and a
photodiode, such as photodiode 72, located in the floor of the main
channel. In this embodiment, the area detectable by the detection
region is the circular portion each channel defined by the
receptive field of the photodiode in that channel. The volume of
the detection region is the volume of a cylinder with a diameter
equal to the receptive field of the photodiode and a height equal
to the depth of the channel above the photodiode.
[0155] The signals from the photodiodes are carried via output
lines 76 to the processor (not shown), which processes the signals
into values corresponding to the length of the polynucleotide
giving rise to the signal. The processor then uses this
information, for example, to control active elements in the
discrimination region. The processor may process the signals into
values for comparison with a predetermined or reference set of
values for analysis or sorting.
[0156] When more than one detection region is used, the photodiodes
in the laser diode chip are preferably spaced apart relative to the
spacing of the detection regions in the analysis unit. That is, for
more accurate detection, the photodiodes are placed apart at the
same spacing as the spacing of the detection region.
[0157] The processor can be integrated into the same chip that
contains the analysis unit(s), or it can be separate, e.g., an
independent microchip connected to the analysis unit-containing
chip via electronic leads that connect to the detection region(s)
and/or to the discrimination region(s), such as by a photodiode.
The processor can be a computer or microprocessor, and is typically
connected to a data storage unit, such as computer memory, hard
disk, or the like, and/or a data output unit, such as a display
monitor, printer and/or plotter.
[0158] The types and numbers of molecules or cells, based on
detection of a reporter associated with or bound to the molecules
or cells passing through the detection region, can be calculated or
determined, and the data obtained can be stored in the data storage
unit. This information can then be further processed or routed to
the data outlet unit for presentation, e.g. histograms, of the
types of molecules or cells (or levels of a cell protein,
saccharide), or some other characteristic. The data can also be
presented in real time as the sample is flowing through the
device.
[0159] With reference to FIG. 3B, the photodiode 78 can be larger
in diameter than the width of the main channel, forming a detection
region 80 that is longer (along the length of the main channel 82)
than it is wide. The volume of such a detection region is
approximately equal to the cross-sectional area of the channel
above the diode multiplied by the diameter of the diode.
[0160] In a preferred sorting embodiment the detection region is
connected by the main channel to the discrimination region. The
discrimination region may be located immediately downstream of the
detection region, or may be separated by a suitable length of
channel. Constraints on the length of channel between the detection
and discrimination regions are discussed below, with respect to the
operation of the device. This length is typically between about 1
.mu.m and about 2 cm. The discrimination region is at the junction
of the main channel and the branch channels. It comprises the
physical location where molecules are directed into a selected
branch channel. The means by which the molecules or cells are
directed into a selected branch channel may (i) be present in the
discrimination region, as in, e.g., electrophoretic or
microvalve-based discrimination, or (ii) be present at a distant
location, as in, e.g., electroosmotic or flow stoppage-based
discrimination.
[0161] If desired, the device may contain a plurality of analysis
units, i.e., more than one detection and discrimination region, and
a plurality of branch channels which are in fluid communication
with and branch out from the discrimination regions. It will be
appreciated that the position and fate of molecules or cells in the
discrimination region can be monitored by additional detection
regions installed, for example, immediately upstream of the
discrimination region and/or within the branch channels immediately
downstream of the branch point. The information obtained by the
additional detection regions can be used by a processor to
continuously revise estimates of the velocity of the molecules or
cells in the channels and to confirm that molecules or cells having
a selected characteristic enter the desired branch channel.
[0162] A group of manifolds (a region consisting of several
channels which lead to or from a common channel) can be included to
facilitate movement of sample from the different analysis units,
through the plurality of branch channels and to the appropriate
solution outlet. Manifolds are preferably microfabricated into the
chip at different levels of depth. Thus, devices of the invention
having a plurality of analysis units can collect the solution from
associated branch channels of each unit into a manifold, which
routes the flow of solution to an outlet. The outlet can be adapted
for receiving, for example, a segment of tubing or a sample tube,
such as a standard 1.5 ml centrifuge tube. Collection can also be
done using micropipettes.
6.3. Valve Structures
[0163] In an embodiment where pressurized flow is used, valves can
be used to block or unblock the pressurized flow of molecules or
cells through selected channels. A thin cantilever, for example,
may be included within a branch point, as shown in FIGS. 4A and 4B,
such that it may be displaced towards one or the other wall of the
main channel, typically by electrostatic attraction, thus closing
off a selected branch channel. Electrodes are on the walls of the
channel adjacent to the end of the cantilever. Suitable electrical
contacts for applying a potential to the cantilever are also
provided in a similar manner as the electrodes. Because the
cantilever in FIG. 4B is parallel to the direction of etching, it
may be formed of a thin layer of silicon by incorporating the
element into the original photoresist pattern. The cantilever is
preferably coated with a dielectric material such as silicon
nitride, as described in Wise, et al., 1995 (35), for example, to
prevent short circuiting between the conductive surfaces.
[0164] Alternatively, a valve may be situated within each branch
channel, rather than at the branch point, to close off and
terminate pressurized flow through selected channels. Because the
valves are located downstream of the discrimination region, the
channels in this region may be formed having a greater width than
in the discrimination region, which simplifies the formation of
valves.
[0165] A valve within a channel may be microfabricated, if desired,
in the form of an electrostatically operated cantilever or
diaphragm. Techniques for forming such elements are well known in
the art (e.g., 24, 29, 35, 36, 37). Typical processes include the
use of selectively etched sacrificial layers in a multilayer
structure or, for example, the undercutting of a layer of silicon
dioxide via anisotropic etching. For example, to form a cantilever
within a channel, as illustrated in FIGS. 4A and 4B, a sacrificial
layer 168 may be formed adjacent to a small section of a
non-etchable material 170, using known photolithography methods, on
the floor of a channel, as shown in FIG. 4A. Both layers can then
be coated with, for example, silicon dioxide or another
non-etchable layer, as shown at 172. Etching of the sacrificial
layer deposits the cantilever member 174 within the channel, as
shown in FIG. 4B. Suitable materials for the sacrificial layer,
non-etchable layers and etchant include undoped silicon, p-doped
silicon and silicon dioxide, and the etchant EDP (ethylene
diamine/pyrocatechol), respectively. Because the cantilever in FIG.
4B is parallel to the direction of etching, it may be formed of a
thin layer of silicon by incorporating the element into the
original photoresist pattern. The cantilever is preferably coated
with a dielectric material such as silicon nitride, as described in
(35) for example, to prevent short circuiting between the
conductive surfaces.
[0166] The width of the cantilever or diaphragm should
approximately equal that of the channel, allowing for movement
within the channel. If desired, the element may be coated with a
more malleable material, such as a metal, to allow for a better
seal. Such coating may also be employed to render a non-conductive
material, such as silicon dioxide, conductive.
[0167] As above, suitable electrical contacts are provided for
displacing the cantilever or diaphragm towards the opposing surface
of the channel. When the upper surface is a glass cover plate, as
described below, electrodes and contacts may be deposited onto the
glass.
[0168] It will be apparent to one of skill in the field that other
types of valves or switches can be designed and fabricated, using
well known photolithographic or other microfabrication techniques,
for controlling flow within the channels of the device. Multiple
layers of channels can also be prepared.
[0169] Operation of the valves or charging of the electrodes, in
response to the level of fluorescence measured from an analyte
molecule, is controlled by the processor, which receives this
information from the detector. All of these components are operably
connected in the apparatus, and electrical contacts are included as
necessary, using standard microchip circuitry.
[0170] In preferred embodiments, an integrated semiconductor laser
and/or an integrated photodiode detector are included on the
silicon wafer in the vicinity of the detection region. This design
provides the advantages of compactness and a shorter optical path
for exciting and/or emitted radiation, thus minimizing
distortion.
[0171] The silicon substrate containing the microfabricated flow
channels and other components is covered and sealed, preferably
with a thin glass or quartz cover, although other clear or opaque
cover materials may be used. when external radiation sources or
detectors are employed, the interrogation region is covered with a
clear cover material to allow optical access to the analyte
molecules. Anodic bonding to a "PYREX" cover slip may be
accomplished by washing both components in an aqueous
H.sub.2SO.sub.4/H.sub.2O.sub.2 bath, rinsing in water, and then
heating to about 350.degree. C. while applying a voltage of, e.g.,
450V.
6.4. Exemplary Microchip Architectures for Sorting
[0172] As illustrated with respect to FIGS. 5A-5D, there are a
number of ways in which cells can be routed or sorted into a
selected branch channel.
[0173] FIG. 5A shows a discrimination region 102, which is suitable
for electrophoretic discrimination as the sorting technique. The
discrimination region is preceded by a main channel 104. A junction
divides the main channel into two branch channels 106 and 108. The
discrimination region 102 includes electrodes 110 and 112,
positioned on outer side walls of the branch channels 106 and 108,
and which connect to leads 114 and 116. The leads are connected to
a voltage source (not shown) incorporated into or controlled by a
processor (not shown), as described, infra. The distance (D)
between the electrodes is preferably less than the average distance
separating the cells during flow through the main channel. The
dimensions of the electrodes are typically the same as the
dimensions of the channels in which they are positioned, e.e such
that the electrodes are as high and wide as the channel.
[0174] The discrimination region shown in FIG. 5B is suitable for
use in a device that employs electro-osmotic flow, to move the
molecules or cells and bulk solution through the device. FIG. 4B
shows a discrimination region 122 which is preceded by a main
channel 124. The main channel contains a junction that divides the
main channel into two branch channels 126 and 128. An electrode 130
is placed downstream of the junction of the main channel, for
example near the sample inlet of main channel. Electrodes are also
placed in each branch channel (electrodes 132 and 134). The
electrode 130 can be negative and electrodes 132 and 134 can be
positive (or vice versa) to establish bulk solution flow according
to well-established principles of electro-osmotic flow (25). See,
also, U.S. patent application Ser. No. 09/325,667 filed May 21,
1999.
[0175] After a molecule or cell passes the detection region (not
shown) and enters the discrimination region 122 (e.g. between the
main channel and the two branch channels) the voltage to one of the
electrodes 132 or 134 can be shut off, leaving a single attractive
force that acts on the solution and the molecule or cell to
influence it into the selected branch channel. As above, the
appropriate electrodes are activated after the molecule or cell has
committed to the selected branch channel in order to continue bulk
flow through both channels. In one embodiment, the electrodes are
charged to divert the flow into one branch channel, for example
channel 126, which can be called a waste channel. In response to a
signal indicating that a molecule or cell has been identified or
selected for collection, the charge on the electrodes can be
changed to divert the selected molecule or cell into the other
channel (channel 128), which can be called a collection
channel.
[0176] In another embodiment of the invention, shown in FIG. 5C,
the molecules or cells are directed into a predetermined branch
channel via a valve 140 in the discrimination region. The valve 140
comprises a thin extension of material to which a charge can be
applied via an electrode lead 142. The valve 140 is shown with both
channels open, and can be deflected to close either branch channel
by application of a voltage across electrodes 144 and 146. A
molecule or cell is detected and chosen for sorting in the
detection region (not shown), and can be directed to the
appropriate channel by closing off the other channel, e.g. by
applying, removing or changing a voltage applied to the electrodes.
The valve can also be configured to close one channel in the
presence of a voltage, and to close the other channel in the
absence of a voltage.
[0177] FIG. 5D shows another embodiment of a discrimination region
of the invention, which uses flow stoppage in one or more branch
channels as the discrimination means. The sample solution moves
through the device by application of positive pressure at an end
where the solution inlet is located. Discrimination or routing of
the molecules or cells is affected by simply blocking a branch
channel (145 or 148) or a branch channel sample outlet using valves
in a pressure-driven flow (147 or 149). Due to the small size scale
of the channels and the incompressibility of liquids, blocking the
solution flow creates an effective "plug" in the non-selected
branch channel, thereby temporarily routing the molecule or cell
together with the bulk solution flow into the selected channel.
Valve structures can be incorporated downstream from the
discrimination region, which are controlled by the detection
region, as described herein.
[0178] Alternatively, the discrimination function represented in
FIG. 50 may be controlled by changing the hydrostatic pressure at
the sample outlets of one or both branch channels 145 or 148. If
the branch channels in a particular analysis unit have the same
resistance to fluid flow, and the pressure at the sample inlet of
the main channel of an analysis unit is P, then the fluid flow out
of any selected branch channel can be stopped by applying a
pressure P/n at the sample outlet of the desired branch channel,
where n is the number of branch channels in the analysis unit.
Accordingly, in an analysis unit having two branch channels, the
pressure-applied at the outlet of the branch to be blocked is
P/2.
[0179] As shown in FIG. 5D, a valve is situated within each branch
channel, rather than at the branch point, to close off and
terminate pressurized flow through selected channels. Because the
valves are located at a point downstream from the discrimination
region, the channels in this region may be formed having a greater
width than in the discrimination region in order to simplify the
formation of valves. The width of the cantilever or diaphragm
should approximately equal the width of the channel, allowing for
movement within the channel. If desired, the element may be coated
with a more malleable material, such as a metal, to allow for a
better seal. Such coating may also be employed to render a
non-conductive material, such as silicon dioxide, conductive. As
above, suitable electrical contacts are provided for displacing the
cantilever or diaphragm towards the opposing surface of the
channel. When the upper surface is a glass cover plate, electrodes
and contacts may be deposited onto the glass.
6.5. A Cascade Device
[0180] FIG. 6 shows a device with analysis units containing a
cascade of detection and discrimination regions suitable for
successive rounds of polynucleotide or cell sorting. Such a
configuration may be used, for example, with a polynucleotide or
cellsorting device to generate a series of samples containing
"fractions" of polynucleotides, where each fraction contains a
specific size range of polynucleotide fragments (e.g., the first
fraction contains 100-500 by fragments, the next 500-1000 by
fragments, and so on). In a cell sorting device, such a cascade
configuration may be used to sequentially assay the cell for, e.g.,
three different fluorescent dyes corresponding to expression of
three different molecular markers. Samples collected at the outlets
of the different branch channels contain pools of cells expressing
defined levels of each of the three markers. The number of
reporters employed, and therefore the number of markers of
interest, can be varied as desired, e.g. to meet the needs of a
particular experiment or application.
6.6. Microfabricated Polynucleotide Analysis Device
[0181] Also included in the present invention is a microfabricated
polynucleotide analysis device suitable for quantitation and
analysis of the size distribution of polynucleotide fragments in
solution. Such a device is a simplified version of the sorting
device described above, in that analysis units in the device need
not contain a discrimination region or branch channels, and the
device need not contain a means for directing molecules to selected
branch channels. Each analysis unit comprises a single main channel
containing a detection region as described above. Since the optics
which collect the optical signal (e.g., fluorescence) can be
situated immediately adjacent the flow stream (e.g., diode embedded
in the channel of a microscope objective adjacent a glass coverslip
covering the channel), the signal-to-noise ratio of the signal
collected using a microfabricated polynucleotide analysis device of
the invention is high relative to other types of devices.
Specifically, the invention allows, e.g., the use of oil-immersion
high numerical apperature (N.A.) microscope objectives to collect
the light (e.g., 1.4 N.A.). Since the collection of light is
proportional to the square of the N.A., a 1.4 N.A. objective
provides about a four-fold better signal than an 0.8 N.A.
objective.
6.7. Microfabricated Cell Sorting Device
[0182] The invention also includes a microfabricated device for
sorting reporter-labeled cells by the level of reporter they
contain. The device is similar to polynucleotide-sorting devices
described above, but is adapted for handling particles on the size
scale of cells rather than molecules. This difference is manifested
mainly in the dimensions of the microfabricated channels, detection
and discrimination regions. Specifically, the channels in the
device are typically between about 20 .mu.m and about 500 .mu.m in
width and between about 20 .mu.m and about 500 .mu.m in depth, to
allow for an orderly flow of cells in the channels. Similarly, the
volume of the detection region in a cell sorting device is larger
than that of the polynucleotide sorting device, typically being in
the range of between about 10 pl and 100 nl. To prevent the cells
from adhering to the sides of the channels, the channels (and
coverslip) preferably contain a coating which minimizes cell
adhesion. Such a coating may be intrinsic to the material from
which the device is manufactured, or it may be applied after the
structural aspects of the channels have been microfabricated. An
exemplary coating has the surface properties of a material, such as
"TEFLON".
[0183] The device may be used to sort any procaryotic (e.g.,
bacterial) or eukaryotic (e.g., mammalian) cells which can be
labeled (e.g., via antibodies) with optically-detectable reporter
molecules (e.g., fluorescent dyes). Exemplary mammalian cells
include human blood cells, such as human peripheral blood
mononuclear cells (PBMCs). The cells can be labeled with antibodies
directed against any of a variety of cell marker antigens (e.g.,
HLA DR, CD3, CD4, CD8, CD11a, CD11c, CD14, CD16, CD20, CD45,
CD45RA, CD62L, etc.), and the antibodies can in turn be detected
using an optically-detectable reporter (either via directly
conjugated reporters or via labeled secondary antibodies) according
to methods known in the art.
[0184] It will be appreciated that the cell sorting device and
method described above can be used simultaneously with multiple
optically-detectable reporters having distinct optical properties.
For example, the fluorescent dyes fluorescein (FITC), phycoerythrin
(PE), and "CYCHROME" (Cy5-PE) can be used simultaneously due to
their different excitation and emission spectra. The different dyes
may be assayed, for example, at successive detection and
discrimination regions. Such regions may be cascaded as shown in
FIG. 6 to provide samples of cells having a selected amount of
signal from each dye.
6.8. Microfabrication of a Silicon Device
[0185] Analytical devices having microscale flow channels, valves
and other elements can be designed and fabricated from a solid
substrate material. Silicon is a preferred substrate material
because of the well developed technology permitting its precise and
efficient fabrication, but other materials may be used, including
polymers such as polytetrafluoroethylenes. Micromachining methods
well known in the art include film deposition processes, such as
spin coating and chemical vapor deposition, laser fabrication or
photolithographic techniques, or etching methods, which may be
performed by either wet chemical or plasma processes. (See, for
example, Angell et al. (37) and Manz et al. (38).
[0186] FIGS. 7A-7D illustrate the initial steps in microfabricating
the discrimination region portion of a nucleic acid sorting device
(e.g. Device 20 in FIG. 1) by photolithographic techniques. As
shown, the structure includes a silicon substrate 160. The silicon
wafer which forms the substrate is typically washed in a 4:1
H.sub.2SO.sub.4/H.sub.2O bath, rinsed in water and spun dry. A
layer 162 of silicon dioxide, preferably about 0.5 .mu.m in
thickness, is formed on the silicon, typically by heating the
silicon wafer to 800-1200.degree. C. in an atmosphere of steam. The
oxide layer is then coated with a photoresist layer 164, preferably
about 1 .mu.m inch-thickness. Suitable negative or positive resist
materials are well known. Common negative resist materials include
two-component bisarylazide/rubber resists. Positive resist
materials include polymethyl-methacrylate (PMMA) and two component
diazoquinone/phenol is resin materials. See, e.g., "Introduction to
microlithography", Thompson (36).
[0187] The coated laminate is irradiated through a photomask 166
imprinted with a pattern corresponding in size and layout to the
desired pattern of the microchannels. Methods for forming
photomasks having desired photomask patterns are well known. For
example, the mask can be prepared by printing the desired layout on
an overhead transparency using a high resolution (3000 dpi)
printer. Exposure is carried out on standard equipment such as a
Karl Suss contact lithography machine.
[0188] In the method illustrated in FIGS. 7A-5D, the photoresist is
a negative resist, meaning that exposure of the resist to a
selected wavelength, e.g., UV, light produces a chemical change
that renders the exposed resist material resistant to the
subsequent etching step. Treatment with a suitable etchant removes
the unexposed areas of the resist, leaving a pattern of bare and
resist-coated silicon oxide on the wafer surface, corresponding to
the layout and dimensions of the desired microstructures. In this
example, because a negative resist was used, the bare areas
correspond to the printed layout on the photomask. The wafer is now
treated with a second etchant material, such as a reactive ion etch
(RIE), effective to dissolve the exposed areas of silicon dioxide.
The remaining resist is removed, typically with hot aqueous
H.sub.2SO.sub.4. The remaining pattern of silicon dioxide (162) now
serves as a mask for the silicon (160). The channels are etched in
the unmasked areas of the silicon substrate by treating with a KOH
etching solution. Depth of etching is controlled by time of
treatment. Additional microcomponents may also be formed within the
channels by further photolithography and etching steps, as
discussed below.
[0189] Depending on the method to be used for directing the flow of
molecules through the device, electrodes and/or valves are
fabricated into the flow channels. A number of different techniques
are available for applying thin metal coatings to a substrate in a
desired pattern. These are reviewed in, for example, Krutenat,
Kirk-Othmer 3rd ed., Vol. 15, pp. 241-274 (32), incorporated herein
by reference. A convenient and common technique used in fabrication
of microelectronic circuitry is vacuum deposition. For example,
metal electrodes or contacts may be evaporated onto a substrate
using vacuum deposition and a contact mask made from, e.g., a
"MYLAR" sheet. Various metals such as platinum, gold, silver or
indium/tin oxide (ITO) may be used for the electrodes.
[0190] Deposition techniques allowing precise control of the area
of deposition are preferred for application of electrodes to the
side walls of the channels in the device. Such techniques are
described, for example, in Krutenat (32), above, and references
cited therein. They include plasma spraying, where a plasma gun
accelerates molten metal particles in a carrier gas towards the
substrate, and physical vapor deposition using an electron beam,
where atoms are delivered on line-of-sight to the substrate from a
virtual point source. In laser coating, a laser is focused onto the
target point on the substrate, and a carrier gas projects powdered
coating material into the beam, so that the molten particles are
accelerated toward the substrate.
[0191] Another technique allowing precise targeting uses an
electron beam to induce selective decomposition of a previously
deposited substance, such as a metal salt, to a metal. This
technique has been used to produce sub-micron circuit paths (e.g.,
26).
6.9. Elastomeric Microfabricated Device
[0192] This Example demonstrates the manufacture of a disposable
microfabricated device, which can function as a stand-alone device
or as a component of an integrated microanalytical chip, in sorting
molecules or cells.
[0193] Preparation of the Microfabricated Device. A Silicon Wafer
was Etched and fabricated as described above and in (15). After
standard contact photolithography techniques to pattern the oxide
surface of the silicon wafer, a C.sub.2F.sub.2/CHF.sub.3 gas
mixture was used to etch the wafer by RIE. The silicon wafer was
then subjected to further etch with KOH to expose the silicon
underneath the oxide layer, thereby forming a mold for the silicone
elastomer. The silicon mold forms a "T" arrangement of channels.
The dimensions of the channels may range broadly, having
approximately 5.times.4 .mu.m dimension.
[0194] The etching process is shown schematically in FIG. 8.
Standard micromachining techniques were used to create a negative
master mold out of a silicon wafer. The disposable silicone
elastomer chip was made by mixing General Electric RTV 615
components (20) together and pouring onto the etched silicon wafer.
After curing in an oven for two hours at 80.degree. C., the
elastomer was peeled from the wafer and bonded hermetically to a
glass cover slip for sorting. To make the elastomer hydrophilic the
elastomer chip was immersed in HCl (pH=2.7) at 60 degrees C. for 40
to 60 min. Alternatively, the surface could have been coated with
polyurethane (3% w/v in 95% ethanol and diluted 10.times. in
ethanol). It is noted that the master wafer can be reused
indefinitely. The device shown has channels that are 100 .mu.m wide
at the wells, narrowing to 3 .mu.m at the sorting junction
(discrimination region). The channel depth is 4 .mu.m, and the
wells are 2 mm in diameter. These dimensions can be modified
according to the size range of the molecules or cells to be
analyzed or sorted.
[0195] Detection Apparatus. In this embodiment the device was
mounted on an inverted optical microscope (Zeiss Axiovert 35) as
shown in FIG. 9. In this system, the flow control can be provided
by voltage electrodes for electro-osmotic control or by capillaries
for pressure-driven control. The detection system can be
photomultiplier tubes or photodiodes, depending upon the
application. The inlet well and two collection wells were
incorporated into the elastomer chip on three sides of the "T"
forming three channels (FIG. 7). The chip was adhered to a glass
coverslip and mounted onto the microscope.
6.10. Operation of a Polynucleotide Analysis Device
[0196] The operation of a polynucleotide analysis chip is
described. This example refers to polynucleotides, but it will be
appreciated that other molecules may be analyzed or sorted using
similar methods and devices. Likewise, cells can be processed using
similar methods and devices, adapted to the appropriate size.
[0197] A solution of reporter-labeled polynucleotides is prepared
as described below and introduced into the sample inlet end(s) of
the analysis unit(s). The solution may be conveniently introduced
into a reservoir, such as reservoir 48 of FIG. 1, via a port or
connector, such as connector 70 in FIG. 2, adapted for attachment
to a segment of tubing, such as liquid chromatography or HPLC
tubing.
[0198] It is typically advantageous to "hydrate" the device (i.e.,
fill the channels of the device with the solvent, e.g., water or a
buffer solution, in which the polynucleotides will be suspended)
prior to introducing the polynucleotide-containing solution. Such
hydrating can be achieved by supplying water or the buffer solution
to the device reservoir and applying hydrostatic pressure to force
the fluid through the analysis unit(s).
[0199] Following such hydration, the polynucleotide-containing
solution is introduced into the sample inlets of the analysis
unit(s) of the device. As the stream of labeled polynucleotides
(e.g., tagged with a reporter such as a fluorescent dye) is passed
in a single file manner through the detection region, the optical
signal (e.g., fluorescence) from the optically-detectable reporter
moieties on each molecule are quantitated by an optical detector
and converted into a number used in calculating the approximate
length of polynucleotide in the detection region.
[0200] Exemplary reporter moieties, described below in reference to
sample preparation, include fluorescent moieties which can be
excited to emit light of characteristic wavelengths by an
excitation light source. Fluorescent moieties have an advantage in
that each molecule can emit a large number of photons (e.g., upward
of 106) in response to exciting radiation. Suitable light sources
include lasers, laser diodes, high-intensity lamps, e.g., mercury
lamps, and the like. In embodiments where a lamp is used, the
channels are preferably shielded from the light in all regions
except the detection region, to avoid bleaching of the label. In
embodiments where a laser is used, the laser can be set to scan
across a set of detection regions from different analysis units.
Other optically-detectable reporter moieties include
chemiluminescent moieties, which can be used without an excitation
light source.
[0201] Where laser diodes are used as a light source, the diodes
may be microfabricated into the same chip that contains the
analysis units. Alternatively, the laser diodes may be incorporated
into a second chip (laser diode chip; LDC) that is placed adjacent
to the chip such that the laser light from the diodes shines on the
detection regions. The photodiodes in the LDC are preferably placed
at a spacing that corresponds to the spacing of the detection
regions in the chip.
[0202] The level of reporter signal is measured using an optical
detector, such as a photodiode (e.g., an avalanche photodiode), a
fiber-optic light guide leading, e.g., to a photomultiplier tube, a
microscope with a high numerical apperature (N.A.) objective and an
intensified video camera, such as a SIT camera, or the like. The
detector may be microfabricated or placed into the PAC itself
(e.g., a photodiode as illustrated in FIGS. 3A and 3B), or it may
be a separate element, such as a microscope objective.
[0203] In cases where the optical detector is a separate element,
it is generally necessary to restrict the collection of signal from
the detection region of a single analysis unit. It may also be
advantageous to scan or move the detector relative to the
polynucleotide analysis unit ("PAC"), preferably by automation. For
example, the PAC can be secured in a movable mount (e.g., a
motorized/computer-controlled micromanipulator) and scanned under
the objective. A fluorescence microscope, which has the advantage
of a built-in excitation light source (epifluorescence), is
preferably employed for detection of a fluorescent reporter.
[0204] Since current microfabrication technology enables the
creation of sub-micron structures employing the elements described
herein, the dimensions of the detection region are influenced
primarily by the size of the molecules under study. These molecules
can be rather large by molecular standards. For example, lambda DNA
(.about.50 kb) in solution has a diameter of approximately 0.5
.mu.m. Accordingly, detection regions used for detecting
polynucleotides in this size range have a cross-sectional area
large enough to allow such a molecule to pass through without being
substantially slowed down relative to the flow of the solution
carrying it and causing a "bottle neck". The dimensions of a
channel should therefore be at least about twice, preferably at
least about five times as large per side or in diameter as the
diameter of the largest molecule that will be passing through
it.
[0205] Another factor important to consider in the practice of the
present invention is the optimal concentration of polynucleotides
in the sample solution. The concentration should be dilute enough
so that a large majority of the polynucleotide molecules pass
through the detection region one by one, with only a small
statistical chance that two or more molecules pass through the
region simultaneously. This is to insure that for the large
majority of measurements, the level of reporter measured in the
detection region corresponds to a single molecule, rather than two
individual molecules.
[0206] The parameters which govern this relationship are the volume
of the detection region and the concentration of molecules in the
sample solution. The probability that the detection region will
contain two or more molecules (P.sub..gtoreq.2) can be expressed
as
P.sub..gtoreq.2=1-{1+[DNA]*V}*e.sup.-[DNA]*.sup.V
where [DNA] is the concentration of polynucleotides in units of
molecules per .mu.m.sup.3 and V is the volume of the detection
region in units of .mu.m.sup.3.
[0207] It will be appreciated that P.sub..gtoreq.2 can be minimized
by decreasing the concentration of polynucleotides in the sample
solution. However, decreasing the concentration of polynucleotides
in the sample solution also results in increased volume of solution
processed through the device and can result in longer run times.
Accordingly, the objectives of minimizing the simultaneous presence
of multiple molecules in the detection chamber (to increase the
accuracy of the sorting) needs to be balanced with the objective of
generating a sorted sample in a reasonable time in a reasonable
volume containing an acceptable concentration of polynucleotide
molecules.
[0208] The maximum tolerable P.sub..gtoreq.2 depends on the desired
"purity" of the sorted sample. The "purity" in this case refers to
the fraction of sorted polynucleotides that are in the specified
size range, and is inversely proportional to P.sub..gtoreq.2.
[0209] For example, in applications where high purity is not
required, such as the purification of a particular restriction
fragment from an enzymatic digest of a portion of vector DNA, a
relatively high P.sub..gtoreq.2 (e.g., P.sub..gtoreq.2=0.2) may be
acceptable. For most applications, maintaining P.sub..gtoreq.2 at
or below about 0.1 provides satisfactory results.
[0210] In an example where P.sub..gtoreq.2 is equal 0.1, it is
expected that in about 10% of measurements, the signal from the
detection region will be due to the presence of two or more
polynucleotide molecules. If the total signal from these molecules
is in the range corresponding to the desired size fragment, these
(smaller) molecules will be sorted into the channel or tube
containing the desired size fragments.
[0211] The DNA concentration needed to achieve a particular value
P.sub..gtoreq.2 in a particular detection volume can be calculated
from the above equation. For example, a detection region in the
shape of a cube 1 .mu.m.sup.3 per side has a volume of 1 femtoliter
(fl). A concentration of molecules resulting, on average, in one
molecule per fl, is about 1.7 nM. Using a P.sub..gtoreq.2 of about
0.1, the polynucleotide concentration in a sample analyzed or
processed using such a 1 fl detection region volume is
approximately 0.85 nM, or roughly one DNA molecule per 2 detection
volumes ([DNA]*V=.about.5). If the concentration of DNA is such
that [DNA]*V is 0.1, P.sub..gtoreq.2 is less than 0.005; i.e.,
there is less than a one half of one percent chance that the
detection region will at any given time contain two of more
fragments.
[0212] The signal from the optical detector is routed, e.g., via
electrical traces and pins on the chip, to a processor, which
processes the signals into values corresponding to the length of
the polynucleotide giving rise to the signal. These values are then
compared, by the processor, to pre-loaded instructions containing
information on which branch channel molecules of a particular size
range will be routed into. Following a delay period that allows the
molecule from which the reporter signal originated to arrive at the
discrimination region, the processor sends a signal to actuate the
active elements in the discrimination region such that the molecule
is routed into the appropriate branch channel.
[0213] The delay period is determined by the rate at which the
molecules move through the channel (their velocity relative to the
walls of the channel) and the length of the channel between the
detection region and the discrimination region. In cases where the
sample solution is moved through the device using hydrostatic
pressure (applied, e.g., as pressure at the inlet end and/or
suction at the outlet end), the velocity is typically the flow rate
of the solution. In cases where the molecules are pulled through
the device using some other means, such as via electro-osmotic flow
with an electric field set up between the inlet end and the outlet
end, the velocity as a function of molecule size can be determined
empirically by running standards, and the velocity for a specific
molecule calculated based on the size calculated for it from the
reporter signal measurement.
[0214] A relevant consideration with respect to the velocity at
which the polynucleotide molecules move through the device is the
shear force that they may be subject to. At the channel dimensions
contemplated herein, the flow through the channels of the device is
primarily laminar flow with an approximately parabolic velocity
profile. Since the cross-sectional area of the channels in the
device can be on the same order of magnitude as the diameter of the
molecules being analyzed, situations may arise where a portion of a
particular molecule is very near the wall of the channel, and is
therefore in a low-velocity region, while another portion of the
molecule is near the center of the channel, i.e., in a
high-velocity region. This situation creates a shear force (F) on
the molecule, which can be estimated using the following
expression:
F=6.pi..eta.R.sub..lamda.V
where R.sub..lamda. is the radius of the molecule and .eta. is the
viscosity of the solution. This expression assumes that the
molecule is immobilized on a stationary surface and subject to
uniform flow of velocity V.
[0215] The amount of force necessary to break a double stranded
fragment of DNA is approximately 100 pN. Accordingly, the maximal
shear force that the molecules are subjected to should preferably
be kept below this value. Substituting appropriate values for the
variables in the above expression for lambda DNA yields a maximum
velocity of about 1 cm/sec for a channel 1.mu.m in radius (i.e., a
channel of a dimension where one portion of the lambda molecule can
be at or near the wall of the channel with the opposite side in the
center of the channel). Since devices designed for use with such
large molecules will typically have channels that are considerably
larger in diameter, the maximum "safe" velocity will typically be
greater than 1 cm/sec.
[0216] As discussed above, the sample solution introduced into a
device of the invention should be dilute enough such that there is
a high likelihood that only a single molecule occupies the
detection region at any given time. It follows then that as the
solution flows through the device between the detection and
discrimination regions, the molecules will be in "single file"
separated by stretches of polynucleotide-free solution. The length
of the channel between the detection and discrimination region
should therefore not be so long as to allow random thermal
diffusion to substantially alter the spacing between the molecules.
In particular, the length should be short enough that it can be
traversed in a time short enough such that even the smallest
molecules being analyzed will typically not be able to diffuse and
"switch places" in the line of molecules.
[0217] The diffusion constant of a 1 kb molecule is approximately 5
.mu.m.sup.2/sec; the diffusion equation gives the distance that the
molecule diffuses in time t as:
<X.sup.2>.about.Dt
Using this relationship, it can be appreciated that a 1 kbp
fragment takes about 0.2 seconds to diffuse 1 .mu.m. The average
spacing of molecules in the channel is attraction of the
cross-sectional area of the channel and the molecule concentration,
the latter being typically determined in view of acceptable values
of P.sub..gtoreq.2 (see above). From the above relationships, it is
then straightforward to calculate the maximum channel length
between the detection and discrimination region which would ensure
that molecules don't "switch places". In practice, the channel
length between the detection and discrimination regions is between
about 1 .mu.m and about 2 cm.
[0218] As illustrated above with respect to FIGS. 5A-D, there are a
number of ways in which molecules can be routed or sorted into a
selected branch channel. For example, in a device employing the
discrimination region shown in FIG. 4A, the solution is preferably
moved through the device by hydrostatic pressure. Absent any field
applied across electrodes 110 and 112, a molecule would have an
equal probability of entering one or the other of the two branch
channels 106 and 108. The sorting is accomplished by the processor
temporarily activating a voltage source connected to the electrode
leads 114 and 116 just before or at the time the molecule to be
routed enters the junction of the main channel and the two branch
channels. The resulting electric field exerts a force on the
negatively-charged DNA molecule biasing it toward the
positively-charged electrode. The molecule will then be carried
down the branch channel containing the positively-charged electrode
by the bulk solution flow. The electric field is turned off when
the molecule has committed itself to the selected channel. As soon
as the molecule clears the corner from the discrimination region
and into the branch channel, it escapes effects of the electric
field that will be applied to the next molecule in the solution
stream.
[0219] The discrimination region shown in FIG. 5B is designed for
use in a device that employs electroosmotic flow, rather than flow
induced by hydrostatic pressure, to move both the polynucleotides
and bulk solution through the device. Electrodes are setup in the
channels at the inlet and outlet ends of the device. Application of
an electric field at the ends of the channels (with electrode 130
being negative, and electrodes 132 and 134 being positive) sets up
bulk solution flow according to well-established principles of
electroosmotic flow (see, e.g., 25). When a specific polynucleotide
molecule enters the junction region between the main channel and
the two branch channels, the voltage to one of either electrodes
132 or 134 is shut off, leaving a single attractive force, acting
on the solution and the DNA molecule, into the selected branch
channel. As above, both branch channel electrodes are activated
after the molecule has committed to the selected branch channel in
order to continue bulk flow through both channels.
[0220] In another embodiment of the invention the polynucleotides
are directed into a selected branch channel via a valve in the
discrimination region. An exemplary valve is shown in FIG. 5C. The
valve consists of a thin extension of material 140 which can be
charged via an electrode 142. The extension can then be deflected
to close one or the other of the branch channels by application of
an appropriate voltage across electrodes 144 and 146. As above,
once the molecule has committed, the voltage can be turned off.
[0221] In a device in which the sample solution is moved through
the device by application of positive pressure at the sample inlet
end(s) of the analysis unit(s), the discrimination function may be
affected by simply blocking branch channel sample outlets into
which the sample is not supposed to go, and leaving the selected
outlet open. Due to the small size scale of the channels and the
incompressibility of liquids, blocking the solution flow creates an
effective "plug" in the unselected branch channels, routing the
molecule along with the bulk solution flow into the selected
channel. This embodiment is illustrated in FIG. 4D. It can be
achieved by, for example, incorporating valve structures downstream
of the discrimination region.
[0222] Alternatively, the discrimination function may be affected
by changing the hydrostatic pressure at the sample outlets of the
branch channels into which the sample is not supposed to go.
Specifically, if the branch channels in a particular analysis unit
all offer the same resistance to fluid flow, and the pressure at
the sample inlet of the main channel of an analysis unit is P, then
the fluid flow-out of any selected branch channel can be stopped by
applying a pressure P/n at the sample outlet of that branch
channel, where n is the number of branch channels in that analysis
unit. Accordingly, in an analysis unit having two branch channels,
the pressure applied at the outlet of the branch to be blocked is
P/2.
[0223] It will be appreciated that the position and fate of the
molecules in the discrimination region can be monitored by
additional detection regions installed, e.g., immediately upstream
of the discrimination region and/or in the branch channels
immediately downstream of the branch point. This information be
used by the processor to continuously revise estimates of the
velocity of the molecules in the channels and to confirm that
molecules having selected size characteristics end up in the
selected branch channel.
[0224] Solution from the branch channels is collected at the outlet
ends of the analysis units. As described above, devices with a
plurality of analysis units typically collect the solution from
corresponding branch channels of each unit into a manifold, which
routes the solution flow to an outlet port, which can be adapted
for receiving, e.g., a segment of tubing or a sample tube, such as
a standard 1.5 ml centrifuge tube.
[0225] The time required to isolate a desired quantity of
polynucleotide depends on a number of factors, including the size
of the polynucleotide, the rate at which each analysis unit can
process the individual fragments, and the number of analysis units
per chip, and can be easily calculated using basic formulas. For
example, a chip containing 1000 analysis units, each of which can
sort 1000 fragments per second, could isolate 0.1 .mu.g of 10 kb
DNA in about 2.5 hours.
6.11. Other Microfabricated Devices
[0226] Operation of a microfabricated cell sorting device is
essentially as described above with respect to the polynucleotide
sorting device. Since cells typically do not have predictable a net
charge, the directing means are preferably ones employing a valve
in the discrimination region as described above, or flow stoppage,
either by valve or hydrostatic pressure.
[0227] Operation of a microfabricated analysis device is
accomplished essentially as is described above, except that
functions relating to sorting polynucleotide molecules into branch
channels don't need to be performed. The processor of such analysis
devices is typically connected to a data storage unit, such as
computer memory, hard disk or the like, as well as to a data output
unit, such as a display monitor, printer and/or plotter. The sizes
of the polynucleotide molecules passing through the detection
region are calculated and stored in the data storage unit. This
information can then be further processed and/or routed to the data
output unit for presentation as, e.g., histograms of the size
distribution of DNA molecules in the sample. The data can, of
course, be presented in real time as the sample is flowing through
the device, allowing the practitioner of the invention to continue
the run only as long as is necessary to obtain the desired
information.
[0228] In preferred molecular (e.g. DNA, polynucleotide or
polypeptide) analysis and sorting embodiments, a microfabricated
chip of the invention has a detection volume of about 10 to about
5000 femtoliters (fl), preferably about 50 to about 1000 fl, and
most preferably on the order of about 200 fl. In preferred cell
analysis and sorting embodiments, a microfabricated chip of the
invention has a detection volume of approximately 1 to 1,000,000
femtoliters (fl), preferably about 200 to 500 fl, and most
preferably about 375 fl.
6.12. Exemplary Embodiment and Additional Parameters
[0229] Microfluidic Chip Fabrication. In a preferred embodiment,
the invention provides a "T" on "Y" shaped series of channels
molded into optically transparent silicone rubber or
PolyDiMethylSiloxane (PDMS), preferably PDMS. This is cast from a
mold made by etching the negative image of these channels into the
same type of crystalline silicon wafer used in semiconductor
fabrication. As described above, the same techniques for patterning
semiconductor features are used to form the pattern of the
channels. The uncured liquid silicone rubber is poured onto these
molds placed in the bottom of a Petri dish. To speed the curing,
these poured molds are baked. After the PDMS has cured, it is
removed from on top of the mold and trimmed. In a chip with one set
of channels forming a "T", three holes are cut into the silicone
rubber at the ends of the "T", for example using a hole cutter
similar to that used for cutting holes in cork, and sometimes
called cork borers. These holes form the sample, waste and
collection wells in the completed device. In this example, the hole
at the bottom end of the T is used to load the sample. The hole at
one arm of the T is used for collecting the sorted sample while the
opposite arm is treated as waste. Before use, the PDMS device is
placed in a hot bath of HCl to make the surface hydrophilic. The
device is then placed onto a No. 1 (150 .mu.m thick) (25.times.25
mm) square microscope cover slip. The cover slip forms the floor
(or the roof) for all three channels and wells. The chip has a
detection region as described above.
[0230] Any of or all of these manufacturing and preparation steps
can be done by hand, or they can be automated, as can the operation
and use of the device.
[0231] The above assembly is placed on an inverted Zeiss
microscope. A carrier holds the cover slip so that it can be
manipulated by the microscope's x-y positioning mechanism. This
carrier also has mounting surfaces which support three electrodes,
which implement the electro-osmotic and/or electrophoretic
manipulation of the cells or particles to be analyzed and sorted.
The electrodes are lengths of platinum wire taped onto a small
piece of glass cut from a microscope slide. The wire is bent into a
hook shape, which allows it to reach into one of the wells from
above. The cut glass acts as a support platform for each of the
electrodes. They are attached to the custom carrier with
double-sided tape. This allows flexible positioning of the
electrodes. Platinum wire is preferred for its low rate of
consumption (long life) in electrophoretic and electro-osmotic
applications, although other metals such as gold wire may also be
used.
[0232] Device Loading. To operate the device for sorting, one of
the wells, e.g. the collection or waste well, is first filled with
buffer. All three channels, starting with the channel connected to
the filled well, wick in buffer via capillary action and gravity.
Preferably, no other well is loaded until all the channels fill
with buffer, to avoid the formation of air pockets. After the
channels fill the remaining wells can be loaded with buffer, as
needed, to fill or equilibrate the device. The input or sample well
is typically loaded last so that the flow of liquid in the channels
is initially directed towards it. Generally, equal volumes of
buffer or sample are loaded into each well. This is done in order
to prevent a net flow of liquid in any direction once all of the
wells are loaded, including loading the sample well with sample. In
this embodiment, it is preferred that the flow of material through
the device (i.e. the flow of sample) be driven only by the
electrodes, e.g. using electro-osmotic and/or electrophoretic
forces. The electrodes may be in place during loading, or they can
be placed into the wells after loading, to contact the buffer.
[0233] Electrodes. Two of the above electrodes are driven by high
voltage operational amplifiers (op-amps) capable of supplying
voltages of +-150 V. The third electrode is connected to the
electrical ground (or zero volts) of the high voltage op-amp
electronics. For sorting operation the driven electrodes are placed
in the collection and waste wells. The ground electrode is placed
in the sample well. The op-amps amplify, by a factor of 30, a
control voltage generated by two digital to analog converters
(DACs). The maximum voltage these DACs generate is +-5 V, which
determines the amplification factor of 30. The 150 V limit is
determined by the power supply to the amplifiers, which are rated
for +-175 V. These DACs reside on a card (a Lab PC 1200 Card,
available from National Instruments, Austin, Tex.) mounted in a
personal computer. The card also contains multiple channels of
analog to digital converters (ADC's) one of which is used for
measuring the signal generated by photomultiplier tubes (PMTs).
This card contains two DACs. A third DAC can be used to drive the
third electrode with an additional high voltage op amp. This would
provide a larger voltage gradient, if desired, and some additional
operational flexibility.
[0234] Without being bound by any theory, it is believed that the
electrodes drive the flow of sample through the device using
electro-osmotic or electrophoretic forces, or both. To start the
movement of molecules, cells or particles to be sorted, a voltage
gradient is established in the channels. This is done by generating
a voltage difference between electrodes.
[0235] In this example, the voltage of the two driven electrodes is
raised or lowered with respect to the grounded electrode. The
voltage polarity depends on the charge of the molecules, cells or
particles to be sorted (if they are charged), on the ions in the
buffer, and on the desired direction of flow. Because the electrode
at the sample well in the examples is always at zero volts with
respect to the other two electrodes, the voltage at the "T"
intersection or branch point will be at a voltage above or below
zero volts, whenever the voltage of the other two electrodes is
raised or lowered. Typically, the voltage is set or optimized,
usually empirically, to produce a flow from the sample well, toward
a downstream junction or branch point where two or more channels
meet. In this example, where two channels are used, one channel is
typically a waste channel and terminates in a waste well; the other
channel is a collection channel and terminates in a collection
well.
[0236] To direct the molecules, particles or cells to a particular
channel or arm of the "T" (e.g. collection or waste), the voltage
at the electrode in one well (or multiple wells, in multi-channel
embodiments) is made the same as the voltage at the junction or
branch point, where the channels meet. The voltage of the electrode
at one well of the two or more wells is raised or lowered, to
produce a gradient between that well and the branch point. This
causes the flow to move down the channel towards and into the well,
in the direction produced by the gradient. Typically, the voltage
of the electrode at the waste well is raised or lowered with
respect to the voltage at the collecting well, to direct the flow
into the waste channel and the waste well, until a molecule,
particle or cell is identified for collection. The flow is diverted
into the collection channel and collection well by adjusting the
voltages at the electrodes to eliminate or reduce the gradient
toward the waste well, and provide or increase the gradient toward
the collection well. For example, in response to a signal
indicating that a molecule or cell has been detected for sorting,
by examination in a detection region upstream of the branch point,
the voltage at the waste and collection points can be switched, to
divert the flow from one channel and well to the other.
[0237] The voltage at the branch point (the intersection voltage)
is determined by the voltage gradient desired (e.g. Volts/mm) times
the distance from the sample well electrode to the branch point
(gradient x distance), which in this example is placed where all of
the channels of the "T" intersect. The gradient also determines the
voltage at the waste or collection electrode (gradient x distance
from sample well to collection well).
[0238] Conceptually, the channels and wells of the "T" can be
treated as a network of three resistors. Each segment of the "T"
behaves as a resistor whose resistance is determined by the
conductivity of the buffer and the dimensions of the channel. A
voltage difference is provided across two of the resistors, but not
the third. If the electrodes in each of the three wells is
equidistant from the branch point, then each channel will have the
same resistance.
[0239] For example, assume that each section of the "T" has 100 K
ohms of resistance. If 100 volts is applied across two of the
resistors and the third resistor is left unconnected, the current
at the junction of the two resistors would be 50 volts. If a
voltage source of 50 volts is connected to the end of the third
resistor, the voltage at the junction does not change. That is, a
net of zero volts is established across the third resistor; there
is no voltage gradient and a flow is not initiated or changed. If a
different voltage is applied, a gradient can be established to
initiate or direct the flow into one channel or another. For
example, to change the direction of flow from one arm of the "T" to
the other, the voltage values of the two driven electrodes are
swapped. The junction voltage remains the same. If the electrode
distances from the "T" intersection are not equal, then the
voltages can be adjusted to accommodate the resulting differences
in the effective channel resistance. The end result is still the
same. The electrode in the well of the channel which is temporarily
designated not to receive particles or cells is set at the voltage
of the "T" intersection. The voltage at the other driven electrode
is set to provide a gradient that directs molecule, cell or
particle flow into that well. Thus, cells or particles can be sent
down one channel or another, and ultimately into one well or
another, by effectively opening one channel with a net or relative
voltage gradient while keeping the other channel or channels closed
by a net or relative voltage gradient of zero.
[0240] In a preferred embodiment for sorting according to the
invention, a slight flow down the channel that is turned "off" is
desired. This keeps the molecules or cells moving away from the
branch point (the "T" junction), particularly those which have
already been directed to that channel. Thus, a small non-zero
gradient is preferably established in the "off" or unselected
channel. The selected channel is provided with a significantly
higher gradient, to quickly and effectively divert the desired
molecules or cells into that channel.
[0241] The placement of the wells and their electrodes with respect
to the branch point, and in particular their distance from the
branch point, is an important factor in driving the flow of
molecules or cells as desired. As the wells and electrodes are
brought closer to the branch point, it becomes more important to
precisely place the electrodes, or precisely adjust the
voltages.
[0242] Detection Optics. In this example, a Ziess Axiovert 35
inverted microscope is used for detection of molecules or cells for
sorting. The objective lens of this microscope faces up, and is
directed at the detection region of the described microfluidic
chip, through the coverslip which in this example is the floor of
the device. This microscope contains all the components for
epifluorescence microscopy. See, Inoue pp 67-70, 91-97 (52). In
this embodiment a mercury arc lamp or argon ion laser is used as
the light source. The mercury lamp provides a broad spectrum of
light that can excite many different fluorophores. The argon ion
laser has greater intensity, which improves the detection
sensitivity but is generally restricted to fluorophores that excite
at 488 or 514 nm. The mercury tamp is used, for example, to sort
beads as described elsewhere herein. The laser is used for sorting
GFP E. coli bacterial cells as described elsewhere herein. The high
power argon ion beam is expanded to fill the illumination port of
the microscope, which matches the optical characteristics of the
mercury arc lamp and provides a fairly uniform illumination of the
entire image area in a manner similar to the mercury lamp. However,
it is somewhat wasteful of the laser light. If a lower powered
laser is used, the laser light is focused down to coincide with the
detection region of the chip, to achieve the same or similar
illumination intensity and uniformity with less power
consumption.
[0243] The objective used in the example is an Olympus PlanApo
60.times.1.4 N.A. oil immersion lens. The optics are of the
infinity corrected type. An oil immersion lens enables collecting a
substantial percentage of the 180 degree hemisphere of emitted
light from the sample. This enables some of the highest sensitivity
possible in fluorescence detection. This microscope has 4 optical
ports including the ocular view port. Each port, except the ocular,
taps .about.20% of the available light collected from the sample
when switched into the optical path. Only the ocular port can view
100% of the light collected by the objective. In this embodiment, a
color video camera is mounted on one port, another has a Zeiss
adjustable slit whose total light output is measured with a
photomultiplier tube (PMT). The fourth port is not used.
[0244] The microscope focuses the image of the sample onto the
plane of the adjustable slit. An achromatic lens collimates the
light from the slit image onto the active area of the PMT.
Immediately in front of the PMT window an optical band pass filter
is placed specific to the fluorescence to be detected. The PMT is a
side on-type and does not have a highly uniform sensitivity across
its active area. By relaying the image to the PMT with the
achromat, this non-uniformity is averaged and its effect on the
measured signal is greatly minimized. This also enables near ideal
performance of the bandpass filter. A 20% beam splitter has been
placed in the light path between the achromat and filter. An ocular
with a reticle re-images this portion of the collimated light. This
enables viewing the adjustable slit directly, to insure that the
detection area that the PMT measures is in focus and aligned. The
adjustable slit allows windowing a specific area of the channel for
detection. Its width, height, and x,y position are adjustable, and
conceptually define a detection region on the chip. In this
embodiment, the microscope is typically set to view a 5 .mu.m
(micron) length of the channel directly below the "T"
intersection.
[0245] The PMT is a current output device. The current is
proportional to the amount of light incident on the photocathode. A
transimpedance amplifier converts this photo-current to a voltage
that is digitized by the Lab PC 1200 card. This allows for
interpreting the image to select cells or particles having an
optical reporter for sorting, as they pass through the detection
region, based for example on the amount of light or fluorescence
measured as an indication of whether a cell or particle has a
predetermined level of reporter and should be chosen for
collection. Voltages at the electrodes of the chip can be adjusted
or switched according to this determination, for example by signals
initiated by or under the control of a personal computer acting in
concert with the Lab PC1200 card.
[0246] Absorbence Detection. In another embodiment for detecting
cells or molecules, absorbence detection is employed, which
typically uses relatively longer wavelengths of light, e.g.,
ultraviolet (UV). Thus, for example, a UV light source can be
employed. Additional objective lenses can be used to image a
detection region, such that the lenses are preferably positioned
from the top surface if the PDMS device is made reasonably thin.
Measurement of the light transmitted, i.e., not absorbed by the
particle or cell, using an adjustable slit, e.g., a Zeiss
adjustable slit as described above, is similar to that used in
fluorescence detection. A spectrophotometer may also be used. As
molecules, particles or cells pass through the detection window
they attenuate the light, permitting detection of a desired
characteristic or the lack thereof. This can improve the accuracy
of the particle sorting, for example, when sorting based on an
amount of a characteristic, rather than presence of the
characteristic alone, or to confirm a signal.
[0247] It is noted that in some cases, detection by absorbence may
be detrimental at certain wavelengths of light to some biological
material, e.g., E. coli cells at shorter (UV) wavelengths.
Therefore, biological material to be sorted in this manner should
first be tested first under various wavelengths of light using
routine methods in the art. Preferably, a longer wavelength can be
selected which does not damage the biological material of interest,
but is sufficiently absorbed for detection.
[0248] Optical trapping. In another embodiment, an optical trap, or
laser tweezers, may be used to sort or direct molecules or cells in
a PDMS device of the invention. One exemplary method to accomplish
this is to prepare an optical trap, methods for which are well
known in the art, that is focused at the "T" intersection proximate
to, and preferably downstream of, the detection region. Different
pressure gradients are established in each branch. A larger
gradient at one branch channel creates a dominant flow of
molecules, particles or cells, which should be directed into the
waste channel. A second, smaller gradient at another branch channel
should be established to create a slower flow of fluid from the "T"
intersection to another channel for collection. The optical trap
remains in an "off" mode until a desired particle is detected at
the detection region. After detection of a desired characteristic,
the particle or cell is "trapped", and thereby directed or moved
into the predetermined branch channel for collection. The molecule
or cell is released after it is committed to the collection channel
by turning off the trap laser. The movement of the cell or molecule
is further controlled by the flow into the collection well. The
optical trap retains its focus on the "T" intersection until the
detection region detects the next molecule, cell or particle.
[0249] Flow control by optical trapping permits similar flexibility
in buffer selection as a pressure driven system. In addition, the
pressure gradients can be easily established by adjusting the
volume of liquid added to the wells. However, it is noted that the
flow rate will not be as fast when the pressure in one channel is
above ambient pressure and pressure in another is below.
[0250] Forward Sorting. In an electrode-driven embodiment, prior to
loading the wells with sample and buffer and placing the
electrodes, the electrode voltages are set to zero. Once the sample
is loaded and the electrodes placed, voltages for the driven
electrodes are set, for example using computer control with
software that prompts for the desired voltages, for example the
voltage differential between the sample and waste electrodes. If
the three wells are equidistant from the "T" intersection, one
voltage will be slightly more than half the other. In a typical
run, the voltages are set by the program to start with directing
the molecules, particles or cells to the waste channel. The user is
prompted for the threshold voltage of the PMT signal, to identify a
molecule, particle or cell for sorting, i.e. diversion to the
collection channel and well. A delay time is also set. If the PMT
voltage exceeds the set threshold, the driven electrode voltages
are swapped and then, after the specified delay time, the voltages
are swapped back. The delay allows the selected molecule, particle
or cell enough time to travel down the collection channel so that
it will not be redirected or lost when the voltages are switched
back. As described above, a slight gradient is maintained in the
waste channel; when the voltages are switched, to provide
continuity in the flow. This is not strong enough to keep the
molecule, particle or cell moving into the other or "off" channel
it if is too close to or is still at the branch point.
[0251] The value of this delay depends primarily on the velocity of
the molecules, particles or cells, which is usually linearly
dependent on the voltage gradients. It is believed that momentum
effects do not influence the delay time or the sorting process. The
molecules, particles or cells change direction almost
instantaneously with changes in the direction of the voltage
gradients. Unexpectedly, experiments have so far failed to vary the
voltages faster than the particles or cells can respond. Similarly,
experiments have so far shown that the dimensions of the channels
do not effect the delay, on the distance and time scales described,
and using the described electronics. In addition the speed with
which the cells change direction even at high voltage gradients is
significantly less than needed to move them down the appropriate
channel, when using a forward sorting algorithm.
[0252] Once the voltage and delay value are entered the program, it
enters a sorting loop, in which the ADC of the Lab PC 1200 card is
polled until the threshold value is exceeded. During that time, the
flow of particles or cells is directed into one of the channels,
typically a waste channel. Once the threshold is detected, the
above voltage switching sequence is initiated. This directs a
selected cell or particle (usually and most preferably one at a
time) into the other channel, typically a collection channel. It
will be appreciated that the cells or particles are being sorted
and separated according to the threshold criteria, without regard
for which channel or well is considered "waste" or "collection".
Thus, molecules or cells can be removed from a sample for further
use, or they can be discarded as impurities in the sample.
[0253] After the switching cycle is complete (i.e. after the
delay), the program returns to the ADC polling loop. A counter has
also been implemented in the switching sequence which keeps track
of the number of times the switching sequence is executed during
one run of the program. This should represent the number of
molecules, cells or particles detected and sorted. However, there
is a statistical chance that two molecules, cells or particles can
pass through simultaneously and be counted as one. In this
embodiment, the program continues in this polling loop indefinitely
until the user exits the loop, e.g. by typing a key on the computer
keyboard. This sets the DACs (and the electrodes) to zero volts,
and the sorting process stops.
[0254] Reverse Sorting. The reverse sorting program is similar to
the forward sorting program, and provides additional flexibility
and an error correction resource. In the event of a significant
delay in changing the direction of flow in response to a signal to
divert a selected molecules, cell or particle, for example due to
momentum effects, reversible sorting can change the overall
direction of flow to recover and redirect a molecule, cell or
particle that is initially diverted into the wrong channel.
Experiments using the described electrode array show a delay
problem and an error rate that are low enough (i.e. virtually
non-existent), so that reversible sorting does not appear
necessary. The algorithm and method may be beneficial, however, for
other embodiments such as those using pressure driven flow, which
though benefiting from an avoidance of high voltages, may be more
susceptible to momentum effects.
[0255] If a molecule or cell is detected for separation from the
flow, and switching is not fast enough, the molecule or cell will
end up going down the waste channel with all of the other
undistinguished cells. However, if the flow is stopped as soon as
possible after detection, the molecule or cell will not go too far.
A lower driving force can then be used to slowly drive the particle
in the reverse direction back into the detection window. Once
detected for a second time, the flow can be changed again, this
time directing the molecule or cell to the collection channel.
Having captured the desired molecule or cell, the higher speed flow
can be resumed until the next cell is detected for sorting. This is
achieved by altering the voltages at the electrodes, or altering
the analogous pressure gradient, according to the principles
described above.
[0256] To move molecules or cells at higher velocities, for faster
and more efficient sorting, higher voltages may be needed, which
could be damaging to molecules or cells, and can be fatal to living
cells. Preliminary experiments indicate that there may be a limit
to the trade-off of voltage and speed in an electrode driven
system. Consequently, a pressure driven flow may be advantageous
for certain embodiments and applications of the invention.
Reversible sorting may be advantageous or preferred in a pressure
driven system, as hydraulic flow switching may not be done as
rapidly as voltage switching. However, if a main or waste flow can
move fast enough, there may be a net gain in speed or efficiency
over voltage switching even though the flow is temporarily reversed
and slowed to provide accurate sorting. Pressure driven
applications may also offer wider flexibility in the use of buffers
or carriers for sample flow, for example because a response to
electrodes is not needed.
6.13. Diagnosis of Tuberculosis
[0257] A method for DNA fingerprinting is disclosed, and is
particularly suitable for forensic identification (e.g. by VNTR),
bacterial typing and human or animal pathogen diagnosis. The method
applies restriction length polymorphism using synthetically
generated polynucleotide fragments. This method may be used with
PCR, but in preferred embodiments PCR is not required.
[0258] In this example, the invention is applied to diagnosing the
presence of the tuberculosis bacteria (TB). A short sequence of the
TB genome, e.g. 20-50 bp (base pairs), is selected that is a fixed
distance from a restriction site. This sequence and its
relationship to the restriction site are known or statistically
predicted to be unique or strongly characteristic of TB and can
serve to distinguish TB from other organisms, alone or in
combination with other sequences and/or restriction sites. Thus,
additional short sequences can be selected in relation to the same
or different restriction sites, in order to increase statistical
discrimination. In this way, a unique fingerprint of DNA fragments
can be constructed.
[0259] To identify the bacteria, a single-stranded DNA
oligonucleotide is synthesized to complement the sequence of each
short fragment. The sample DNA to be identified is denatured, and
is combined with the oligonucleotides, triphosphates and DNA
polymerase. Some of the nucleotides should be fluorescently labeled
to serve as a reporter. A strand of fluorescent DNA will be
synthesized, which can be cut at the restriction site to yield a
fragment of fixed length. The resulting DNA fragments can then be
sized in any suitable way, for example by gel electrophoresis, or
the microfabricated technologies described herein, or both. The use
of micro fabricated devices is preferred. These devices are fast
(10 minutes) and require only femtograms of sample DNA, i.e. only a
few thousand molecules. If multiple oligonucleotides are made, the
reactions can be multiplexed, for example all of the
oligonucleotides can be combined with the sample DNA in one test
tube.
6.14. DNA Fingerprinting
[0260] This example demonstrates construction a synthetic
fingerprint of a DNA sample and identification of a sample. The
method can be varied in a number of ways that will be apparent to
the practitioner. For example, post-staining with an intercalating
dye can be used in place of labeled nucleotides. Suitable dyes
include those that are specific to double stranded polynucleotides
or DNA (e.g. picogreen from Molecular Probes, Inc.). Alternatively,
single stranded DNA can be digested, for example using a
single-strand specific nuclease. Another variation is to use
affinity purification to pull down the fragment of interest, for
example using biotinylated oligonucleotides and streptavidin
magnetic coated beads.
[0261] DNA samples are denatured or digested with a specific
restriction enzyme, such as Bgl II, EcoR I, Hind III or Xho I in
the presence of a buffer, according to the instructions from the
manufacturer. This can be done at about 34.degree. C. for about 1
minute. Multiple digestions may be done and a final mixture of
thousands of base pairs is preferred. The DNA fragments are then
extended with primers and fluorescent nucleotides. If the buffer
used for digestion is not compatible with DNA extension, another
buffer may be used, or dialysis or ethanol precipitation can be
conducted.
[0262] The DNA extension is accomplished by first preparing a
10.times. primer solution (1 .mu.M) and a 10.times. nucleotide mix
comprising 250 .mu.M dATP, 500 .mu.M fluorescein-dATP (from NEN),
and 750 .mu.M dTTP, dCTP and dGTP. The DNA sample is then diluted
in TE buffer to get a final 10.times. concentration of about 5-50
ng/.mu.l. This strongly depends on the average size of DNA
fragments in the digestion sample and also the relative amount of
DNA fragments (templates) that will hybridize with the primers.
Generally speaking, about InM of DNA templates (10.times.) gives
optimum results. DNA extension can be done at about 68.degree. C.
for about 2 hours.
[0263] The following solutions are made:
TABLE-US-00001 Mix 2 (Gibco Elongase Enzyme Mix) Mix 1 (total 100
.mu.l) 2 .mu.l ultra-pure water 16 .mu.l 5X Buffer A 1 .mu.l of 10x
nucleotide mix 24 .mu.l Buffer B 1 .mu.l of 10x primer 4 .mu.l
Elongase Enzyme Mix (Gibco) 1 .mu.l of 10x DNA samples 56 .mu.l of
ultra-pure water
Buffer A and Buffer B are from the Gibco Elongase Enzyme Package.
Then, combine equal volumes of Mix 1 and Mix 2 and mix well. 100
.mu.l of Mix 2 can be used for about 20 different DNA samples (in
Mix 1). Therefore, the total volume of Mix 2 can be adjusted
according to the number of DNA samples to be run.
[0264] Following these procedures, a polymerase reaction is run for
1 minute at 96.degree. C. for denaturing, for 1 minute at
55.degree. C. for annealing and for 1-hour at 68.degree. C. for
extension. The annealing time and temperature may vary, depending
on the primer used in Mix 1. The PCR reaction and conditions can be
optimized or modified according to techniques known in the art.
[0265] Preferably, only one reaction is performed. Successive
rounds of PCR amplification are not needed. Dialysis or a spin
column is then used to clean out unused fluorescent single
nucleotides (fluorescein-dATP).
[0266] After the extension reaction is run the fluorescent-labeled
DNA samples are diluted to about 100 fM (1,000 times for a
10.times. template with a concentration of 1 nM). Then 10 .mu.l of
the diluted sample is run on the SMS system described above, using
10 mW laser power and 2,450-volt APD bias for 10 to 30 minutes. The
data is analyzed using a DNA size distribution or threshold method
according to a pre-selected fluorescent level, as described
above.
6.15. Identification of T7 DNA
[0267] This example demonstrates construction of a synthetic
fingerprint from the genome of T7 phage, and identification of a
sample, according to the methods of Example 14. An oligonucleotide
primer specific to T7 phage was used, fluorescent fragments were
generated, and these were sized in an SMS device of the invention.
Control tests using the T7 olignucleotide primer as the fingerprint
with a lambda phage sample showed virtually no signal in the SMS
device of Example 9.
[0268] In this example, with T7 and .lamda., digestion was not
needed and was not done, because the DNA fragments were already of
fixed length. The DNA fragments are extended with primers and
fluorescent nucleotides. Dialysis was done at 4.degree. C. for 3
hours using a 5 .mu.l sample in 10 ml of TE buffer. After dialysis
a 10.times. dilution in TE buffer was done (i.e. by adding 45 .mu.l
of TE buffer).
[0269] The DNA extension is accomplished by first preparing a
10.times. primer solution 1 .mu.M and a 10.times. nucleotide mix
comprising 250 .mu.M dATP, 500 .mu.M fluorescein-dATP (from NEN),
and 750 .mu.M dTTP, dCTP and dGTP. The DNA sample is then diluted
in TE buffer to get a final 10.times. concentration of about 5-50
ng/.mu.l. This strongly depends on the average size of DNA
fragments in the digestion sample and also the relative amount of
DNA fragments (templates) that will hybridize with the primers.
Generally speaking, about 1 nM of DNA templates (10.times.) gives
optimum results. DNA extension can be done at about 68.degree. C.
for about 2 hours.
[0270] Mix 1 and Mix 2 were made as in Example 14. Then, 5 .mu.l of
Mix 2 was added to Mix 1 and they are mixed well.
[0271] In this example the following T7 primer was used, which
binds to T7 at base position 588:
TABLE-US-00002 (SEQ. ID. NO. 1)
5'-CATTGACAACATGAAGTAACATGCAGTAAGA-3'.
[0272] Following these procedures, a polymerase reaction is run for
1 minute at 96.degree. C. for denaturing, for 1 minute at
60.degree. C. for annealing and for 2 hours at 68.degree. C. for
extension. In this example, 0.5 .mu.l of a 100 .mu.l solution of
Taq polymerase was used. Only one reaction is performed. Successive
rounds of PCR amplification are not needed. Dialysis or spin column
is then used to clean out unused fluorescent single nucleotides
(fluorescein-dATP). After the extension reaction is run the
fluorescent-labeled DNA samples are diluted to about 100 fM. Then
10 .mu.l of the diluted sample is run on the SMS system, using 10
mW laser power and 2,450-volt APD bias for 10 to 30 minutes. The
data is analyzed using DNA size distribution or threshold method
according to a pre-selected fluorescent level.
[0273] For analysis using a microfabricated device of the
invention, a sample solution was diluted by a factor of two. The
device comprised a T-shaped set of channels about 3 .mu.m wide and
about 1.75 .mu.m deep in an elastomer substrate. This chip was
treated with HCl to render it hydrophillic. The device, as
described in Example 9, used a laser with 3.5 mW of power (488 nm)
with a 2,450 V APD detector bias.
[0274] The above procedure was repeated with an unknown test
sample, which comprised either T7 phage DNA (which should match) or
lambda phage (which should not match). The sizing results for the
test sample were compared against the T7 phage standard. The
results of the T7 v. T7 experiment are shown in FIG. 13. The
results of the T7 v. lambda phage experiment are shown in FIG. 14.
These results are compared in FIG. 15, showing strong signals for
the T7 matching T7, and weak signals for lambda, which does not
match T7. As shown, there is a clearly a distinguishable signal
between the T7 and lambda results. FIG. 13 shows many brightly
fluorescent DNA fragments that are relatively long (match),
compared to the relatively short and dull fragments of FIG. 14 (no
match). These differences can be readily detected based on a preset
threshold, as shown in FIG. 15. The results shown in the figures
were obtained with 5-10 minutes of run time on the SMS device of
the invention.
6.16. Preferred Systems and Embodiments for Molecular
Fingerprinting
[0275] This Example describes, in general terms, preferred
embodiments of the molecular fingerprinting assays that are
described and demonstrated elsewhere in this application (see, for
instance, the Examples presented in Sections 6.13-6.15, supra). The
description of these methods is made by way of non-limiting
examples. Accordingly, the skilled artisan will appreciate that
many variations of these methods may be practiced without departing
from the spirit of the present invention. For example, many of the
specific steps described above may be eliminated and, moreover, the
steps need not necessarily be performed in the particular
sequential order(s) recited herein.
[0276] Using the methods described herein, a skilled artisan may
readily analyze any sample, e.g., to detect a particular nucleic
acid and to thereby determine whether that particular nucleic acid
is present in the sample. For example, the methods of the invention
may be used to analyze samples of cells or tissue (or samples of
nucleic acid derived therefrom) to determine whether a particular
nucleic acid is present in the cells or tissue. In one embodiment,
the methods may be used to determine whether the cells or tissue
express a particular nucleic acid. In another embodiment the
particular nucleic acid may be a nucleic acid from a pathogen
(e.g., from an infectious agent such as a virus or bacteria), and
the fingerprinting methods of this invention may be used to
determine whether the cells or tissues are infected with that
pathogen.
[0277] In other embodiments, the methods of the invention may be
used to analyze a sample derived from an individual (e.g., a
clinical sample derived from a patient). For example, the molecular
fingerprinting methods of this invention may be used to analyze a
cell or tissue sample from an individual or, more preferably, a
sample or nucleic acids derived from such cells or tissue. In such
embodiments the molecular fingerprinting methods of the invention
may be used, e.g., as a diagnostic method (e.g., to detect a
nucleic acid or nucleic acids characteristic of a particular
pathogen), as part of a therapeutic methods (e.g., to monitor
expression of a certain gene or genes during a therapy) or as a
forensic method (for example, paternity testing) to name a few
applications.
[0278] In preferred embodiments, the methods of this invention are
used to detect or analyze single stranded nucleic acids within a
sample. Accordingly, a nucleic acid sample analyzed according to
the invention will preferably be a sample of single-stranded
nucleic acid molecules. However, samples containing double-stranded
nucleic acids may also be analyzed. In such embodiments, the sample
is preferably denatures (e.g., by heat) or otherwise exposed to
conditions in which the complementary nucleic acids separate to
produce single-stranded nucleic acid.
[0279] The skilled artisan will readily appreciate that the methods
of this invention may be used to analyze and/or detect any type of
nucleic acid in a sample. Thus, although the specific examples
presented herein frequently describe the invention in terms of
detecting or analyzing DNA, the invention may also be used to
detect and/or analyze RNA. Indeed, any type of synthetic or
naturally occurring nucleic acid may be analyzed and/or detected
using the invention, including but not limited to the various types
recited, supra, in Section 5.1.
[0280] In preferred embodiments, therefore, the invention is used
to detect a particular nucleic acid, referred to here as the
"target" nucleic acid, in a sample. Preferably, the base sequence
of the target nucleic acid detected with this invention will be at
least partly known. More preferably, the known (or partly known)
sequence will comprise at least one sequence that is recognized by
a particular "cleavage agent". The term cleavage agent, as used
herein, refers to any agent (e.g., any enzyme, chemical or other
substance) that is able to cut or cleave a nucleic acid molecule
into two or more fragments. In preferred embodiments, each cleavage
agent used in the methods and compositions of this invention will
recognize a specific (and preferably different) nucleic acid
sequence; i.e., each cleavage agent preferably has a specific
recognition site (also referred to herein as the "cleavage site" or
the "restriction site"). Recognition sites that are four bases in
length are preferred. However, the invention is not limited to
recognition sites of any particular length. Indeed, in embodiments
where a plurality of different cleavage agents are used the
different cleavage agents may have recognition sites of different
lengths.
[0281] In particularly preferred embodiments, a cleavage agent used
in the invention is a restriction enzyme. Restriction enzymes are
well known in the art and may be readily obtained, e.g., from a
variety of commercial sources (for example, Promega Corp., Madison,
Wis.). Similarly, methods for using restriction enzymes are also
generally well known and routine in the art. See, for example, the
references cited in Section 5.1, supra, for general molecular
biology techniques. Preferred restriction enzymes are ones that cut
nucleic acids by recognizing a specific sequence of bases (i.e.,
the recognition site). Typically, the recognition site for a
restriction enzyme will be about 4, 5 or 6 nucleotides in length. A
cut is typically made within the recognition site. Therefore the
location of the cut in a nucleic acid having a known recognition
site will also be known.
[0282] In one preferred, exemplary embodiment of the invention, a
primer is selected or chosen (e.g., by a user) which is able to
bind or hybridize to the target nucleic acid under suitable
conditions and at a specific, known or predetermined location in
the target nucleic acid sequence. In particular, the primer
preferably binds or hybridizes to the target nucleic acid at a
location that is a known or predetermined distance from the
restriction site; i.e., at a site that is a specific number of
bases away from the restriction site. Generally, the primer is a
nucleic acid that is complementary to a particular sequence of the
target nucleic acid molecule and is therefore capable of
hybridizing to that complementary sequence under appropriate
hybridization conditions. In preferred embodiments, the primer is
an oligonucleotides between about 4 and about 100 bases in length,
more typically about 10-100 bases in length and preferably between
about 20-50 bases in length.
[0283] A suitable primer having been selected, chosen or otherwise
obtained, the primer is then contacted to the nucleic acid sample
under suitable conditions so that the primer binds to the target
nucleic acid at the predetermined location. The sample, with the
primer, is then incubated with a polymerase and a plurality of
nucleotides under conditions such that primer extension can occur,
e.g., by adding nucleotides to the primer and using the target
nucleic acid as a template, thereby generating a second nucleic
acid molecule that is complementary to the target nucleic acid.
Typically, the plurality of nucleotides will include one or more
nucleotides that are detectably labeled (e.g., with a reporter) so
that the primer extension product, or a fragment thereof, may be
detected by detecting the reporter.
[0284] In one embodiment, the primer extension reaction continues
to the end of the target nucleic acid. However, in preferred
embodiments primer extension need only continue up to at least the
restriction site so that the complementary nucleic acid that is
generated by the primer extension also contains the restriction
site. Generally, polymerases synthesize polynucleotides beginning
at the 3'-end of a polynucleotide primer and moving in the
3'-direction. Thus, in preferred embodiments of the invention, a
primer is chosen that hybridizes to a sequence of the target
nucleic acid that is situated a particular known distance upstream
(i.e., in the 3'-direction) from the restriction site on the
single-stranded target nucleic acid.
[0285] Having generated a nucleic acid that is complementary to the
target nucleic acid and contains the target nucleic acid's
restriction site (or a complement thereof), the complementary
nucleic acid may also be cut or cleaved by a cleavage agent (e.g.,
a restriction enzyme) which recognizes the particular restriction
site. Because synthesis of the complementary nucleic acid beings a
fixed, predetermined distance from the restriction site, using the
cleavage agent to cut or cleave the complementary nucleic acid
gives rise to a fragment having a known length; namely, the length
of the known, fixed distance from the restriction site to the
position on the target sequence where the primer hybridizes and
primer extension begins.
[0286] Accordingly, in preferred embodiments the cleavage agent is
next contacted to the sample so that the target sequence and/or the
complementary sequence are cut or cleaved, thereby generating a
fragment or fragments having the fixed known length. In one
embodiment, the cleavage agent is contact to the sample without
denaturing the polynucleotides; i.e., so that the target and
complementary nucleic acids are hybridized to each other and are
therefore double stranded. However, in an alternative embodiment
the cleavage agent is able to specifically recognize the
restriction site in the single-stranded complementary nucleic acid,
and the two strands are separated (e.g., by heating the sample)
before contacting the cleavage agent.
[0287] A user may then readily determine whether the target nucleic
acid is present in the sample by detecting fragments of the known
fixed length. For instance, in preferred embodiments where primer
extension is performed using detectably labeled nucleotides (e.g.,
with a reporter), the fragments may be readily detected by
separating polynucleotides in the sample according to length and
detecting the reporter. A variety of techniques are known in the
art for separating polynucleotides by their length or size and any
of these techniques may be used in the present invention.
Exemplary, non-limiting techniques include gel electrophoresis,
high performance liquid chromatography (HPLC) and mass
spectroscopy. However, in particularly preferred embodiments
polynucleotides are sorted according to size using a microfluidic
device of the present invention, e.g., according to any of the
polynucleotide sorting algorithms described supra.
[0288] A skilled artisan will recognize that the above-described
methods may be readily adapted to various nucleic acid
amplification techniques such as the polymerase chain reaction
(PCR). For example, many different copies of a single primer may be
repeatedly contacted to the sample followed by repeated primer
extension reactions so that a plurality of complementary nucleic
acids are obtained. Alternatively, sense and antisense primers may
be designed to amplify a particular subsequence of the target
nucleic acid which contains the restriction site. Contacting the
cleavage agent with the resulting amplification products will then
produce fragments having a specific, predetermined size and these
fragments may be detected as described above.
[0289] The methods of the invention are ideally suited however, to
performing only a single round of primer extension so that a single
complementary nucleic acid is created and a single fragment is
detected. Single nucleic acid fragments may be readily detected,
e.g., using a microfluidic device of the invention to sort
polynucleotides one molecular at a time, as described supra.
Moreover, these methods are particularly advantageous since
analysis may be performed using small samples and without
undergoing complicated thermocycling that is required, e.g., for
PCR. As result, the molecular fingerprinting methods of this
invention are ideally suited for "lab on a chip" devices (described
supra) that comprise a single microfluidic device. In such devices,
the entire sequence of primer extension, strand cleavage and
fragment detection may be performed using a single microfluidic
device or kit and such kits are therefore considered to be part of
the present invention.
[0290] A skilled artisan will also readily appreciate that steps of
these molecular fingerprinting methods may be performed in a
variety of different sequences. For example, in one alternative
embodiment the nucleic acid sample may be contacted with a cleavage
agent before implementing primer extension. In embodiments, the
primer will then hybridize to a nucleic acid fragment generated
when the target nucleic acid is cut or cleaved with the cleavage
agent. In particular, the primer preferably hybridizes to this
fragment a known, predetermined distance from the fragment's end
(i.e., from the cut site). As a result, primer extension occurs
from the primer and along the target sequence to the cut site, and
a complementary fragment having the known, predetermined length is
thereby obtained.
[0291] Those skilled in the art will further appreciate that
variations of the above-described methods may be performed using a
plurality of different primers. Preferably, each primer will
hybridize to a different sequence within the target nucleic acid,
each different sequence being a known but different distance from
the target nucleic acid's restriction site. In such variations of
the invention, a plurality of primer extension reactions may be
implemented (preferably at least one for each different primer)
either sequentially or at the same time, and the
different-extension products may then be cleaved with the cleavage
agent. A plurality of fragments having different predetermined
lengths are then produced, and these fragments may be separated
according to their size and detected as described, above.
[0292] It will be appreciated by persons of ordinary skill in the
art that the examples and preferred embodiments herein are
illustrative, and that the invention may be practiced in a variety
of embodiments which share the same inventive concept.
7. REFERENCE CITED
[0293] Numerous references, including patents, patent applications
and various publications, are cited and discussed in the
description of this invention. The citation and/or discussion of
such references is provided merely to clarify the description of
the present invention and is not an admission that any such
reference is "prior art" to the invention described herein. All
references cited and discussed in this specification and/or listed
here below are incorporated herein by reference in their entirety
and to the same extent as if each reference was individually
incorporated by reference.
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