U.S. patent application number 11/075682 was filed with the patent office on 2005-07-28 for fractionation of macro-molecules using asymmetric pulsed field electrophoresis.
Invention is credited to Austin, Robert Hamilton, Huang, Lotien Richard, Sturm, James Christopher.
Application Number | 20050161331 11/075682 |
Document ID | / |
Family ID | 22971713 |
Filed Date | 2005-07-28 |
United States Patent
Application |
20050161331 |
Kind Code |
A1 |
Huang, Lotien Richard ; et
al. |
July 28, 2005 |
Fractionation of macro-molecules using asymmetric pulsed field
electrophoresis
Abstract
A method and apparatus for fractionation of charged
macro-molecules such as DNA is provided. DNA solution is loaded
into a matrix including an array of obstacles. An alternating
electric field having two different fields at different
orientations is applied. The alternating electric field is
asymmetric in that one field is stronger in duration or intensity
than the other field, or is otherwise asymmetric. The DNA molecules
are thereby fractionated according to site and are driven to a far
side of the matrix where the fractionated DNA is recovered. The
fractionating electric field can be used to load and recover the
DNA to operate the process continuously.
Inventors: |
Huang, Lotien Richard;
(Princeton, NJ) ; Sturm, James Christopher;
(Princeton, NJ) ; Austin, Robert Hamilton;
(Princeton, NJ) |
Correspondence
Address: |
MCCARTER & ENGLISH, LLP
FOUR GATEWAY CENTER
100 MULBERRY STREET
NEWARK
NJ
07102
US
|
Family ID: |
22971713 |
Appl. No.: |
11/075682 |
Filed: |
March 9, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11075682 |
Mar 9, 2005 |
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10022189 |
Dec 18, 2001 |
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6881317 |
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60256298 |
Dec 18, 2000 |
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Current U.S.
Class: |
204/547 ;
204/643 |
Current CPC
Class: |
C12N 15/101 20130101;
Y10T 436/10 20150115; Y10T 436/25 20150115; Y10T 436/11
20150115 |
Class at
Publication: |
204/547 ;
204/643 |
International
Class: |
G01N 027/453 |
Goverment Interests
[0002] The present invention has been made under Federal Contract
Grant No. MDA 972-00-1-0031 and the government may have certain
rights to the subject invention.
Claims
What is desired to be protected by Letters Patent is set forth in
the appended claims:
1. A method of continuously fractionating charged macro-molecules
comprising: loading molecules into a matrix of obstacles; applying
an assymetric electric field to the matrix to separate the
molecules according to size along a horizontal direction of the
matrix; and collecting separated molecules at a plurality of
locations along a bottom edge of the matrix.
2. (canceled)
3. The method of claim 1 wherein the step of applying an asymmetric
electric field to the matrix comprises applying to the matrix
time-dependent electric fields (t) whose odd-order integrals over
time, .intg..vertline.(t).vertline..sup.n(t)dt, are not at the
time-average field orientation for every n, where n is any positive
even integer.
4. The method of claim 1 wherein the step of applying an asymmetric
electric field comprises: alternating first and second electric
pulses of first and second waveforms; maintaining the integral of
one of the first or second pulses' amplitude over time larger than
that of the other pulse; varying the orientation of the first
electric pulse within first and second orientations, and the
orientation of the second electric pulse within third and forth
orientations.
5. The method of claim 4 wherein the first and second waveforms are
square pulses.
6. The method of claim 5 wherein one of the square pulses is of
higher amplitude than the other.
7. The method of claim 5 wherein one of the square pulses is of
longer duration than the other.
8. The method of claim 1 wherein the step of applying an asymmetric
electric field comprises: alternating first and second electric
pulses of first and second waveforms; maintaining the integral over
time of one of the first or second pulses' amplitudes larger thank
that of the other pulse; and applying the first and second electric
pulses at first and second fixed orientations.
9. The method of claim 8 wherein the first and second waveforms are
square pulses.
10. The method of claim 9 wherein one of the square pulses is of
higher amplitude than the other.
11. The method of claim 9 wherein one of the square pulses is of
longer duration than the other.
12. The method of claim 1 wherein the charged macro-molecules are
deoxyribonucleic acid (a.k.a. DNA).
13. (canceled)
14. (canceled)
15. The method of claim 1 wherein the molecules are loaded using
electric fields.
16. The method of claim 1 wherein the molecules are extracted from
the array of obstacles using electric fields.
17. The method of claim 1 wherein the molecules are routed to the
next processing step after fractionation.
18. A method of continuously fractionating charged macro-molecules
comprising: loading molecules into a matrix with an array of
obstacles; applying to the matrix electric fields whose amplitudes
are constant in time; varying field orientations of the electric
fields with time to create an asymmetrical electric field to
separate the molecules according to size along a horizontal
direction of the matrix; and collecting separated molecules at a
plurality of locations along a bottom edge of the matrix.
19. (canceled)
20. The method of claim 18 wherein the fields alternate between two
fixed orientations.
21. The method of claim 18 wherein the charged macro-molecules are
deoxyribonucleic acid (a.k.a. DNA).
22. (canceled)
23. (canceled)
24. The method of claim 18 wherein the molecules are loaded using
electric fields.
25. The method of claim 18 wherein the molecules are extracted from
the array of obstacles using electric fields.
26. The method of claim 18 wherein the molecules are routed to the
next processing step after fractionation.
27. An apparatus for continuously fractionating charged
macro-molecules comprising: an array of obstacles; asymmetrically
alternating electric fields applied to the array of obstacles to
separate molecules according to size along a horizontal direction
of the array; and a plurality of locations along a bottom edge of
the array for collecting separated molecules.
28. The apparatus of claim 27 wherein the asymmetrically
alternating electric fields comprise: an electric field which is
alternating in direction as a function of time at a location in the
matrix, and which has a time average of an electric field vector
over many cycles, whereby the time integral of the vector at the
same location over a part of the cycles when the electric field is
instantaneously pointing to one side of the vector is not spatially
symmetric about the vector with the time integral of the vector
over another part of the cycles at the same location when the
electric field is instantaneously pointing to another side of the
vector.
29. The apparatus of claim 27 wherein the asymmetrically
alternating electric fields comprise: time-dependent electric
fields (t) whose odd-order integrals over time,
.intg..vertline.(t).vertline..sup.n(t)dt, are not at the
time-average field orientation for every n, where n is any positive
even integer.
30. The apparatus of claim 27 wherein the asymmetrically
alternating electric fields comprise: first and second electric
pulses of first and second waveforms; the integral over time of one
of the first or second pulses' amplitude larger than that of the
other pulse; the orientation of the first electric pulse varying
between a first orientation and second orientation, and the
orientation of the second electric pulse varying between a third
orientation and forth orientation.
31. The apparatus of claim 30 wherein the first and second
waveforms are square pulses.
32. The apparatus of claim 31 wherein one of the square pulses is
of higher amplitude than the other.
33. The apparatus of claim 31 wherein one of the square pulses is
of longer duration than the other.
34. The apparatus of claim 27 wherein the asymmetrically
alternating electric fields comprise: first and second alternating
electric pulses of first and second waveforms; the integral over
time of one of the first or second pulses' amplitudes larger than
that of the other pulse; the first and second electric pulses
applied at first and second fixed orientations.
35. The apparatus of claim 34 wherein the first and second
waveforms are square pulses.
36. The apparatus of claim 35 wherein one of the square pulses is
of higher amplitude than the other.
37. The apparatus of claim 35 wherein one of the square pulses is
of longer duration than the other.
38. The apparatus of claim 27 wherein the asymmetrically
alternating electric fields comprise: electric fields whose
amplitudes are constant in time; the field orientation varying with
time in such a manner that .intg.[.theta.(t)].sup.n+1dt are not
zero for every n, where .theta.(t) is field orientation with
respect to the time-average field orientation, and n is any even
integer larger than zero.
39. The apparatus of claim 38 wherein the fields alternate between
two fixed orientations.
40. The apparatus of claim 27 wherein the charged molecules are
deoxyribonucleic acid (a.k.a. DNA).
41. (canceled)
42. The apparatus of claim 27 further comprising extraction
structures for extracting fractionated molecules from the array of
obstacles.
43. The apparatus of claim 27 further comprising one or more
loading channels for loading molecules.
44. The apparatus of claim 27 wherein the molecules are extracted
from the array of obstacles using electric fields.
45. The apparatus of claim 27 wherein the molecules are loaded into
the array of obstacles using electric fields.
46. The apparatus of claim 27 wherein the molecules are routed to
the next processing step after fractionation.
Description
RELATED APPLICATIONS
[0001] This application claims the priority of Provisional
Application Ser. No. 60/256,298, filed Dec. 18, 2000, the entire
disclosure of which is expressly incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to a method and apparatus for
fractionating charged macro-molecules such as DNA using asymmetric
pulsed field electrophoresis.
[0005] 2. Related Art
[0006] The analysis and fractionation of large DNA molecules is a
central step in large scale sequencing projects. Conventionally,
gel electrophoresis is used to fractionate DNA molecules according
to their sizes. This method includes two steps: sample loading and
fractionation. First, sample solution containing DNA is loaded into
loading wells in the gel slab before the electric field is turned
on. Then, an electric field is applied. The DNA molecules move in
the opposite direction of the electric field because they are
negatively charged. As the electric field is applied, DNA molecules
travel at different speeds according to their sizes, but the
directions in which they migrate are always the same. Eventually,
sample DNA molecules are separated into different bands, each of
which contains DNA molecules of the same size, as shown in FIG. 1.
Shorter DNA fragments move faster than longer ones. Therefore, they
are separated according to their sizes. However, this standard
method only works effectively for DNA molecules smaller than 40
kbp. Above this range, the standard method has to be modified. In
particular, the applied electric field can no longer be DC, but is
made to alternate between two different orientations. This modified
scheme (pulsed-field gel electrophoresis) is routinely used in
modern molecular biology laboratories, but it typically takes a few
days to fractionate one set of DNA samples.
[0007] What is needed, and has not heretofore been provided, is a
method and apparatus for quickly, or even continuously,
fractionating charged macro-molecules.
OBJECTS AND SUMMARY OF THE INVENTION
[0008] It is an object of the present invention to provide a method
and apparatus for quickly fractionating charged
macro-molecules.
[0009] It is an additional object of the present invention to
provide a method and apparatus for continuously fractionating
charged macro-molecules.
[0010] It is a further object of the present invention to provide a
method and apparatus for fractionating macro-molecules using
asymmetric pulsed electrophoresis wherein an alternating electric
field having two different orientations is applied, and one of the
fields is stronger than the other in terms of duration or
intensity, or the field is otherwise asymmetric.
[0011] The present invention relates to a method and apparatus for
fractionation of charged macro-molecules such as DNA. DNA solution
is loaded into a matrix including an array of obstacles. An
alternating electric field having two different fields at different
orientations is applied. The alternating electric field is
asymmetric in that one field is stronger in duration or intensity
than the other field, or is otherwise asymmetric. The DNA molecules
are thereby fractionated according to size and are driven to a far
side of the matrix where the fractionated DNA is recovered. The
fractionating electric field can be used to load and recover the
DNA to operate the process continuously.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Other important objects and features of the invention will
be apparent from the following Detailed Description of the
Invention taken in connection with the accompanying drawings in
which:
[0013] FIG. 1 shows conventional gel electrophoresis.
[0014] FIG. 2 is a diagram showing asymmetric pulsed-field
electrophoresis in micro/nano-fabricated matrices according to the
present invention.
[0015] FIG. 3 is a diagram showing the basic principle of
asymmetrical pulsed electrophoresis of the present invention.
[0016] FIG. 4 shows the way stretched DNA molecules move under
asymmetrical pulsed electric field.
[0017] FIG. 5 shows a support material (matrix) for use in
fractionation of DNA according to the present invention.
[0018] FIG. 6A is a top view and FIG. 6B is a side view of the
microfabricated support material shown in FIG. 5.
[0019] FIG. 7 shows fractionation of T4 and T7 DNA.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention relates to a method and apparatus for
fractionation of charged macro-molecules such as DNA. DNA solution
is loaded into a matrix including an array of obstacles. An
alternating electric field having two different fields at different
orientations is applied. The alternating electric field is
asymmetric in that one field is stronger in duration or intensity
than the other field, or is otherwise asymmetric. The DNA molecules
are thereby fractionated according to size and are driven to a far
side of the matrix where the fractionated DNA is recovered. The
fractionating electric field can be used to load and recover the
DNA to operate the process continuously.
[0021] The present invention provides a method and apparatus for
the fractionation of macro-molecules on micro/nano-fabricated
support materials (a.k.a. matrices). Because the motion of DNA
molecules can be accurately controlled in micro/nano-fabricated
environments, the fractionation of DNA can be achieved with very
high resolution in a short time (i.e. seconds), even for DNA
molecules larger than 100 kbp. In addition, the process can be
operated continuously, i.e., DNA is loaded, fractionated, and
recovered at the same time. Moreover, because this method exploits
micro/nano-fabricated structure, it can be readily integrated into
lab-on-a-chip devices as a component.
[0022] According to the present invention, DNA molecules enter from
one point or loading channel 14 on the boundary 12 of the matrix 10
as shown in FIG. 2. The molecules are subsequently fractionated
into different bands at different orientations, according to their
sizes, as they are driven towards the other side 13 of the matrix
10, where the purified DNA molecules 30 are finally recovered. The
DNA molecules are fractionated into short fragments 32 at one end,
long fragments 36 at the other end, and medium fragments 34
therebetween. The electric field (E.sub.1 and E.sub.2) that
fractionates the DNA sample can also be used to load and recover
the sample, enabling the process to be operated continuously.
[0023] A mixture of DNA molecules emerges continuously from the
loading channel. The support material comprises a
micro/nano-fabricated porous structure, in which DNA molecules can
move. An alternating electric field, shown in E.sub.1 and E.sub.2,
is applied across the whole matrix. E.sub.1 and E.sub.2 are at an
angle with respect to each other, preferably an obtuse angle, and
have different intensities and/or durations. Because DNA molecules
are stretched and moving in a zigzag way under the alternating
field, shorter fragments move at an angle to longer fragments.
[0024] When DNA molecules are subject to an alternating electric
field between two orientations at an angle such as an obtuse angle,
they are stretched to different lengths according to their
molecular weight. Referring to FIG. 3, let the end-to-end length of
a stretched DNA molecule be x. Assume that electric field E.sub.1
displaces every DNA molecules by approximately the same
displacement .alpha. e.sub.1, whereas E.sub.2 displaces every DNA
molecules by approximately .beta. e.sub.2 (e.sub.1 and e.sub.2 are
unit vectors, and both .alpha. and .beta. are positive numbers,
since DNA molecules are negatively charged and move opposite to an
applied electric field). This is a valid assumption because it is
known that all DNA molecules have virtually the same mobility due
to the fact that the long range hydrodynamic interaction is
shielded by the counter ion layers. For the simplicity, let .alpha.
be larger than .beta.. This can be achieved by pulsing along
-e.sub.1 longer than along -e.sub.2, and/or by making the electric
field stronger along -e.sub.1 than along -e.sub.2. Because the
electric field is alternating between two different directions, the
DNA molecules will move in a zigzag way. Ideally, the electric
field is chosen so that x<.beta.<.alpha.. The net motion of
very short DNA molecules (x<<.beta.) in one pulsing cycle (a
cycle refers to applying E.sub.1, then E.sub.2) is simply .alpha.
e.sub.1+.beta. e.sub.2. On the contrary, very long molecules
(x>.beta.) travel (.alpha.-.beta.)e.sub.1 in a cycle. Even
though this could be rather surprising at first glance, it is not
hard to understand if it is realized that when the field is
switched from one to the other, the tails 40 of DNA strands become
the ends that lead the motion and the heads 42 follow, as shown in
FIG. 4. In principle, we can predict the angles of the bands into
which DNA mixtures are fractionated by this technique, if the
stretched lengths of DNA molecules are smaller than or equal to
.beta.. Within this range (x<.beta. or x=.beta.), the net motion
of DNA molecules in one cycle is
(.alpha.-x)e.sub.1+(.beta.-x)e.sub.2. Purified DNA molecules can be
recovered at the bottom of the support material, after many cycles.
In one cycle, a DNA molecule stretched to length x will travel
(.alpha.-x)e.sub.1+(.beta.-x)e.sub.2.
[0025] As shown in FIG. 4, an alternating electric field not only
stretches DNA molecules to a linear conformation, but also makes
them to move in a zigzag way. The initial position of a DNA
molecule is labeled as 0. The big dot on one end of the DNA
represents the "head" 42 of the molecule. The other end of the
molecule is referred to as the "tail" 40. When E.sub.1 is applied,
the DNA molecule moves to position 1. The tail 40 leads the motion
as the electric field is switched to E.sub.2. By the end of one
cycle, the molecule moves to position 2, and the net displacement
in one cycle (.alpha.-x)e.sub.1+(.beta.-x)e.sub.2.
[0026] By electric field, what is meant is the spatial average of
the field around a location over a length scale of several
obstacles, not the microscopic field distribution around a single
obstacle. Any electric field at a given location, whose direction
varys with time, can be resolved uniquely into two sequences of
electric pulses according to the instantaneous direction of the
field. The first sequence of electric pulses comprises the electric
field pointing to one side of the average field vector over the
whole period of time when the field is applied to fractionate the
molecules. The second sequence of electric pulses comprises the
electric field pointing to the other side of the average field
vector. If the field vector at a moment is at the same direction or
at the opposite direction of the average field vector, it is
excluded in either of the pulse sequence. By asymmetrical electric
field, what is meant is that the two sequences of electric pulses,
resolved from a given electric field, as a function of time, have
vector integrals over time that is not symmetric about the
time-averaged field direction. Said another way, the electric
fields, fields (t) whose odd-order integrals over time,
.intg..vertline.(t).vertline..sup.n(t)dt, are not at the
time-average field orientation for every n, where n is any positive
even integer. As such, by applying electric fields with different
orientations and different strengths, i.e. different durations or
different intensities or both, one applies an asymmetric field.
Asymmetric fields can also be generated by sweeping signals in
terms of orientation, duration and intensity. In the past, the
field has first and second pulse sequences whose vector integrals
over time are symmetrical about the average field.
EXPERIMENTAL RESULTS
[0027] The following example uses a microfabricated matrix 10. As
shown in FIG. 5, the matrix 10 consists of two parts: a
microfabricated array of obstacles 20 in quartz, and a cap layer 18
that is hermetically bonded to the microfabricated side of the
quartz substrate 16. The quartz substrate 16 is
surface-micromachined using standard microfabrication techniques.
The substrate is subsequently bonded to a glass cap layer 18
hermetically. The cavities between the substrate and the cap layer
become microfluidic channels in which DNA molecules are
fractionated. The dimensions of this microfabricated device are
depicted in FIGS. 6a and 6b. FIG. 6a is the top view of the matrix
10, and FIG. 6b is a side view of the matrix 10. The matrix 10 in
this case is a hexagonal array of obstacles 20. Each obstacle 20
comprises a cylindrical post 2 .mu.m in diameter. The
center-to-center distance between neighboring obstacles is 4 .mu.m.
The uniformity of the electric field across the whole matrix is
controlled accurately by the peripheral structures surrounding the
matrix. FIG. 7 shows the fractionation of T4 (169 kbp) and T7 (40
kbp) DNA molecules. The pulse condition is E.sub.1=120 V/cm at
60.degree. with respect to the horizontal boundary, and E.sub.2=60
V/cm at -60.degree.. The DNA injected into the matrix is 10
.mu.g/ml of T4 DNA and 10 .mu.g/ml of T7 DNA in 1/2 TBE buffer. The
duration of E.sub.1 is identical to that of E.sub.2, which is 166
msec. The frequency at which the electric field alternates is 3 Hz.
Clearly, the DNA mixture separates into two bands.
[0028] Having thus described the invention in detail, it is to be
understood that the foregoing description is not intended to limit
the spirit and scope thereof.
* * * * *