U.S. patent number 5,314,829 [Application Number 07/992,601] was granted by the patent office on 1994-05-24 for method for imaging informational biological molecules on a semiconductor substrate.
This patent grant is currently assigned to California Institute of Technology. Invention is credited to L. Stephen Coles.
United States Patent |
5,314,829 |
Coles |
May 24, 1994 |
Method for imaging informational biological molecules on a
semiconductor substrate
Abstract
Imaging biological molecules such as DNA at rates several times
faster than conventional imaging techniques is carried out using a
patterned silicon wafer having nano-machined grooves which hold
individual molecular strands and periodically spaced unique bar
codes permitting repeatably locating all images. The strands are
coaxed into the grooves preferably using gravity and pulsed
electric fields which induce electric charge attraction to the
molecular strands in the bottom surfaces of the grooves.
Differential imaging removes substrate artifacts.
Inventors: |
Coles; L. Stephen (Marina Del
Rey, CA) |
Assignee: |
California Institute of
Technology (Pasadena, CA)
|
Family
ID: |
25538517 |
Appl.
No.: |
07/992,601 |
Filed: |
December 18, 1992 |
Current U.S.
Class: |
436/165; 850/18;
850/59; 422/504; 435/6.11; 422/82.05; 356/244; 250/440.11; 250/311;
436/164; 436/94; 436/56; 250/307; 422/939; 435/6.12; 977/853 |
Current CPC
Class: |
B01L
9/56 (20190801); B01L 3/502761 (20130101); B01L
3/508 (20130101); C07K 1/047 (20130101); B01J
2219/00547 (20130101); B01J 2219/00608 (20130101); B01J
2219/00612 (20130101); B01J 2219/00621 (20130101); B01J
2219/00659 (20130101); B01L 2200/0663 (20130101); B01L
2200/0668 (20130101); B01L 2300/021 (20130101); B01L
2300/0803 (20130101); B01L 2300/0819 (20130101); B01L
2300/0893 (20130101); B01L 2400/0415 (20130101); B01L
2400/0472 (20130101); B82Y 15/00 (20130101); C40B
70/00 (20130101); G01N 2035/00237 (20130101); Y10S
977/853 (20130101); Y10T 436/143333 (20150115); Y10T
436/13 (20150115); B01J 2219/00549 (20130101) |
Current International
Class: |
C07B
61/00 (20060101); B01L 3/00 (20060101); C07K
1/00 (20060101); C07K 1/04 (20060101); G01N
35/00 (20060101); G01N 021/03 () |
Field of
Search: |
;436/56,63,89,94,164,165,527,422 ;422/55,58,82.05,104,88,92,102
;250/306,307,311,440.11 ;435/6,91 ;204/129.3 ;935/77 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Lindsay et al. "Images of the DNA Double Helix in Water"-Science,
vol. 24, Jun. 2, 1989, pp. 1063-1064. .
Watson, et al., Recombinant DNA: A Short Course, W. H. Freeman and
Cinoabtl New York, 1983, appendix A, pp. 248-253. .
Ayesha Sitlani, "Sequence Specific Recognition and Photocleavage of
DNA by Phenanthrenequinone Diimine Complexes or Rhodium (III)" PHD
Thesis, California Institute of Technology, 1992. .
David M. Glover, Gene Cloning: The Mechanics of DNA Nabuoykatuibm
Chapman and Hall New York, 1984, Chapter 6 pp. 128-157..
|
Primary Examiner: Alexander; Lyle A.
Assistant Examiner: Wallenhorst; Maureen M.
Attorney, Agent or Firm: Wallace; Robert M. Keller; Michael
L.
Government Interests
BACKGROUND OF THE INVENTION
Origin of the Invention
The invention described herein was made in the performance of work
under a NASA contract, and is subject to the provisions of Public
Law 96-517 (35 USC 202) in which the Contractor has elected to
retain title.
Claims
What is claimed is:
1. A method of imaging molecular strands using a substrate having a
plurality of straight grooves in a top surface of said substrate,
each of said grooves being of a size capable of holding only a
single individual one of said molecular strands along any one
section of a length of the groove, and being capable of forcing
single individual molecular strands into a straight position when
positioned in one of said plurality of grooves, said method
comprising:
preparing a specimen of said molecular strands and depositing said
specimen onto said top surface;
aligning and depositing individual ones of said molecular strands
into individual ones of said plurality of grooves whereby the shape
and size of each groove forces the molecular strands into a
straight position; and
recording a first image of a selected section of said top surface
of said substrate having at least one of said molecular strands in
one of said grooves using an atomic imaging device.
2. The method of claim 1 wherein:
said aligning and depositing of said specimen onto said top surface
of said substrate is preceded by recording a preliminary image of
said selected section of said substrate in the absence of any
molecular strand specimen thereon using said atomic imaging device;
and
said recording of said first image is followed by substrating said
preliminary image from said first image to produce a second image
of a molecular strand by itself substantially free of image
artifacts of said substrate.
3. The method of claim 2 wherein said aligning and depositing
individual ones of said molecular strands into individual ones of
said plurality of grooves comprises using gravity to draw said
individual ones of said molecular strands into individual ones of
said plurality of grooves.
4. The method of claim 2 wherein said molecular strands are of the
type which are attracted by one of (i) a positive electrical field,
and (ii) a negative electrical field and wherein said aligning and
depositing individual ones of said molecular strands into
individual ones of said plurality of grooves comprises placing an
electric field of a type which attracts said molecular strands
perpendicular to said top surface of said substrate so as to draw
said individual ones of said molecular strands into individual ones
of said plurality of grooves.
5. The method of claim 4 wherein:
said substrate comprises a crystalline semiconductor.
6. The method of claim 4 wherein said placing an electric field
perpendicular to said top surface is preceded by aligning said
molecular strands in a direction parallel to at least some of said
grooves.
7. The method of claim 6 wherein said aligning of said molecular
strands parallel to at least some of said grooves comprises placing
an electric field across said top surface of said substrate in a
direction parallel to at least some of said grooves.
8. The method of claim 7 wherein said electric field parallel to at
least some of said grooves is a pulsed electric field.
9. The method of claim 6 wherein said electric field perpendicular
to said top surface is a pulsed electric field.
10. The method of claim 1 wherein said preparing said specimen
comprises preparing a specimen of denatured DNA molecular
strands.
11. The method of claim 10 wherein said preparing includes marking
each of the different bases of said DNA molecular strands with a
particular and unique heavy metal atom.
12. The method of claim 1 wherein said substrate further comprises
address markers located at periodic locations associated with said
plurality of grooves across said top surface of said substrate and
wherein a different address marker uniquely identifies each of said
periodic locations in said top surface of said substrate, and
wherein said method of recording a first image includes recording
an image of said address markers associated with said periodic
locations within said selected section, whereby the location and
orientation of each recorded image of a molecular strand is
deducible from the address markers visible in said first image.
13. The method of claim 12 wherein each of said address markers
comprises a different computer-readable bar code.
Description
Technical Field
The invention relates to sequencing of biological informational
molecules at atomic resolution such as DNA and in particular to
apparatus for holding individual DNA strands stationary and
straight at known locations.
Background Art
Biologically interesting images tend to be lucky accidents and are
not routinely repeatable. This occurs because the state of the art
scanning tunneling electron microscope (STM) and the atomic force
microscope (AFM) do not have the capacity to pan over an image in
real-time. On a uniform crystal lattice, such capabilities are not
required, but for non-homogeneous biological molecules, alignment
of the image is essential.
Atomic resolution is achievable down to a few angstroms using
either STM of AFM. However, the imaging of non-homogeneous
(informational) biological molecules using these techniques has
been disappointing thus far, for at least two reasons: (1) The
non-reproducability of the images, preventing one from doing before
and after studies to prove the absence of substrate artifacts in a
fuzzy image and (2) the inability to lay the molecule down flat on
a plate and have it stick rigidly to the surface for reproducible
scans in different directions to enhance the signal-to-noise ratio,
much as CAT Scans improve images of target tissues over a single
X-Ray.
With respect to the first problem, a good metaphor to appreciate
the problem is to imagine a parachutist regularly bailing out at
night at 10,000 feet and trying to hit the same ground spot on
successive dives. It just can't be done. The reason physicists are
successful with STM or AFM imaging of crystals is that it normally
doesn't matter "where you happen to touch down." For example, in
the case of a metallurgist imaging the crystal lattice of an alloy,
the surface is uniformly isotropic, essentially infinitely, in all
directions (N, E, S, W). It doesn't matter where you look, you
always see the same thing. Furthermore, there are no simple
landmarks to allow one to pan over an image in real time with "x-y"
micrometers and find a reproducible location, as is now routinely
done in light microscopy by histologists examining specially
stained pathology slides. Biologically interesting STM images
published in text books tend to be lucky accidents and are not
routinely repeatable.
With respect to the second problem, when DNA is stretched out in
its non-coiled primary structure (double-stranded helix), it is a
long fragile ungainly molecule. In its native form in the nucleus
of a cell, it is normally hypercoiled in association with disklike
proteins (histones) and further folded into metaloops that
ultimately appear in human cells as 23 pairs of chromosomes, which
during the metaphase of mitosis (cell division) assemble in a
delicate structure called the spindle apparatus. In its denatured
form, however, DNA and RNA tends to lie on a flat surface in a
random configuration like "a plate of spaghetti," making it
exceedingly difficult to image, let alone sequence.
Finally, even after one obtains a reasonable image with
appropriately distributed unique markers (such as heavy metal atoms
or unusual easy-to-visualize side chains) to discriminate the four
basic alphabetic letters or nucleotide bases (A, G, T, C), a third
problem is to "read-off" the sequence automatically.
Specimen preparation is one of the most delicate aspects of all
automatic DNA sequencing technologies. Native double stranded
helical DNA consists of two strands of bases paired together along
the rungs of a twisted step-ladder structure. One strand is called
the primary strand (for transcription of the messenger RNA), while
the other strand is called the secondary strand. Although the
thickness of such a double helix is only about 20 angstroms, if
fully stretched out, a typical molecule could literally extend for
miles. Of course, native DNA is spooled around basic protein
molecules or histones, the spools being super coiled into
chromosomes, referred to hereinabove, whose dimensions are in the
submicron range. The information content of this long biological
molecule is contained in the base-pair sequence, like the bits
encoded along the length of a strip of magnetic computer tape
wrapped around a spool. This is the data which is to be read. In
order to do so, the DNA primary and secondary strand pair are
preferably separated using well-known techniques.
SUMMARY OF THE INVENTION
Grooves etched in a semiconductor surface are used to hold
biological molecules such as individual DNA strands at known
reproducible locations. A grid of grooves is etched into the
surface of a silicon substrate. The coordinates of each
intersection of grooves in the grid is marked with, for example, a
computer-readable bar code etched into the substrate surface
adjacent to the intersection. A section of the grid is then imaged
with an STM or AFM before a DNA specimen is placed thereon and
recorded as a "before" image. Denatured DNA is then placed on the
substrate and individual DNA strands are coaxed into individual
grooves. Coaxing the individual DNA strands into individual grooves
may entail, for example, applying an electric field across the
substrate to align the strands parallel to one set of grooves and
then applying an electric field through the substrate to draw the
strands downwardly into the grooves. In some cases, gravity may be
sufficient to coax a number of DNA strands into individual grooves.
The section of the grid is again imaged to record an "after" image
of both the substrate and a DNA specimen. The "before" image is
then digitally subtracted from the "after" image to produce a final
image of the DNA specimen alone. Confirmation that the correct
"before" and "after" images are subtracted is provided by the bar
codes etched near each intersection in the grid. Furthermore, the
bar codes permit the STM or AFM to scan across the substrate and
then return to the originally imaged section of the grid for a
repeat image, whenever desired.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a plan view of a semiconductor wafer including diceable
chips with a grid of grooves for holding DNA strands.
FIG. 2 is an enlarged perspective cross-sectional view of a portion
of the semiconductor wafer of FIG. 1 showing one DNA strand lying
in a groove.
FIG. 3 is an enlarged plan view of a portion of the semiconductor
wafer of FIG. 1 illustrating the bar codes at each intersection of
the grid.
DETAILED DESCRIPTION OF THE INVENTION
Referring to FIG. 1, a standard 3-inch silicon wafer 10 of crystal
orientation (1,0,0) has many 1-cm square dies 15 separated by 1 mm
borders. Each die 15 has etched into it an orthogonal grid 20 of
grooves, a typical groove 30 thereof being illustrated in the
enlarged view of FIG. 2. Each 1-cm square die has 1000 grooves 30
or lines running in each orthogonal direction so that there are
1000.times.1000 intersections, the distance between groove centers
being one micron, thus forming a 1 mm square grid within the die
15. The grid is shown proportionally oversized within the die 15 in
FIG. 1 for clarity purposes. As indicated in FIG. 2, each groove is
V-shaped, being about 50 nanometers in width at the top, the two
sides thereof descending downwardly toward the apex at opposing 57
degree angles with respect to the vertical. This angle is the
result of the reactive ion etching process employed and the
substrate material.
FIG. 3 illustrates the labelling of each intersection of orthogonal
grooves 30 using a computer-readable bar code 35
photolithographically etched into the substrate surface adjacent to
the intersection of a pair of orthogonal grooves 30.
The wafer 10 and die 15 of FIGS. 1-3 are fabricated as follows.
Silicon wafers are cleaved in a vacuum from an extruded silicon
crystal rod, and their top surfaces are highly polished to atomic
smoothness (or about 5 angstroms, corresponding to the instrinsic
"bumpiness of the silicon atoms). Then, a thin layer of silicon
dioxide is formed on the top surface of each wafer. An electron
beam direct "write step" is employed to define the grid 20 of
orthogonal grooves 30 in each die 15 in a conventional reactive ion
etching step. After application of the electron beam, each wafer is
anisotropically etched in a bath of potassium hydroxide or ethylene
diamine pyrocatechol to form the 50 nanometer grooves 30 of FIG. 2
in a conventional "nano-machining" step.
The computer-readable bar codes 35 are photolithographically etched
into the top surface of the substrate using conventional
photoresist and etching techniques.
Each wafer is then diced along the die borders separating the
individual die 15.
A DNA specimen is prepared for depositing on the die 15 as follows.
The initial step of DNA specimen preparation is to cleave the DNA
into one micron fragments (containing between one thousand and two
thousand DNA bases) using conventional techniques such as either
ultrasound or with an appropriate restriction endonuclease, one of
about 50 well-known enzymes that cuts DNA at precise locations.
(See Watson et al., Recombinant DNA: A Short Course, W. H. Freeman
and Company, New York, 1983, Appendix A, pages 348-353.) The DNA is
then denatured into its single stranded form using the conventional
techniques discussed earlier herein. Then, using established
techniques such as the one described in Ayesha Sitlani, "Design of
Rhodium Complexes to Probe Site-Specific Recognition of DNA," Ph.D
Thesis, California Institute of Technology, 1992, particular
heavy-metal atoms are affixed to particular bases of the DNA strand
as marker-identifiers. This completes the specimen preparation
process.
An eyedropper is employed to place a small drop of the prepared DNA
specimen onto the top surface of the die 15. Individual DNA strands
are coaxed into individual grooves 30 using a variety of
techniques. Specifically, gravitational forces will help the DNA
strands to drop into the grooves 30. Also, a pair of external
capacitor plates may be placed along opposing sides of the die 15
and a pulsed voltage applied thereto to help aligning the DNA
strands on the substrate surface in one of the two orthogonal
directions of the grid of grooves 30. Finally, the DNA strands are
pulled into the grooves 30 by applying an appropriate electrical
field (using external capacitor plates) across the thickness of the
die 15 so that the bottom surfaces of the V-shaped grooves acquire
a positive charge as indicated in FIG. 2. The DNA strands have a
native negative charge, and are therefore attracted by the positive
charge on the bottom groove surfaces as illustrated in FIG. 2. The
result is that a DNA strand 40 is pulled into a groove 30 as
illustrated in FIG. 2.
The DNA strand 40 is imaged while in the groove 30 as follows. An
Atomic Force Microscope (AFM) is used in accordance with the
following assumptions: (1) Real time data analysis is to be
performed; (2) There is a 0.5 angstrom raster line separation; (3)
a 100 angstrom image width is sufficient; (4) the ATM scanning
movement is parallel to the direction of the groove 30; and (5)
successive bases in the DNA strand 40 are separated by 6 angstroms.
The latter assumption is supported by David M. Glover, Gene
Clonino: The Mechanics of DNA Manipulation (Chapman and Hall, New
York 1986).
Before a DNA specimen is deposited onto the substrate surface of
the die 15, the surface (or at least a small selected section
thereof) is imaged without any DNA specimen, to record a "before"
image of the substrate surface only. After the DNA specimen has
been deposited onto the substrate surface, individual strands
thereof are coaxed into the grooves 30. This coaxing is
accomplished in several ways. First, the substrate surface is
maintained in a face-up position so that gravity assists in drawing
the DNA strands in to the grooves. Secondly, the DNA strands may be
aligned parallel to one of the two orthogonal groove directions by
applying an electric field across the substrate surface in the
appropriate direction, by means of external capacitor plates
temporarily held at opposing edges of the die 15. A pulsed voltage
is applied across the capacitor plates. Finally, in order to pull
the DNA strands into the grooves, a voltage on the order of
micro-volts is applied through the thickness of the substrate
(i.e., from the top surface to the bottom surface) so that the
bottom surfaces of the grooves acquire a positive charge, as
indicated in FIG. 2. This voltage is applied, for example, by
placing external capacitor plates near the top and bottom substrate
surfaces and applying a pulsed voltage across the capacitor plates
to induce a voltage difference between the top and bottom of each
groove 30 on the order of several microvolts.
After a sufficient coaxing of the DNA strands into the grooves in
the substrate surface, the same selected area of the substrate
surface is imaged to record an "after" image of a DNA strand in a
groove. The "before and "after images are digitally subtracted from
one another pixel-by-pixel using conventional digital image
processing techniques to produce a final image of a DNA strand by
itself, free of any substrate artifacts.
Before images of many sections of the grid 15 may be obtained in
case it is not known which one of the sections will have an
interesting DNA specimen aligned in a groove. The grid may be
searched by the AFM or STM throughout those sections of the grid
for which a "before" image was obtained prior to deposition of the
DNA specimen. Each section of the grid 15 being unambiguously
defined by the computer-readable bar codes it contains, each
"after" image is readily associated with the exact "before" image
for precise digital subtraction and removal of substrate artifacts
in a "final" image of the DNA strand alone.
In performing the image analysis of the "final" image of the DNA
strand alone, a conventional pattern recognition algorithm may be
employed to automatically identify the different-sized heavy-metal
atoms affixed as markers on the different DNA bases. This is but
one example of an application of the present invention. In other
applications, molecules other than DNA may be imaged. The main
advantage of the invention is the repeatability of any image of a
particular specimen by using the bar codes to re-locate previously
imaged sections of the grid.
The sequencing rate (the rate at which individual DNA bases are
identified along the strand) is determined by the raster length and
the scan rate (linear speed of the AFM tip), as follows:
If we assume a tip speed or raster rate of 100 angstroms/sec in
high-resolution imaging mode, we can obtain a sequencing rate of 6
bases per minute. As a frame of reference, the theoretical limit of
Fluorescence Sequencing, such as the commercially-available
machines from Applied Biosystems, Inc. of Foster City, Calif., is
about 10 bases per minute. On the other hand, if we increase the
tip speed of the AFM to 35,000 angstroms/sec, which is the highest
rate demonstrated for "topographic mode" atomic resolution imaging,
the sequencing rate of the invention is 1800 bases/minute. A
further increase is obtained by increasing the tip speed to the
highest rate demonstrated for "current imaging mode" atomic
resolution, which is a tip speed of 100 microns/sec. This
corresponds to a sequencing rate of 60,000 bases/minute. Thus, the
invention offers a revolutionary improvement over the current state
of the art.
While the invention has been described in detail by specific
reference to preferred embodiments, it is understood that
variations and modifications thereof may be made without departing
from the true spirit and scope of the invention.
* * * * *