U.S. patent application number 13/200940 was filed with the patent office on 2012-02-02 for binding method and apparatus for sorting objects.
This patent application is currently assigned to ARRYX, INC.. Invention is credited to Tania Chakrabarty.
Application Number | 20120028848 13/200940 |
Document ID | / |
Family ID | 40432262 |
Filed Date | 2012-02-02 |
United States Patent
Application |
20120028848 |
Kind Code |
A1 |
Chakrabarty; Tania |
February 2, 2012 |
Binding method and apparatus for sorting objects
Abstract
The present invention relates to a method and apparatus of
sorting objects including, providing a sample having wanted objects
and unwanted objects; coating a surface of a sample holder with an
antibody; placing an eluted sample on the sample holder; binding an
antigen in the wanted objects with the antibody on the surface of
the sample holder to sort the objects into wanted objects and
unwanted objects; separating the wanted objects; and performing
PCR-based STR analysis on the wanted objects. In one embodiment,
holographic optical trapping is used to further sort the wanted
objects. In other embodiments, the wanted objects are sperm and the
antibody is a human sperm specific antibody, and the PCR is single
cell PCR-based STR analysis. In still other embodiments, the
binding is direct or indirect, ligands are used to bind to
object-specific organomolecules, and protein A or protein G are
used to bind the antibody.
Inventors: |
Chakrabarty; Tania;
(Chicago, IL) |
Assignee: |
ARRYX, INC.
Chicago
IL
|
Family ID: |
40432262 |
Appl. No.: |
13/200940 |
Filed: |
October 5, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12232164 |
Sep 11, 2008 |
8067170 |
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13200940 |
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60960004 |
Sep 11, 2007 |
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60960059 |
Sep 13, 2007 |
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Current U.S.
Class: |
506/39 ;
435/287.2 |
Current CPC
Class: |
G01N 33/5091 20130101;
G01N 33/56966 20130101; G01N 2800/367 20130101 |
Class at
Publication: |
506/39 ;
435/287.2 |
International
Class: |
C12M 1/40 20060101
C12M001/40; C40B 60/12 20060101 C40B060/12 |
Claims
1. An apparatus for sorting objects comprising: a sample holder
coated with an antibody, said sample holder containing a sample of
wanted objects and unwanted objects, said wanted objects containing
an antigen; wherein said antibody coated on said sample holder
binds with said antigen on said wanted objects; a chip, including a
microfluidics chip, on which said wanted objects are sorted; an
apparatus for performing PCR-based STR analysis on said wanted
objects and/or said unwanted objects.
2. The apparatus of claim 1, further comprising: a holographic
optical trapping apparatus which optically traps and sorts said
wanted objects from said unwanted objects in said sample.
3. The apparatus of claim 1, wherein said wanted objects are cells,
including sperm cells, and said unwanted objects are
contaminants.
4. The apparatus of claim 1, wherein said PCR apparatus performs
one of bulk PCR on multiple sorted objects of a same type, or
single cell PCR-based STR analysis; and wherein said apparatus for
performing said PCR is automated and multiplexed.
5. The apparatus of claim 1, wherein said sample holder and a
substrate on which said apparatus performs said PCR-based STR
analysis, are a single chip.
6. The apparatus of claim 2, further comprising: a plurality of
ligands disposed on said sample holder; wherein cell surface
receptors on a surface of said wanted objects bind with said
ligands.
7. The apparatus of claim 2, further comprising: a plurality of
ligands disposed on said sample holder; wherein said ligands are
bound to object-specific organomolecules; and wherein said ligand
bound object uses said antibody against said ligand.
8. The apparatus of claim 2, wherein said sample holder is a bead
coated with said antibody to bind one of directly or indirectly to
said wanted objects.
9. The apparatus of claim 2, wherein said sample holder is coated
with one of Protein A or Protein G.
10. The apparatus of claim 8, wherein said antibody further
comprises a primary antibody and a secondary antibody; wherein said
primary antibody is bound to said antigen; and wherein said primary
antibody is bound to a secondary antibody; wherein said bead is
coated with one of Protein A or Protein G and wherein said bead
recognizes and captures said wanted objects bound to said primary
antibody.
Description
[0001] The present application claims priority from U.S.
Provisional Patent Applications No. 60/960,004, filed Sep. 11,
2007, and 60/960,059, filed Sep. 13, 2007, the contents of both of
which are herein incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to methods and apparatuses for
sorting objects in DNA analysis. More particularly, the present
invention relates to the sorting of sperm in a binding method using
antibody or other sperm-recognizing biomolecules, and an apparatus
thereof. In one embodiment, a polymerase chain reaction (PCR)
method is performed after the objects are sorted--in some cases, a
single cell PCR method--to identify persons/assailants in sexual
assault cases in forensic DNA analysis, or for other applications
in medical diagnostics.
[0004] 2. Description of the Related Art
[0005] In conventional forensic DNA analysis, specimens are
commonly matched to alleged criminal suspects in modern law
enforcement, using human identification systems commonly based on
short tandem repeats (STR) analysis which involve the amplification
of the query DNA by polymerase chain reaction (PCR). PCR is a
powerful tool which allows for replicating/amplifying trace amounts
of DNA fragments into quantities that can be analyzed in a
meaningful way. This technology has been adapted for DNA
sequencing, DNA fingerprinting etc., and has the ability to detect
specific DNA fragments in samples.
[0006] Thus, forensic DNA analysis is accomplished using the high
power of discrimination and rapid analysis speed of STR markers in
the human genome, and has now become the most popular method of
choice in forensic DNA analysis.
[0007] Although STR analysis is commonly used, it suffers from
several pitfalls, the most significant of which arises from
contamination of the DNA samples prior to PCR (Polymerase Chain
Reaction) based STR analysis, and the time it takes to perform the
entire STR analysis on a given sample.
[0008] For example, the DNA to be analyzed for STRs from sexual
assault evidence should ideally come from the sperm of the
assailant. However, the sperm sample is often commonly contaminated
with (1) epithelial cells lining the vagina, and occasionally, with
(2) epithelial cells from the mouth (buccal cells), and (3) cells
from the skin, as well as cells in the urine sample. One might also
expect to see erythrocytes, neutrophils, foam cells (non-descript
epithelial cells), etc., in sexual assault crime scene samples as
well.
[0009] Thus, it is clear that better and more accurate STR analysis
will be achieved if the sperm cells could be separated from any or
all of the contaminating cells before PCR is performed.
[0010] Commonly used methods of differential extraction cannot
completely separate male (assailant) sperm and female (victim)
epithelial cell DNA in a forensic sample. For example, initial
lysis using reductant free solution, lyses epithelial cells (the
most common contaminant in a sexual assault forensic sample), and
leaves sperm cells intact for effective separation of DNA
fractions. However, differential lysis causes immature sperm cell
lysing thereby causing unwanted DNA to be coamplified along with
the query DNA (from sperm alone). This leads to mixed STR profile
generation which are hard to analyze and cannot identify a unique
individual. Such problems in STR analysis causes 50% of the STR
analysis based human identification to fail.
[0011] In addition, another limitation in solving forensic cases
comes from the limited availability of cells for analysis. This may
be due to limited evidence samples being present, degradation of
the DNA and cell samples in general over time, and/or the presence
of very few sperm cells in a sexual assault crime sample, to be
able to solve the case based on standard PCR.
[0012] Thus, a method that would prevent or alleviate the above
problems is desired.
SUMMARY OF THE INVENTION
[0013] The present invention relates to methods and apparatuses for
sorting objects using a binding method, using antibody or other
sperm-recognizing biomolecules, in DNA analysis, and specifically
relates to separating sperm from non-sperm contaminants.
[0014] In one embodiment, an antibody that recognizes sperm is
coated on a substrate to sort and separate sperm from other
contaminants. In general, one method which can operate either in
stand-alone mode or in conjunction with holographic optical
trapping to separate sperm from contaminating cells, is to use an
antibody-coated substrate where the antibody selectively recognizes
a surface antigen on the human sperm, for example.
[0015] Alternatively, another choice of antibody could be one that
targets the H-Y antigen typically found on male determining
spermatozoa.
[0016] In another embodiment, the antibody on the substrate could
be an anti-immunoglobulin antibody which in turn recognizes the
monoclonal antibody targeted against the sperm specific surface
antigen. This involves first using a sperm specific antibody to
recognize and bind to sperm in a forensic sample and subsequently
allows the antibody labeled sperm to be recognized and captured by
anti-immunoglobulin.
[0017] In cases where very few sperm are present in a forensics
sample containing other contaminating cells, one can even use
multiple sperm specific antibodies in tandem to select and sort the
sperm for downstream analysis.
[0018] Standard bioconjugation chemistries are available for
attaching antibodies on a substrate which can be glass or other
materials like plastic. Also this invention can be utilized on
substrates of varying geometries such as a flat substrate (in
single or multi well format) or a curved surface such as that of an
Eppendorf tube. Antibody conjugation on the substrate can be via
utilization of common covalent or non-covalent linkage or via
adsorption.
[0019] One can also envision an extension of this technique to use
beads in sperm sorting where instead of coating a substrate with
the antibody/antibodies, one utilizes antibody coated beads such as
those made of silica, polystyrene or magnetic beads to recognize
and bind sperm.
[0020] The antibodies which can be utilized to target sperm
antigens can be full length or cleaved or even short peptides that
recognize the epitope on the sperm surface. In another embodiment
that relies on binding sperm but does not involve antibody based
sperm recognition, is one where one uses binding partners of the
sperm surface receptors to capture sperm from a mixed cell
sample.
[0021] The present invention will resolve the long-standing problem
of co-amplification of female DNA in the sperm cell fraction which
has been suggested to occur in .about.40% of forensic samples
relevant to sexual assault (see Korf BR, in "Current Protocols in
Human Genetics", Wiley: New York 1999). Recent reports suggest that
only 25% of all sexual assault cases lead to the identification of
the perpetrator because the problem of co-amplification of DNA in
such samples causes most STR based human identification to be
ambiguous.
[0022] In a complementary embodiment where very few sperm are
present in the sample but the dominant cell type are epithelial
cells from the vagina, then one can use the antibody approach (with
or without holographic optical trapping (HOT)) to first separate
out the epithelial cells from sperm by causing the epithelial cells
to adhere to the epithelial cell specific antibody coated
substrate. In such a situation, the sperm will remain in the
supernatant and can be either directly used for cell lysis and
extraction. If further purity is needed, they can be separated by
using HOT in parallel with the antibody approach. A variety of
epithelial cell specific markers especially those from the human
vagina, are present for use in this approach. New and novel markers
which are constantly being discovered to target/identify various
cell types including vaginal cells, can be used. A combination of
antibodies might also be used to separate the epithelial cells from
sperm in this embodiment. Similarly an antibody free binding
approach using common ligand receptor binding may also be used.
[0023] The present invention will improve purity in forensic
samples to be analyzed through better separation of sperm from
contaminating cells, thereby increasing the efficacy in downstream
PCR-based STR readouts. In addition, the proposed method is
amenable to automation which current methodologies do not
allow.
[0024] Thus, the present invention relates to a method and
apparatus of sorting objects including providing a sample having
wanted objects and unwanted objects; coating a surface of a sample
holder with an antibody; eluting the sample and placing the eluted
sample on the sample holder; binding an antigen in the wanted
objects with the antibody on the surface of the sample holder to
sort the objects into wanted objects and unwanted objects;
separating the wanted objects; removing the unwanted objects; and
performing PCR-based STR analysis on the wanted objects. In one
embodiment, holographic optical trapping is used to sort the wanted
objects from the unwanted objects. In one embodiment, the wanted
objects are sperm and the antibody is a human sperm specific
antibody. In another embodiment, the STR readout uses single cell
PCR based amplification. In other embodiments, the binding is
direct or indirect such as using secondary antibodies instead of
using primary antibodies alone. In another embodiment, ligands are
used to bind to object-specific macromolecules (such as cell
surface receptors).
[0025] There has thus, been outlined, some features consistent with
the present invention in order that the detailed description
thereof that follows may be better understood, and in order that
the present contribution to the art may be better appreciated.
There are, of course, additional features consistent with the
present invention that will be described below and which will form
the subject matter of the claims appended hereto.
[0026] In this respect, before explaining at least one embodiment
consistent with the present invention in detail, it is to be
understood that the invention is not limited in its application to
the details of construction and to the arrangements of the
components set forth in the following description or illustrated in
the drawings. Methods and apparatuses consistent with the present
invention are capable of other embodiments and of being practiced
and carried out in various ways. Also, it is to be understood that
the phraseology and terminology employed herein, as well as the
abstract included below, are for the purpose of description and
should not be regarded as limiting.
[0027] As such, those skilled in the art will appreciate that the
conception upon which this disclosure is based may readily be
utilized as a basis for the designing of other structures, methods
and systems for carrying out the different purposes of the present
invention. It is important, therefore, that the claims be regarded
as including such equivalent constructions insofar as they do not
depart from the spirit and scope of the methods and apparatuses
consistent with the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 is a flow chart of the steps in a method of sorting
objects, according to one embodiment consistent with the present
invention.
[0029] FIG. 2 depicts the apparatus and method of sorting objects
of FIG. 1.
[0030] FIGS. 3A and 3B depict the apparatus and method of sorting
objects according to other embodiments consistent with the present
invention.
[0031] FIG. 4 is a schematic of a method and apparatus of sorting
objects using holographic optical trapping, in another embodiment
consistent with the present invention.
DESCRIPTION OF THE INVENTION
[0032] The present invention relates to a method and apparatus of
sorting objects in DNA analysis, using binding methods, and/or
holographic optical trapping (HOT).
[0033] Antibody Method
[0034] In one embodiment, an antibody coated substrate is used to
sort objects such as sperm cells. In general, the present
embodiment can operate either in stand-alone mode or in conjunction
with holographic optical trapping (HOT) to separate sperm from
contaminating cells, and uses an antibody-coated substrate where
the antibody selectively recognizes a surface antigen on the human
sperm.
[0035] Alternatively, another choice of antibody could be one that
targets the H-Y antigen typically found on male determining
spermatozoa.
[0036] Specifically, according to one embodiment of the present
invention, the following steps can be taken, as shown in FIGS. 1
and 2, to use an antibody method and apparatus to sort sperm from
contaminants and to determine the DNA of a person(s) in a forensics
case.
[0037] In step 100, a sample 200 is prepared by taking a swab 201
of a forensics specimen, from a victim of sexual assault, for
example, and the sample 200 is eluted using a buffer solution 212
(see FIG. 1) in step 101.
[0038] The eluted samples 200 typically contain sperm cells 202
from the assailant(s), epithelial cells from the victim, and other
contaminants 203, for example.
[0039] In step 102, the surface of a test tube 204, an Eppendorf
container 205, glass slide 206, microfluidic chip 207, or other
container (platforms) intended for sperm separation, are coated
with a human sperm specific antibody or another biomolecule such as
ligands, peptides, proteins, 208 etc., that would bind to the
corresponding binding partner on the sperm. The sperm specific
antibody coating 208 can be applied to any surface or sample
chamber--i.e., test tube 204, Eppendorf container 205, glass
coverslip/coverslide 206, chip 207. Various well established
techniques exist for attaching antibodies and peptides, proteins,
ligands or other biomolecules to a glass substrate. The surface
coating may be applied over the entire internal wall/area of a
container (i.e., test tube 204, Eppendorf 205) or on only specific
areas or patches on the glass coverslip 206 or chip 207, for
example.
[0040] In step 103, the eluted forensic sample 200 to be analyzed
is placed in the antibody-coated sample chamber/container 204-206
via pipetting or other active mechanism such as a pump, or passive
mechanism such as gravity flow, for example.
[0041] In step 104, the antigen 300 (see FIG. 3A(i)) (on the sperm
202) and sperm specific antibody 208 (as might be on the surface of
the container 204-206), are allowed to bind. Thus, the antibody 208
directly recognizes the antigen(s) 300 on the cell surface of the
sperm 202, and selectively binds to the surface antigen 300 while
epithelial and other contaminating cells 203 remain unbound. The
antibodies 208 which can be utilized to target sperm antigens 300
can be full length or cleaved or even short peptides that recognize
the epitope on the sperm 202 surface. In addition, one can use
binding partners of the sperm 202 surface receptors to capture
sperm 202 from a mixed cell sample 200.
[0042] In step 105, the non-sperm (i.e., non-adhering cells or
contaminants 203) are gently washed away from the surface of the
sample chamber 204-206 using a suitable buffer solution of desired
pH and salt concentration or sperm elution buffer 209 (a couple of
washes may be needed), and collected in step 106 if desired,
leaving behind only antibody bound sperm cells 202 for downstream
analysis (see also FIG. 3A(i)). Depending on the number of sperm
202 captured on the surface of the substrate 204-206 which is
required for analysis, the wash steps may be repeated.
[0043] In step 107, the sorted contents (i.e., antibody bound sperm
202) are inspected, scanned, and the quality (purity) visualized.
This can be done (although not limited to) using either brightfield
microscopy (morphology-based identification) or using fluorescent
tags to identify sperm cells 202 where morphology may have been
altered during the handling process. Fluorescence will also make
image processing of the sperm 202 identification easier (since
fluorescence offers better contrast than brightfield images) and
faster.
[0044] In step 108, the sperm cells 202 can then be lysed and the
sperm-containing sample chamber 204-206 can be transferred to a
station 210 in step 109, where in situ PCR followed by STR forensic
analysis, takes place.
[0045] Alternatively, in step 108, the bound sperm cells 202 can be
released from the antibody coated surface of the sample chambers
204-206 using cleaving agents, or by altering/exchanging buffer
solution (such as altering buffer pH to affect antigen-antibody
binding) or by altering the salt concentration to affect the
effective charge shielding, for example. The sorted sperm 202 can
settle by gravity in the sorting container 204-206, or if needed,
further pelleted down by centrifugation before moving the chamber
204-206 with sorted sperm cells 202 to the STR analysis performing
PCR platform 210, in step 109.
[0046] It is to be noted that the ability to carry out the sorting
in open or closed containers 204-206 offers flexibility in platform
design and use with existing instruments. The ability to carry it
out in closed chambers offers the additional advantage of avoiding
any contamination in the pre-PCR handling process.
[0047] In step 110, a result report may be generated with sperm
number, level of purity of the sample 200 tested, and other
relevant performance parameters. These reports prior to actual
PCR-based STR analysis in step 111 will provide better quality
control in the forensic analysis. A visualization method like HOT
(described further below) offers the advantage of keeping track of
intermediate steps leading up to the final result.
[0048] PCR-based STR analysis is performed (step 111) to identify
the person whose DNA matches the DNA of the sperm 202. PCR-based
STR analysis may also be performed on the contaminants or unwanted
objects, such as the epithelial cells of the victim, for
cross-checking the validity of a filed criminal charge. The
PCR-based STR analysis is in itself well-known in the art and has
been broadly commercialized, and thus, not discussed in any detail
herein.
[0049] Multiple PCR reactions can be carried out at any given time
on one machine 210. However, to avoid DNA loss during transfer from
chip to tubes, the PCR may be carried out on-chip 207 using flatbed
thermocycles.
[0050] In step 112, a final STR report is then generated for the
sperm 202 analyzed, and statistics generated on the STR
profile.
[0051] In step 113, the resulting data is matched with the CODIS
database for human identification if it is meant for forensic use,
for example.
[0052] In another embodiment consistent with the present invention,
steps 100-103 remain the same. However, in step 104, the antibody
208 on the substrate 204-207 is an anti-IgG which then recognizes
the antibody 208 bound to the sperm-specific antigen 300 (see FIG.
3A(ii)(a)). Thus, the antibody 208 on the surface of the substrate
204-207 could be an anti-immunoglobulin antibody 208 which in turn
recognizes the monoclonal antibody targeted against the sperm
specific surface antigen 300. In cases where very few sperm 202 are
present in a forensics sample 200 containing other contaminating
cells 203, one can even use multiple sperm specific antibodies 208
in tandem, to select and sort the sperm 202 for downstream analysis
while the anti-immunoglogulin surface antibody may remain the
same.
[0053] In the two-step binding process of this embodiment, in Step
I, the sperm-specific antigen 300 is recognized by a specific
antibody 208. This antibody 208 which is now bound to the sperm 202
surface will be recognized by a secondary (2.degree.) antibody,
such as an anti-IgG (see FIG. 3A(ii)(b).
[0054] Step I (see FIG. 3A(ii)(a)) can be carried out in buffer
solution in an Eppendorf container, incubating the sperm 202 in the
presence of excess antibody 208. Excess antibody 208 can then be
washed off by a simple centrifugal spin. The antibody 208 labeled
sperm 202 is then recovered for Step II (see FIG. 3A(ii)(b)) which
may be performed on a solid support (substrate) 206, for
example.
[0055] In Step II, the primary antibody)(1.degree.) 208 is received
in the secondary antibody)(2.degree.) 301 on the support 206. In
this second situation where a secondary antibody 301 is used to
capture the sperm 202, the secondary antibody 301 (depending on
available binding sites) could capture multiple sperm cells 202.
The surface density of the secondary antibody 301 can be optimized
by keeping steric hindrance in mind. The secondary antibody 301
could be an IgG or an IgM, for example, where an IgM is a pentamer
with more binding (ten) sites or an IgG which has two binding sites
per molecule.
[0056] Thereafter, the steps, such as steps 105-113, remain
substantially the same, with the sperm 202 being inspected and
prepared, for final STR analysis to determine the DNA of the sperm
holder.
[0057] In another embodiment consistent with the present invention,
steps 100-103 remain the same, but in step 104, instead of
immobilizing antibodies 208 on the substrate 206, for example, to
capture sperm 202 either directly or indirectly, as described in
the previous embodiments, one can immobilize ligands 302 (typically
small molecules) on the substrate 206, for example, using various
methods which are commercially known, such that the ligands 302 are
recognized by cell surface receptors 303 on the sperm 202 surface
(see FIG. 3A(iii)), thus, effectively sorting the sperm 202 from
contaminants 203.
[0058] Thereafter, the steps, such as steps 105-113, remain
substantially the same, with the sperm 202 being inspected and
prepared for PCR-based STR analysis.
[0059] In yet another embodiment consistent with the present
invention, steps 100-103 remain the same, but in step 104, an
indirect approach--as shown in FIG. 3A(ii)(b)--is used to implement
a two-step process for sperm 202 capture. The first step involves
use of a ligand 302 to bind the sperm-specific proteins, peptides,
cell surface molecules (glycopeptides, etc.) 304. Thereafter, the
second step is then used to capture the ligand 302 bound sperm 202
using an antibody 301.
[0060] Thereafter, steps 105-113 remain substantially the same,
with the sperm 202 being inspected and prepared for PCR-STR
analysis.
[0061] It is noted that standard bioconjugation chemistries are
available for attaching antibodies 208 on a substrate 205-207 which
can be glass or other materials like plastic. Antibody conjugation
on the substrate can be via utilization of common covalent or
non-covalent linkage or via adsorption.
[0062] Thus, all the approaches discussed herein can be envisioned
in other formats outside of immobilization on a solid support like
glass or plastic 206. This would include substrates of varying
geometries such as a flat substrate (in single or multi well
format) or a curved surface such as that of an Eppendorf tube.
[0063] In another embodiment, as shown in FIG. 3B(v), an
alternative apparatus may include using beads (i.e., silica,
magnetic, polystyrene beads) 305 coated with antibodies to bind
directly or indirectly to the sperm 202 for sperm capture.
[0064] Further, in yet another embodiment consistent with the
present invention as shown in FIG. 3B(vi), one can utilize the
strong affinity between Protein A 306 and Protein G 307 towards
binding antibodies 208 to provide an alternative form of the
invention. Therefore, in the embodiments discussed above with
respect to indirect methods of binding, instead of using secondary
antibodies 301 to recognize the primary antibodies 208 which are
sperm 202 bound, one can have beads 305 and/or Protein A 306 or
Protein G 307, recognize and capture the sperm 202 bound primary
antibodies 208.
[0065] Thereafter, steps 105-113 remain substantially the same,
with the sperm 202 being inspected and prepared for PCR based STR
analysis.
[0066] Holographic Optical Trapping
[0067] In another embodiment consistent with the present invention,
for forensic samples where an additional level of purity (in
sorting sperm, for example) is needed beyond antigen-antibody
binding based sorting or protein-ligand binding based sorting as
described above, HOT 400 may be utilized after performing steps
101-103 above (see FIG. 4).
[0068] The HOT apparatus is well known in the art, and is described
in detail in, for example, U.S. Pat. No. 6,055,106, to Grier et
al., which is herein incorporated by reference in its entirety.
[0069] Thus, HOT can be used in parallel with the antibody approach
described above, where a variety of cell specific markers,
especially those that recognize and bind human sperm, are present
for use and sorting. New and novel markers which are constantly
being discovered to target/identify various cell types including
epithelial cells can be used. A combination of antibodies might
also be used to separate the epithelial cells from objects/sperm in
this embodiment. This invention will resolve existing challenges in
incomplete separation of sperm DNA from epithelial cell DNA which
leads to coamplification of wanted and unwanted DNA. This in turn
leads to mixed STR profile generation rather than a unique STR
profile.
[0070] Specifically, using HOT is advantageous where very few
objects/sperm are present in the sample, and the dominant cell type
are epithelial cells (contaminants) from the vagina. In that case,
one can use the antibody approach described above (with or without
HOT) to first separate out the epithelial cells from sperm by
causing the epithelial cells to adhere to the epithelial cell
specific antibody coated substrate (i.e., glass slide or
microfluidic chip). In such a situation, the sperm will remain in
the supernatant and can be either directly used for cell lysis and
extraction.
[0071] Thus, after the antibody labeled sperm cells on the
substrate are bound, further sorting of non-adhering contaminants
(which might not have been fully removed via washing with buffer)
will be cleaned out using optical trapping, leaving behind only
sperm cells on the substrate. Thus, in this embodiment, HOT 400 is
used to sort the objects (i.e., sperm) in addition to, or
alternatively to, the antibody method, from contaminating cells in
a sample.
[0072] Thereafter, steps 105-113 remain the same as described in
FIG. 1.
[0073] Microfluidics Chip and Single-Cell PCR-STR Analysis
[0074] Note that in the previous embodiments, a microfluidic chip
207 may be used. The use of a microfluidic chip in sorting objects
is described in detail in copending application entitled "Methods
and Apparatus in Sorting Objects in DNA Analysis" filed Sep. 11,
2008, the contents of which are herein incorporated by reference in
their entirety.
[0075] As described therein, HOT 400 is used to sort objects such
as sperm, and taken with the antibody method described herein (see
FIGS. 2 and 4), and the use of a microfluidics chip 207 containing
an input chamber 401 and individual output chambers 402, the sperm
202 can be sorted into the individual chambers 402 using HOT. In
this embodiment, HOT is used to separate the sperm cells 202 in the
forensics sample 200, by visual (microscope or monitor) inspection,
from other contaminating cells 203, by moving the optically trapped
sperm 202 from one area of the microfluidic chip 207 into
individual chambers 402 on the same chip 207, for example.
[0076] Thereafter, PCR-based STR analysis is performed at a PCR 210
station to identify the person whose DNA signature matches that of
the sperm, similarly to steps 105-113 described above.
[0077] Conventionally, a significant number of cells were required
to get a reliable STR readout signal. However, with the gentle
method of HOT-based sperm separation and improved sensitivity, a
more reliable separation of sperm from contaminating non-sperm
cells can be performed, and one can scale down the sample
collection in terms of number of sperm cells needed for PCR-based
STR analysis from about 200 (as is required in conventional
methods), to a few cells, or even to the level of single cell
PCR--greatly increasing its efficiency (see chambers on chip). This
in turn offers all the advantages of single cell PCR in forensics
analysis which include the higher probablility of identifying
assailants in a multiple sexual assault case or in cases where very
few sperms are available for standard bulk PCR analysis.
[0078] Thus, single-cell PCR can be carried out on each individual
sperm in chambers, using standard PCR methods (lysing individual
sperm in individual chambers in a multi-champber chip, extracting
the DNA and simultaneously amplifying the DNA from single cells in
all chambers).
[0079] In particular, the chip is placed on a flatbed thermocycler,
where the extracted DNA is amplified by PCR using STR primers which
are commercially available for forensic cases. Alternatively, a
custom-designed appropriate primer can be used where forensic cases
are not involved. The number of thermocycles is increased to leave
enough DNA from the individual cells at the end of the PCR
cycling.
[0080] DNA extraction may be performed by centrifugation (post cell
lysis) or by attaching to magnetic beads, where the DNA is eluted
from the beads (magnetic or others) by changing the pH (altering
the charge on the DNA, i.e., to affecting binding of the DNA to the
beads).
[0081] Once PCR is completed, standard STR analysis can be carried
out. This involves running gels on the amplified DNA for STR
readouts where each PCR reaction corresponds to amplified DNA from
a single sperm. Commercially available instruments may be used for
this purpose.
[0082] Thus, in addition to standard PCR/STR analysis in tubes or
on plates, in one embodiment, the analysis can also be performed on
a single chip (on-chip PCR). In this embodiment, the sperm in
sample can be lysed in situ, or the sperm in each well or chamber
can by lysed and passed through a filter to separate the cell
debris from the DNA, and only allow DNA to proceed to the next
chamber, where PCR (bulk) is run (i.e., on DNA from several sperm).
The chamber is connected to an on-chip PCR device with the
capability of STR readout.
[0083] As stated above, a final STR report is then generated for
each individual sperm analyzed, and statistics generated on the STR
profile. The resulting data is matched with the CODIS database for
human identification if it is meant for forensic use, for
example.
[0084] The virtue of individual sperm (single cell) STR readout is
that, it:
[0085] (1) increases the likelihood of detecting multiple
assailants (if involved in a sexual assault case) based on the
statistical significance of the STR readout (well-established by
standard regulations); and
[0086] (2) it offers the ability to analyze those crime cases where
obtaining 200 sperm cells is a challenge due to the limited
availability of sample sperm cells, thus, enhancing the chance of
ruling out incriminated persons in a sexual assault case, for
example.
[0087] Thus, one can envision revolutionizing the nature and scope
of STR based forensics offering solutions to more cases where
sufficient sample collection posed a problem before. Analysis on
one or few cells will cut down on sample collection time as
well.
[0088] In rare cases, where allele dropout is a problem and an STR
profile (based on standard bulk PCR analysis) could not be matched
to the CODIS database (the dominant allele masking the other),
there is a greater likelihood of matching an STR signature as
obtained from single cell PCR to the database with relevant
statistical calculations, to arrive at the needed probability.
[0089] By analyzing STR on individual sperm cells and repeating the
analysis for a number of sperm cells from a given sample on a
one-by-one basis, one can now reliably solve sexual assault cases
where multiple assailants are involved such as in a gang-rape
crime. Thus, no deconvolving is needed in resolving STR profiles of
individuals from a cell-mixture set.
[0090] Further, single-cell PCR based forensics will offer the
ability to perform repeat measurements and more statistically
reliable data can be obtained in solving a crime case. Still
further, as described above In step 108, the primary and/or the
secondary antibodies maybe fluorescently labeled and the throughput
for automated sperm separation via HOT will be faster since image
recognition of fluorescent samples typically work faster due to
better contrast.
[0091] The methods proposed here are compatible with automation and
multiplexing--i.e., running multiple forensic sample analysis
thereby increasing throughput; and further, can be integrated with
robotics where multiple crime samples can be eluted, separated and
tested at the same time thereby increasing throughput. Coupling
with robotics includes additional advantages since it is platform
independent--i.e., can be carried out on glass slides (glass
coverslips) or in test tubes or in Eppendorfs or even in 96 well
format (given that there are now machines such as flat bed
thermocyclers that can carry out PCR in 96 well or higher well
formats, in situ PCR is possible using such separation).
[0092] This methodology is compatible with in situ PCR on forensic
samples. Therefore, all advantages of in situ PCR will be valid
such as:
[0093] (i) to reduce chance of contamination since in situ PCR will
involve fewer steps and avoid transfer of samples from one
container to another; and
[0094] (ii) cut down cost of such forensic analysis by limiting
supply cost that is likely involved with more steps and transfer of
samples.
[0095] The invention described herein is better than the commonly
used method of differential extraction which suffers from several
disadvantages such as mixing of male and female fractions and often
immature cell lysing. In addition, differential extraction which is
the widely practiced method for separating sperm from epithelial
cell DNA is labor intensive, time-consuming and lacks scope for
automation. The current invention circumvents these problems and
offers better quality and reliability in the separation process
prior to STR analysis.
[0096] While alternative techniques such as the use of a double
membrane filter in which a distinctly defined rigid pore size of
the filter was designed to allow DNA from digested epithelial cells
to pass through while trapping sperm (see Ladd Carl et al.,
"Development of a high throughput method to isolate sperm DNA in
sexual assault cases", August 2006), this method was argued to be
too harsh and caused immature lysing of sperm. Antibody-antigen
binding or protein-ligand binding based sperm sorting prevent the
harshness of mechanical separations.
[0097] Other alternate methods have used a similar approach where a
nylon mesh membrane was used instead of one with a rigid pore size.
Such membrane-based separations can be partially automated or offer
better speed than manually operated ones via coupling of a vacuum
pump to the system. However, the addition of the pump decreases the
resolution in separation--i.e., unwanted components from the mixed
cell sample can get sucked into the membrane separated portion.
[0098] Further, the present invention has the advantage that it can
be operated via direct microscopic visualization which offers
better control on the sample quality (purity).
[0099] Another method in the field of forensic sample analysis for
cell type separation is called laser capture micro-dissection.
While this method works better than differential extraction in
separating sperm from epithelial cells, it is expensive, requires
additional intermediate steps for cell fixation and works best when
cells are separated from one another and are in single layer rather
than when they are in clusters. Therefore, none of the existing
techniques/methods satisfy all the criteria for epithelial from
sperm cells separation. The invention described here either in its
stand alone mode or in conjunction with HOT can overcome the
pitfalls and difficulties of existing techniques and is amenable to
complete automation.
[0100] While the invention here is particularly described in the
context of solving sexual assault forensic cases (which comprise
about 2/3 of forensic cases (see reference, "Forensics DNA Typing",
2.sup.nd edition by James Butler), its application in the field of
forensics extends beyond that wherever there is a need for cell
type separation.
[0101] In other embodiments, since most cells have cell type
specific surface markers, it is possible to extend the antibody
based separation into areas outside of forensics such as in basic
research or in cancer diagnostics.
[0102] In the field of forensics, one extension of this invention
is the utilization of this antibody based cell separation method in
DNA-based parentage testing. Such testing is necessary to identify
the biological father of the embryo or fetus in the event of a
failed or aborted pregnancy which occurs frequently after sexual
assault. Fetal remains or aborted material is used as the source of
fetal DNA. However if recognizable fetal parts cannot be
confidently identified for parentage testing, microscopic
examination of fixed tissue from post-mortem samples or genetic
amniocentesis are the only ways to distinguish maternal (decidual)
vs. fetal (chorionic villi) components of the recovered products.
However, once again, conventionally, the cell separation is far
from being perfect. However, with the present invention,
antibody-based separation in conjunction with HOT could separate
the maternal and chorionic villi components before PCR amplication
of the extracted DNA. This approach offers the ability for direct
visualization of the samples wherever needed.
[0103] It should be emphasized that the above-described embodiments
of the invention are merely possible examples of implementations
set forth for a clear understanding of the principles of the
invention. Variations and modifications may be made to the
above-described embodiments of the invention without departing from
the spirit and principles of the invention. All such modifications
and variations are intended to be included herein within the scope
of the invention and protected by the following claims.
* * * * *