U.S. patent application number 14/658449 was filed with the patent office on 2016-02-18 for methods and systems for differential extraction.
The applicant listed for this patent is APPLIED BIOSYSTEMS, LLC. Invention is credited to Jason Yingjie LIU.
Application Number | 20160046927 14/658449 |
Document ID | / |
Family ID | 39970126 |
Filed Date | 2016-02-18 |
United States Patent
Application |
20160046927 |
Kind Code |
A1 |
LIU; Jason Yingjie |
February 18, 2016 |
METHODS AND SYSTEMS FOR DIFFERENTIAL EXTRACTION
Abstract
Methods are provided for differential extraction of DNA from at
least two different cell types. Systems for carrying out the
differential extraction methods are also provided. A kit is also
provided for differential extraction of DNA from at least two
different cell types using a multi-compartment container.
Inventors: |
LIU; Jason Yingjie; (Foster
City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
APPLIED BIOSYSTEMS, LLC |
Carlsbad |
CA |
US |
|
|
Family ID: |
39970126 |
Appl. No.: |
14/658449 |
Filed: |
March 16, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12117579 |
May 8, 2008 |
8993292 |
|
|
14658449 |
|
|
|
|
60916999 |
May 9, 2007 |
|
|
|
Current U.S.
Class: |
435/306.1 |
Current CPC
Class: |
C12N 13/00 20130101;
C12N 1/066 20130101; C12M 41/46 20130101; C12N 15/1006
20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10 |
Claims
1. A system for differential extraction of sperm cells, comprising
a plurality of compartments, wherein at least one compartment
comprises a selective sperm lysis buffer.
2. The system of claim 1, comprising a first compartment comprising
a cell-trapping matrix and a second compartment comprising a
selective sperm lysis buffer.
3. The system of claim 2, further comprising a third. compartment
comprising a plurality of DNA-binding particles.
4. The system of claim 3, further comprising a fourth compartment
comprising an elution buffer.
5. The system of claim 1 comprising: a first compartment comprising
a cell-trapping matrix; a second compartment comprising a cell wash
buffer; a third compartment comprising a selective sperm lysis
buffer; a fourth compartment comprising a plurality of DNA binding
particles; a fifth compartment comprising a DNA wash buffer; and a
sixth compartment. comprising an elution buffer.
6. The system of claim 5, wherein the first, second, third, fourth,
fifth, and sixth compartments are part of a removable
cartridge.
7. The system of claim 5, further comprising a seventh compartment
comprising a DNA-binding buffer.
8. The system of claim 7, wherein the seventh compartment further
comprises a second plurality of DNA-binding particles.
9. The system of claim 7, further comprising an eighth compartment
comprising a second plurality of DNA-binding particles.
10.-27. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. patent application
Ser. No. 12/117,579 filed May 8, 2008, which claims the benefit of
U.S. Provisional Application No. 60/916,999 filed May 9, 2007. This
application is related to U.S. Provisional Application No.
60/880,787 filed Jan. 16, 2007, U.S. Provisional Application No.
60/899,106 filed Feb. 2, 2007, U.S. patent application Ser. No.
12/015,414 filed Jan. 16, 2008 and U.S. patent application Ser. No.
12/032,270 filed Feb. 15, 2008. The disclosures of the above
applications are incorporated by reference in their entirety.
[0002] All literature and similar materials cited in this
application, including but not limited to, patents, patent
applications, articles, books, treatises, and internet web pages,
regardless of the format of such literature and similar materials,
are expressly incorporated by reference in their entirety for any
purpose. In the event that one or more of the incorporated
literature and similar materials differs from or contradicts this
application, including but not limited to defined terms, term
usage, described techniques, or the like, this application
controls.
FIELD
[0003] Methods are provided for differential extraction of DNA from
at least two different cell types. Systems for carrying out the
differential extraction methods are also provided.
INTRODUCTION
[0004] Forensic DNA analysis of sexual assault evidence often
involves analysis of DNA from sperm cells and DNA from other cells
such as epithelial cells. The samples obtained from victims often
contain a mixture of sperm and other cells such as epithelial
cells. Because other cells such as epithelial cells may outnumber
sperm cells by many folds, contamination from one or more other
sources of DNA may occur while sperm DNA is being extracted.
Therefore, it is often desirable to separate the sperm cells and
epithelial cells, or separate the sperm DNA from the epithelial DNA
as much as possible, prior to analysis. In certain instances,
separation and isolation of a particular DNA to create an accurate
profile is important for identification of an assailant.
[0005] Differential extraction is a broad term used to describe
several extraction methods that can be used to separate cells. In
certain instances, unique characteristics of sperm cells allow for
the differential extraction of the epithelial cells from the sperm
cells. One differential extraction procedure was described in 1985
(Gill et al., (1985) Nature 318: 557-9). In certain instances,
separation of the sperm cell fraction from the victim's DNA profile
decreases ambiguity in the results and allows for easier
interpretation of the perpetrator's DNA profile in a rape case.
SUMMARY
[0006] In certain embodiments, a system for differential extraction
of sperm cells is provided. In certain embodiments, the system
comprises a plurality of compartments, wherein at least one
compartment comprises a selective sperm lysis buffer. In certain
embodiments, the system comprises a first compartment comprising a
cell-trapping matrix and a second compartment comprising a
selective sperm lysis buffer. In certain embodiments, the system
further comprises a third compartment comprising a plurality of
DNA-binding particles. In certain embodiments, the system further
comprises a fourth compartment comprising an elution buffer.
[0007] In certain embodiments, a system for differential extraction
of sperm cells comprises a first compartment comprising a
cell-trapping matrix; a second compartment comprising a cell wash
buffer, a third compartment comprising a selective sperm lysis
buffer; a fourth compartment comprising a plurality of DNA binding
particles; a fifth compartment comprising a DNA wash buffer; and a
sixth compartment comprising an elution buffer. In certain
embodiments, the system further comprises a seventh compartment
comprising a DNA-binding buffer. In certain embodiments, the
seventh compartment further comprises a second plurality of
DNA-binding particles. In certain embodiments, the system further
comprises an eighth compartment comprising a second plurality of
DNA-binding particles.
[0008] In certain embodiments, the system comprises a plurality of
compartment, wherein the plurality of compartments are part of a
removable cartridge.
[0009] In certain embodiments, a method of differential extraction
of sperm cells in a biological sample that comprises sperm cells
and non-sperm cells is provided. In certain embodiments, the method
comprises (a) placing the biological sample in a first compartment
of a system, wherein the first compartment comprises a
cell-trapping matrix; (b) capturing the sperm cells and the
non-sperm cells with the cell-trapping matrix; (c) incubating the
cell-trapping matrix and the captured sperm cells and non-sperm
cells in a selective sperm lysis buffer to form a sperm cell
lysate; (d) binding sperm cell DNA from the sperm cell lysate to a
plurality of DNA-binding particles; and (e) eluting the sperm cell
DNA from the DNA-binding particles.
[0010] In certain embodiments, capturing the sperm cells and the
non-sperm cells with the cell-trapping matrix comprises applying a
magnetic force to the cell-trapping matrix. In certain embodiments,
incubating the cell-trapping matrix and the captured sperm cells
and non-sperm cells in a selective sperm lysis buffer occurs in the
first compartment of the system. In certain embodiments, the
binding the sperm cell DNA to a plurality of DNA-binding particles
occurs in a second compartment of the system. In certain
embodiments, eluting the sperm cell DNA from the DNA-binding
particles occurs in the second compartment of the system. In
certain embodiments, the sperm cell lysate is moved from the first
compartment to the second compartment.
[0011] In certain embodiments, incubating the cell-trapping matrix
and the captured sperm cells and non-sperm cells in a selective
sperm lysis buffer occurs in a second compartment of the system. In
certain embodiments, binding the sperm cell DNA to a plurality of
DNA-binding particles occurs in a second compartment of the system.
In certain embodiments, eluting the sperm cell DNA from the
DNA-binding particles occurs in a third compartment of the system.
In certain embodiments, the cell-trapping matrix and the captured
sperm cells and non-sperm cells are moved from the first
compartment to the second compartment using a magnetic force.
[0012] In certain embodiments, the non-sperm cells remain captured
on the cell-trapping matrix after incubating the cell-trapping
matrix in the selective sperm lysis buffer. In certain embodiments,
the non-sperm cells are lysed and the non-sperm cell DNA is bound
to the cell-trapping matrix. In certain embodiments, the non-sperm
cells are lysed and the non-sperm cell DNA is bound to a second
plurality of DNA-binding particles.
FIGURES
[0013] The skilled artisan will understand that the Figures,
described below, are for illustration purposes only. The drawings
are not intended to limit the scope of the claimed invention in any
way.
[0014] FIG. 1 shows an exemplary system for differential extraction
of sperm cell DNA and non-sperm cell DNA according to certain
embodiments. The system of FIG. 1 involves transfer of fluids
between compartments. The system of FIG. 1 does not lyse the
non-sperm cells, but instead collects residual DNA present in the
sample after cell capture on the cell-trapping matrix.
[0015] FIG. 2 shows an exemplary system for differential extraction
of sperm cell DNA and non-sperm cell DNA according to certain
embodiments. The system of FIG. 2 involves transfer of fluids
between compartments. The system of FIG. 2 uses the cell-trapping
matrix as the DNA-binding particles for the non-sperm cell DNA.
[0016] FIG. 3 shows an exemplary system for differential extraction
of sperm cell DNA and non-sperm cell DNA according to certain
embodiments. The system of FIG. 3 involves transfer of fluids
between compartments. The system of FIG. 3 comprises two separate
compartments of DNA-binding particles.
[0017] FIG. 4 shows an exemplary system for differential extraction
of sperm cell DNA and non-sperm cell DNA according to certain
embodiments. The system of FIG. 4 involves the transfer of
particles and, in some instances, liquid between compartments. The
cell-trapping matrix is used as the DNA-binding matrix for the
non-sperm cell DNA in the system shown in FIG. 4.
[0018] FIG. 5 shows an exemplary system for differential extraction
of sperm cell DNA and non-sperm cell DNA according to certain
embodiments. The system of FIG. 5 involves the transfer of
particles between compartments. The cell-trapping matrix is used as
the DNA-binding matrix for the non-sperm cell DNA in the system
shown in FIG. 5.
[0019] FIG. 6 shows an exemplary system for differential extraction
of sperm cell DNA and non-sperm cell DNA according to certain
embodiments. The system of FIG. 6 stores the DNA-binding particles
separately from the DNA-binding buffer.
DESCRIPTION OF VARIOUS EMBODIMENTS
[0020] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited
herein, including but not limited to patents, patent applications,
articles, books, and treatises, are hereby expressly incorporated
by reference in their entirety for any purpose. In the event that
one or more of the incorporated documents or portions of documents
define a term that contradicts that term's definition in this
application, this application controls.
[0021] The use of the singular includes the plural unless
specifically stated otherwise. The word "a" or "an" means "at least
one" unless specifically stated otherwise. The use of "or" means
"and/or" unless stated otherwise. The use of "or" in the context of
multiply dependent claims means the alternative only. The meaning
of the phrase "at least one" is equivalent to the meaning of the
phrase "one or more." Furthermore, the use of the term "including,"
as well as other forms, such as "includes" and "included," is not
limiting. Also, terms such as "element" or "component" encompass
both elements or components comprising one unit and elements or
components that comprise more than one unit unless specifically
stated otherwise.
[0022] In this specification, discussion of detecting "a" moiety,
such as a target analyte, encompasses one or more of that moiety
unless specifically stated otherwise. All ranges discussed herein
include the endpoints and all values between the endpoints.
DEFINITIONS
[0023] The term "biological sample" refers to any sample that
contains at least one biological material. Exemplary biological
materials include, but are not limited to, blood, saliva, skin,
feces, urine, sperm cells, epithelial cells (including, but not
limited to, vaginal epithelial cells), muscle tissue, and bone.
[0024] The term "cartridge" refers to a system that comprises a
plurality of compartments and does not contain sufficient fluid
and/or magnetic particle handling mechanisms to function
independently of a separate fluid-handling and/or magnetic
particle-handling instrument. A cartridge may be, in certain
embodiments, designed for a single use, after which it is
discarded. In certain embodiments, one or more of the compartments
in a cartridge contains a reagent. In certain embodiments, all of
the compartments of a cartridge are contained in a single unit. In
certain embodiments, the compartments of a cartridge are divided
between two or more units that together form the cartridge. In
various embodiments, a single cartridge is designed to process 1,
2, 4, 6, 8, 12, 16, 24, 48, 96 or more than 96 samples. In various
embodiments, a cartridge is designed to process between 1 and 48
samples, or between 1 and 24 samples, or between 2 and 24 samples,
or between 1 and 16 samples, or between 2 and 16 samples. In
certain embodiments, when a cartridge is designed to process at
least two samples, it is designed to process at least two of the
samples simultaneously. In certain embodiments, a cartridge is
designed to process all of the samples simultaneously.
[0025] The term "cell mixture" refers to a heterogeneous collection
of at least two or more different cell types.
[0026] The term "cell-trapping matrix" refers to a matrix that
captures cells, including but not limited to, sperm cells and
epithelial cells. Certain exemplary cell trapping matrices are
described, e.g., in U.S. Provisional Application No. 60/890,460. In
certain embodiments, a cell-trapping matrix captures cells but does
not bind DNA in the presence of a cell wash buffer, but is capable
of binding DNA in a DNA-binding buffer.
[0027] The term "cell wash buffer" refers to a buffer in which
cells are captured by a cell-trapping matrix but are not lysed. In
certain embodiments, DNA does not bind to a cell-trapping matrix in
the presence of a cell wash buffer. Exemplary cell wash buffers
include, but are not limited to, phosphate buffered saline (PBS);
Tris-EDTA (TE), pH 7.5; Tris-Acetate-EDTA (TAE), pH 8.5); and
Tris-Boric acid-EDTA (TBE), pH 8. In various embodiments, one
skilled in the art can select a suitable cell wash buffer according
to the selected cell-trapping matrix and cell types.
[0028] The term "compartment" refers to any containment structure
that defines a discrete space configured to hold fluid. For
example, a compartment may be a stand-alone container or receptacle
that defines an interior space configured to hold fluid.
Alternatively, a compartment may be one of a plurality of
partitioned spaces within a container or receptacle, which is
configured to hold fluid. In addition, the fluid-holding space
defined by a compartment may be substantially enclosed or
alternatively, open, at least partially, to atmosphere.
[0029] The term "differential extraction" refers to extraction
methods utilized to extract a subset of cell types from a
heterogeneous population of cells. In certain embodiments,
differential extraction includes the selective lysis of sperm cells
in a mixture of sperm cells and non-sperm cells, including, but not
limited to, epithelial cells.
[0030] The term "DNA-binding particles" refers to magnetic
particles that are capable of binding DNA under appropriate buffer
conditions. Exemplary magnetic particles include, but are not
limited to, ferromagnetic, paramagnetic, and superparamagnetic
particles. In certain embodiments, DNA-binding particles bind DNA
in the presence of a DNA-binding buffer.
[0031] The term "DNA-binding buffer" refers to a buffer in which
DNA-binding particles and/or a cell-trapping matrix is capable of
binding DNA.
[0032] The term "selective sperm lysis buffer" refers to a buffer
that is capable of preferentially lysing sperm cells in a mixture
comprising sperm cells and at least one type of non-sperm cells.
Certain exemplary selective sperm lysis buffers are described,
e.g., in U.S. Provisional Application Nos. 60/899,106 and
60/890,470. "Preferentially lysing sperm cells" means that
primarily sperm cells are lysed. In certain embodiments, a
negligible amount of non-sperm cells are lysed. In various
embodiments, at least 80%, 85%, 90%, 95%, or 99% of the sperm cells
are lysed. In various embodiments, at least 80%, 85%, 90%, or 95%
of the non-sperm cells are not lysed.
[0033] The term "dilution buffer" refers to a buffer that can be
used to dilute a selective sperm lysis buffer so that it becomes a
DNA binding buffer. Dilution buffers may comprise, in various
embodiments, a chaotropic salt, a monovalent salt, and/or an
alcohol.
[0034] The term "elution buffer" refers to a buffer that releases
DNA from DNA-binding particles and/or cell-trapping matrix. Certain
exemplary elution buffers include, but are not limited to, low-salt
buffers (including, but not limited to, TE and deionized water). In
various embodiments, one skilled in the art can select a suitable
elution buffer according to the DNA binding particles and/or
cell-trapping matrix being used. In certain embodiments, heat is
applied to facilitate elution of DNA in the presence of an elution
buffer.
[0035] The term "forensic sample" refers to a biological sample
obtained for use to address identity issues arising in legal
contexts, including, but not limited to murder, rape, trauma,
assault, battery, theft, burglary, other criminal matters,
identity, parental or paternity testing, and mixed-up samples.
[0036] The term "general lysis buffer" refers to a buffer that
lyses non-sperm cells and may or may not also lyse sperm cells.
Certain exemplary general lysis buffers are known in the art, and
in various embodiments, one skilled in the art can select a general
lysis buffer based on the intended use. A nonlimiting exemplary
general lysis buffer comprises 2% SDS, 20 mM EDTA, 200 mM NaCl, 20
mM Tris (pH 8), and 500 .mu.g/mL proteinase K.
[0037] The term "lysate" refers to a liquid phase with lysed cell
debris and DNA.
[0038] The term "medical sample" refers to a sample obtained to
address medical issues including, but not limited to research,
diagnosis, and tissue and organ transplants.
[0039] The terms "salt" or "salt reagent" or "salt solution" refer
to positively and/or negatively charged ionic reagents. In certain
embodiments, a salt reagent disrupts sperm chromatin. A salt
reagent may, in various embodiments, be a monovalent, bivalent, or
multivalent ion. Exemplary salt reagents include, but are not
limited to LiCl, NaCl, KCl, Li.sub.2SO.sub.4, Na.sub.2SO.sub.4,
K.sub.2SO.sub.4, MgCl.sub.2, CaCl.sub.2, MgSO.sub.4, CaSO.sub.4,
NaNO.sub.3, KNO.sub.3, Mg(NO.sub.3).sub.2, and
Ca(NO.sub.3).sub.2.
[0040] The term "disulfide bond reducing agent" refers to an agent
that reduces disulfide bonds, e.g., in proteins. In certain
embodiments, a disulfide bond reducing agent disrupts protamine
disulfide bridges in sperm cells. Disulfide bond reducing agents
can be water-insoluble or water soluble. Exemplary water-insoluble
agents include, but are not limited to, dithiothreitol (DTT) and
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). Exemplary
water-soluble agents include, but are not limited to, glutathione
(GSH) and mercaptoethanol (ME).
Certain Exemplary Selective Sperm Lysis Buffers
[0041] In certain embodiments, a selective sperm lysis buffer
comprises at least one disulfide bond reducing reagent and at least
one salt reagent.
[0042] In certain embodiment, a disulfide bond reducing agent is
selected from ME, DTT, GSH, and TCEP.
[0043] In certain embodiments, at least one salt reagent is
selected from LiCl, NaCl, KCl. Li.sub.2SO.sub.4, Na.sub.2SO.sub.4,
K.sub.2SO.sub.4, MgCl.sub.2, CaCl.sub.2, MgSO.sub.4, CaSO.sub.4,
NaNO.sub.3, KNO.sub.3, Mg(NO.sub.3).sub.2, and Ca(NO.sub.3).sub.2.
In certain embodiments, at least one salt reagent is selected from
NaCl, KCl, and MgCl.sub.2.
[0044] In certain embodiments, a selective sperm lysis buffer
comprises at least one salt reagent at a concentration of at least
0.1M, 0.25M, 0.5M, 1M, 1.5M, or 2M. In certain embodiments, a
selective sperm lysis buffer comprises at least one salt reagent at
a concentration of between 0.1M and 2M.
[0045] In certain embodiments, a selective sperm lysis buffer
comprises at least one disulfide bond reducing reagent at a
concentration of at least 0.01M, 0.05M, 0.1M, 0.2M, 0.3M, 0.4M,
0.5M, 0.7M or 0.8M.
[0046] In certain embodiments, a selective sperm lysis buffer
comprises at least one disulfide bond reducing reagent selected
from ME, DTT, GSH, and TCEP and at least one salt reagent selected
from NaCl, KCl, MgCl.sub.2, and CaCl.sub.2. In certain embodiments,
the salt and reducing agent concentrations are such that the
selective sperm lysis buffer will preferentially lyse sperm
cells.
[0047] In certain embodiments, a selective sperm lysis buffer
comprises NaCl. In certain embodiments, the NaCl concentration is
at least 0.8M. In certain embodiments, a selective sperm lysis
buffer comprises KCl. In certain embodiments, the KCl concentration
is at least 0.8M. In certain embodiments, a selective sperm lysis
buffer comprises MgCl.sub.2. In certain embodiments, the MgCl.sub.2
concentration is at least 0.25M. In certain embodiments, a
selective sperm lysis buffer comprises DTT at a concentration of at
least 50 mM.
[0048] In various embodiments, one of skill in the art can optimize
the final salt concentration level to preferentially lyse sperm
cells.
[0049] In certain embodiments, a selective sperm lysis buffer is
diluted with a dilution buffer so that it becomes a DNA-binding
buffer. In certain embodiments, a selective sperm lysis buffer is
diluted with a DNA-binding buffer and the resulting buffer is a
DNA-binding buffer. In various embodiments, one skilled in the art
can select an appropriate dilution buffer and/or appropriate
dilution amount in order to create a DNA-binding buffer or maintain
the DNA-binding properties of a DNA binding buffer after mixing
with a selective sperm lysis buffer.
Certain Exemplary Cell-Trapping Matrices and DNA-Binding
Particles
[0050] A cell-trapping matrix is a matrix that captures cells,
including but not limited to, sperm cells and epithelial cells. In
certain embodiments, a cell-trapping matrix binds DNA under
appropriate conditions, which conditions may be the same as, or
different from, the conditions used to capture cells. In various
embodiments, a cell-trapping matrix is comprised of magnetic
particles. Exemplary magnetic particles include, but are not
limited to, ferromagnetic, paramagnetic, and superparamagnetic
particles.
[0051] Exemplary cell-trapping matrices and/or DNA-binding
particles include, but are not limited to, porous silica beads with
supermagnetic cores. Exemplary porous silica beads with
supermagnetic cores include, but are not limited to, MP-50 (6.5
.mu.m) and MP-85 (>8 .mu.m) (W. R. Grace, Columbia, Md.); DNA
IQ.TM. silica particles (Promega, Madison, Wis.); MagPrep.RTM.
silica particles (Novagen, San Diego, Calif.); BcMag.RTM.
silica-modified magnetic beads (5 .mu.m or 1 .mu.m) (Bioclone Inc.,
San Diego, Calif.); and supermagnetic silica particles (1 .mu.m or
0.75 .mu.m, G. Kisker GbR, Steinfurt, Germany). Certain exemplary
non-silica cell-trapping matrices include, but are not limited to,
iron oxide immobilized with streptavidin (Sigma, St. Louis, Mo.),
iron(ill) oxide powder (5 .mu.m) (Sigma), MagMAX.RTM. magnetic
particles (1 .mu.m, Applied Biosystems, Foster City, Calif.); and
Dynabeads.RTM. (Invitrogen, Carlsbad, Calif.), which may comprise
different types of surface functional groups (e.g., Dynabeads.RTM.
MyOne carboxylic acid beads, Dynabeads.RTM. WCX, Dynabeads.RTM.
TALON, and Dynabeads.RTM. MyOne tosylactivated).
Certain Exemplary Methods of Cell Capture
[0052] In various embodiments, a cell-trapping matrix comprises
magnetic particles. In certain embodiments, the cell-trapping
matrix captures cells in the absence of a magnetic field. In
certain embodiments, the cell-trapping matrix captures cells in the
presence of a magnetic field. The timing and mechanism of cell
capture depends, in various embodiments, on the cell-trapping
matrix used and the buffer conditions.
[0053] A cell trapping matrix may capture cells by any of a variety
of mechanisms. In certain embodiments, a cell-trapping matrix
captures cells through a non-covalent interaction. Certain
exemplary non-covalent interactions include, but are not limited
to, hydrogen bonding, cation-.PI. interactions, .PI.-.PI.
interactions, ionic pairing, hydrophobic interactions,
dipole-dipole interactions, dipole-induced dipole interactions,
charge-dipole interactions, and van Der Waals interactions. In
certain embodiments, cells are captured by the cell-trapping matrix
through ionic interactions. In certain embodiments, cells are
captured by the cell-trapping matrix through an antibody/antigen
interaction.
[0054] In certain embodiments, the duration and strength of the
non-covalent interaction is such that cells remain captured by the
cell-trapping matrix while the matrix is moved from one location to
another. In certain such embodiments, the cell-trapping matrix
comprises magnetic particles and the movement is caused by applying
a magnetic field. The type, duration and strength of a non-covalent
association between cells and a cell-trapping matrix is determined,
in various embodiments, by the size, shape, surface properties,
surface morphologies, and/or density of the matrix; the size,
shape, surface properties, surface morphologies, and/or density of
the captured cells; and/or the composition, pH, and/or temperature
of the buffer.
[0055] In certain embodiments, a cell-trapping matrix physically
traps cells, e.g., when a magnetic field is applied. Such physical
trapping may be, in certain embodiments, due to aggregation of
cell-trapping matrix particles in the magnetic field. In certain
embodiments, a cell-trapping matrix comprises irregularly shaped
particles to facilitate such physical trapping of cells. In various
embodiments, the cell-trapping matrix particles may be smaller or
larger than the cells to be captured, or may be larger than some
cells and smaller than other cells to be captured. In certain
embodiments, the cell-trapping matrix may capture cells by pushing
the cells towards the source of the magnetic field, sequestering
them from the supernatant. In certain such embodiments, the
cell-trapping matrix is comprised of particles at a density such
that the spacing between particles is smaller than the size of the
smallest cell in the sample.
[0056] In certain embodiments, more than one type of non-covalent
interaction exists between cells and a cell-trapping matrix. In
certain such embodiments, one type of non-covalent interaction may
contribute more to cell capture, and which interaction predominates
may vary from cell type to cell type in a sample.
[0057] Sperm cells have a diameter of about 5 .mu.m, epithelial
cells have a diameter of about 50 .mu.m. In various embodiments, a
cell-trapping matrix used to capture sperm cells and epithelial
cells comprises particles with a diameter of between 0.5 .mu.m and
100 .mu.m. In certain embodiments, the particles have a diameter of
between 1 .mu.m and 10 .mu.m. In certain embodiments, at least 85%,
at least 90%, at least 95%, or at least 99% of the particles in a
cell-trapping matrix have a diameter of between 0.5 .mu.m and 100
.mu.m, or between 1 .mu.m and 10 .mu.m. One skilled in the art can,
in various embodiments, select the appropriate particles size,
shape, density and surface properties for capturing cells according
to the particular application.
[0058] In certain embodiments, a cell-trapping matrix comprises one
or more antibodies. In certain such embodiments, one or more of the
antibodies binds a cell surface antigen present on at least one
type of cell in a sample. In certain embodiments, a cell-trapping
matrix comprises one or more antibodies that together bind to at
least one cell surface antigen on two or more cell types in a
sample. As a non-limiting example, in certain embodiments, a
cell-trapping matrix may comprise a first set of particles coated
with an antibody that binds a cell-surface antigen on epithelial
cells and a second set of particles coated with an antibody that
binds a cell-surface antigen on sperm cells.
[0059] Certain exemplary antibodies that bind sperm cells and/or
epithelial cells include, but are not limited to, monoclonal
antibody BerEP4, which binds the human epithelial antigen, EpCAM
(epithelial cell adhesion molecule); antibodies to sperm protamine;
antibodies to carbohydrate epitope located on human sperm
agglutination antigen-l (SAGA-l) (see, e.g., U.S. Pat. No.
5,605,803); antibodies to SPAN-X, a sperm protein present in
nuclear vacuoles and sperm nuclear redundant membranes (see, e.g.,
PCT/US99/24973); antibodies to C58 or SMARC32 (see, e.g., U.S.
Published App. 2002/0182751). In certain embodiments, an antibody
binds a cell surface antigen (e.g., a protein or carbohydrate)
present on multiple cell types.
[0060] In certain embodiments, cells are pushed, dragged, or
carried by the cell-trapping matrix to the bottom of a compartment,
when a magnetic field is applied underneath the compartment. In
certain embodiments, cells are pushed, dragged, or carried by the
cell-trapping matrix to the side of the compartment, when a
magnetic field is applied from the side of the compartment. In
certain embodiments, cells are pushed, dragged, or carried by the
cell-trapping matrix to a magnetic bar inserted into the
compartment. In certain such embodiments, the magnetic bar is
covered, e.g., with a removable protectant to prevent contamination
of the magnetic bar. In certain embodiments, the magnetic bar can
be used to transfer the cell-trapping matrix from one compartment
to another.
[0061] In certain embodiments, Dynabeads.RTM. MyOne Carboxylic Acid
beads (Dynal Biotech) are used as a cell-trapping matrix to capture
cells. The particles are 1 .mu.m and are used, in certain
embodiments, at a density of about 10.sup.6 particles/pl for a
sample comprising about 55 cells/.mu.L (e.g., 200 .mu.L containing
about 1000 sperm cells and about 10,000 epithelial cells). In
certain embodiments, more than 10.sup.6 particles/pi can be used.
In certain embodiments, fewer than 10.sup.6 particles/.mu.l are
used.
[0062] In various embodiments, at least 50%, at least 60%, at least
70%, at least 80%, at least 90%, or at least 95% of the intact
cells in a sample are captured by the cell-trapping matrix. In
various embodiments, after one cell type has been lysed, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, or at
least 95% of the remaining intact cells in a sample are captured,
or remain captured, by the cell-trapping matrix.
Certain Exemplary DNA-Binding Buffers
[0063] In certain embodiments, when the DNA-binding particles are
silica beads, a DNA-binding buffer comprises a chaotropic salt.
Certain exemplary chaotropic salts include, but are not limited to,
guanidium isothiocyanate (GuSCN) and guanidium chloride. In certain
embodiments, e.g., when the DNA-binding particles are non-silica
beads, a DNA-binding buffer comprises a chaotropic salt and
alcohol. In certain such embodiments, the DNA-binding buffer
further comprises a monovalent salt. Certain exemplary monovalent
salts include, but are not limited to, sodium acetate, sodium
chloride, potassium acetate, potassium chloride, ammonium acetate,
and ammonium chloride. Certain exemplary alcohols include, but are
not limited to, isopropanol and ethanol. In certain embodiments,
the alcohol is at a concentration of 30% or greater in the
DNA-binding buffer. In certain embodiments, a DNA-binding buffer
that comprises a chaotropic salt and an alcohol is referred to as a
"DNA precipitation buffer." Certain exemplary DNA-binding buffers
are described, e.g., in U.S. Pat. Nos. 5,234,809; 5,523,231; and
5,705,628. One skilled in the art can select a suitable DNA-binding
buffer according to the DNA binding particles and/or cell-trapping
matrix being used.
[0064] Exemplary DNA-binding buffers suitable for silica-based
DNA-binding particles include, but are not limited to,
BloodPrep.TM. DNA purification solution (Applied Biosystems) and
DNA IQ.TM. Lysis Solution (Promega). In various embodiments, for
non-silica DNA-binding particles, one skilled in the art can add a
suitable amount of an alcohol to the BloodPrep.TM. DNA purification
solution (Applied Biosystems) or the DNA IQ.TM. Lysis Solution
(Promega). As a non-limiting example, in various embodiments,
isopropanol is added to one of those solutions to a final
concentration of 30% to create a DNA precipitation buffer. As a
further non-limiting example, in various embodiments, ethanol is
added to one of those solutions to a final concentration of 40-50%
to create a DNA precipitation buffer.
[0065] In certain embodiments, DNA-binding particles are stored in
DNA binding buffer in the systems described herein. In certain
embodiments, DNA-binding particles are stored separately from a
DNA-binding buffer in the systems described herein. DNA-binding
particles are stored separately, in certain embodiments, when the
DNA binding buffer is a DNA precipitation buffer.
[0066] In certain embodiments, non-sperm cells are lysed in a
DNA-binding buffer. In certain embodiments, non-sperm cells are
lysed in a DNA-binding buffer with the application of heat. In
certain embodiments, when a DNA-binding buffer is a DNA
precipitation buffer, non-sperm cells are first lysed in a
DNA-binding buffer without the alcohol. In certain embodiments,
alcohol is then added to the lysate to create the DNA precipitation
buffer.
Certain Exemplary DNA Wash Buffers
[0067] In certain embodiments, a DNA wash buffers is formulated
such that substantially no genomic DNA dissolves in the DNA wash
buffer. In certain embodiments, a DNA wash buffers is formulated
such that substantially no large DNA (e.g., greater than about 1
kb) dissolves in the DNA wash buffer. Certain exemplary DNA wash
buffers are known in the art and generally comprise at least one
alcohol. Certain exemplary alcohols that may be used in DNA wash
buffers include, but are not limited to, ethanol and
isopropanol.
[0068] In certain embodiments, when one DNA wash buffer is used, it
comprises 90% ethanol. In certain embodiments, when the DNA is
washed twice, the same DNA wash buffer is used for both washed. In
certain embodiments, when the DNA is washed twice, two different
DNA wash buffers are used. In certain embodiments, when two
different DNA wash buffers are used, the second DNA wash buffer
comprises a higher alcohol concentration than the first DNA wash
buffer. In certain embodiments, a DNA wash buffer comprises GuSCN
and/or GuCl. In certain embodiments, a DNA wash buffer comprises
GuSCN and isopropanol. In certain embodiments, a DNA wash buffer
comprises 70% ethanol. In certain embodiments, a DNA wash buffer
comprises 90% ethanol.
[0069] In various embodiments, one skilled in the art can formulate
one or more DNA wash buffers according to the selected
application.
Certain Exemplary Methods of DNA Analysis
[0070] After the DNA has been isolated, various methods can be used
for DNA analysis, such as Restriction Fragment Length Polymorphism
(RFLP) analysis, and various Polymerase Chain Reaction (PCR)-based
methods, including, but not limited to, Short Tandem Repeat (STR)
analysis.
[0071] Polymerase Chain Reaction (PCR) refers to a reaction that
can be used to amplify nucleic acids, including, but not limited
to, small amounts of DNA. PCR is a technique in which cycles of
denaturation, annealing with one or more primers, and extension
with one or more DNA polymerases, are used to create additional
copies of a target DNA. In certain embodiments, PCR amplifies the
DNA sequence by more than 10.sup.6 fold. Certain exemplary methods
of PCR are described, e.g., in U.S. Pat. Nos. 4,683,195; 4,965,188;
and 4,683,202; and European Patent Nos. EP201184 and EP200362.
[0072] In certain embodiments, DNA samples are subjected to PCR
amplification using primers specific for each locus that contains,
e.g., an STR of interest. An STR locus is composed of tandemly
repeated sequences, each of which is, e.g., 2 to 7 bp in length. In
various embodiments, loci containing 4 bp (tetranucleotide) and/or
5 bp repeat sequences are used for human identification. Four and 5
bp repeat sequences are found throughout the human genome and are,
in certain instances, highly polymorphic. The number of alleles at
a tetranucleotide repeat STR locus ranges, in various embodiments,
from about 4 to 20.
[0073] In certain embodiments, when isolated DNA is used for
detection of polymorphic STRs, the amplified alleles from the
individual DNA samples can be compared to one or more size
standards, e.g., commercial DNA markers and/or locus-specific
allelic ladders, to determine the alleles present at each locus. In
certain embodiments, allelic ladders comprise two or more distinct
lengths of DNA representing two or more known alleles from a
particular locus. In various embodiments, DNA may be visualized by
any technique, including, but not limited to, silver staining,
radioactive labeling, fluorescent labeling, various dyes and
stains. In certain embodiments, prior to visualization, DNA is
separated using denaturing or native gel electrophoresis, or any
other size separation method.
[0074] In certain embodiments, amplified alleles are subjected to
DNA sequence analysis.
[0075] Certain exemplary methods of DNA amplification and analysis
are known in the art and are described, e.g., in Budowle et al.
(DNA Typing Protocols: Molecular Biology and Forensic Analysis,
Eaton Publishing: Mass., USA (2000)).
Certain Exemplary Magnetic Particle Handling Instruments
[0076] In certain embodiments, a differential extraction system
comprises a magnetic particle handling instrument and a removable
cartridge comprising a plurality of compartments, wherein at least
one of the compartments comprises at least one reagent used in the
differential extraction system.
[0077] In certain embodiments, a magnetic particle handling
instrument is similar to the BioRobot EZ1 Workstation (Qiagen).
See, e.g., EZ1 DNA Handbook, Second Edition (February 2004)
(Qiagen). The BioRobot EZ1 Workstation comprises a stage capable of
moving in a plane parallel to the floor, a pipette capable of
moving upwards and downwards relative to the stage, and a magnet
capable of moving towards and away from the pipette. The BioRobot
EZ1 Workstation is able to move both magnetic particles and liquids
between compartments. In certain instances, the BioRobot EZ1
Workstation functions as follows. A removable cartridge containing
a plurality of compartments comprising a plurality of liquid
reagents is placed on the stage of the BioRobot EZ1 Workstation.
The removable cartridge also comprises at least one compartment
containing magnetic particles. The stage moves the compartment
containing the magnetic particles to a position beneath the
pipette. The pipette lowers into the compartment and sucks up a
liquid solution containing the magnetic particles. The magnet moves
toward the pipette to hold the magnetic particles inside the
pipette while the pipette expels the liquid solution without the
magnetic particles. The stage then moves a second compartment
containing a reagent to a position beneath the pipette containing
the magnetic particles held by the magnet. The pipette sucks up the
reagent from the second compartment, the magnet moves away from the
pipette and the pipette expels the reagent and the magnetic
particles into the second compartment.
[0078] In certain embodiments, a magnetic particle handling
instrument is similar to the Maxwell 16 Instrument (Promega). See,
e.g., Maxwell 16 Instrument Operating Manual (September 2005)
(Promega). The Maxwell 16 Instrument comprises a stage capable of
moving in a plane parallel to the floor, a plunger capable of
moving upwards and downwards relative to the stage, and a magnet
located inside of the plunger and capable of moving upwards and
downwards relative to the end of the plunger. The Maxwell 16
Instrument is able to move magnetic particles, but not liquids,
between compartments. In certain instances, the Maxwell 16
Instrument functions as follows. A removable cartridge containing a
plurality of compartments comprising a plurality of liquid reagents
is placed on the stage of the Maxwell 16 Instrument. The removable
cartridge also comprises at least one compartment containing
magnetic particles. The stage moves the compartment containing the
magnetic particles to a position beneath the plunger. The plunger
pipette lowers into the compartment and the magnet lowers within
the plunger to attract the magnetic particles to the end of the
plunger. The plunger and magnet then rise, and the stage moves a
second compartment containing a reagent to a position beneath the
plunger with the magnetic particles held by the magnet. The plunger
lowers into the second compartment and the magnet rises away from
the end of the plunger, releasing the magnetic particles into the
second compartment.
[0079] In certain embodiments, a magnetic particle handling
instrument comprises a stage capable of moving in a plane parallel
to the floor, a pipette capable of moving upwards and downwards
relative to the stage, and a magnet in a location allowing it to
contact a cartridge placed on the stage. In various embodiments, a
magnet may be located beneath the cartridge or on one or multiple
sides of the cartridge. In certain embodiments, the magnet is
capable of moving towards and away from the cartridge in order to
exert and release a magnetic force on at least one compartment of
the cartridge. In certain embodiments, the magnet is stationery and
can be turned on and off electronically in order to exert and
release a magnetic force on at least one compartment of the
cartridge. Such an exemplary magnetic particle handling instrument
functions as follows. A removable cartridge containing a plurality
of compartments comprising a plurality of liquid reagents is placed
on the stage of the instrument. The removable cartridge also
comprises at least one compartment containing magnetic particles.
The stage moves a compartment containing a reagent to a position
beneath the pipette. The pipette sucks up the reagent and the stage
moves the compartment containing the magnetic particles to a
position beneath the pipette. The pipette then expels the reagent
into the compartment. The magnet attracts the magnetic particles to
one location within the compartment, away from the pipette, and the
pipette then sucks up the liquid without the magnetic particles. In
this manner, the pipette can be used to bring reagents to the
magnetic particles and remove reagents from the magnetic particles,
while a magnet is used to hold the magnetic particles out of the
way of the pipette.
[0080] In various embodiments, one skilled in the art can design
and/or program an instrument to carry out the methods described
herein. In certain embodiments, such an instrument may be similar
to one or more of the instruments described above.
Certain Exemplary Systems and Methods for Differential
Extraction
[0081] In various embodiments, a system for differential extraction
comprises a cartridge comprising a plurality of compartments,
wherein at least one of the compartments comprises a selective
sperm lysis buffer. Certain exemplary systems and methods for
differential extraction are described herein. In certain
embodiments, the method comprises transferring fluids between
compartments. In certain embodiments, the method comprises
transferring DNA-binding particles between compartments. In certain
embodiments, the method comprises transferring fluids and
DNA-binding particles between compartments.
[0082] The following examples of differential extraction systems
are non-limiting. For example, the arrangement of compartments
shown in the Figures and described herein is figurative and
non-limiting. In various embodiments one skilled in the art can
design a system based on the teachings herein, the intended use,
and the selected instrumentation. In various embodiments, such a
system comprises more or fewer compartments than any of the
following exemplary systems described herein. In various
embodiments, such a system separates reagents into separate
compartments that are shown combined in one or more of the
following exemplary systems.
Certain Exemplary Systems and Methods Using Fluid Transfer Between
Compartments
[0083] FIG. 1 shows a first non-limiting exemplary system for
differential extraction of sperm cells and non-sperm cells, e.g.,
epithelial cells, according to certain embodiments. The system
shown in FIG. 1 is used, for example, when a sample comprises an
excess of non-sperm cells relative to sperm cells (e.g., at least
10-fold more non-sperm cells than sperm cells). The system of FIG.
1 does not lyse the non-sperm cells, but instead collects residual
DNA present in the sample after cell capture on the cell-trapping
matrix. Such residual DNA may be present due to cell breakage prior
to placing the sample in the system. Such cell breakage may occur,
in various embodiments, prior to collection, during collection,
during storage, during transport, and/or during transfer of the
sample from one container to another. Because of the excess of
non-sperm cells relative to sperm-cells in the sample, the residual
DNA comprises an excess of non-sperm cell DNA relative to sperm
cell DNA.
[0084] In embodiments depicted in FIG. 1, a sample comprising a
mixture of sperm cells and non-sperm cells is placed in compartment
1. Compartment 1 comprises a cell-trapping matrix, which captures
the sperm cells and the non-sperm cells. The supernatant from
compartment 1 is then removed to a collection vessel (not shown).
If the original sample contained an excess of non-sperm cells,
e.g., epithelial cells, relative to sperm cells (for example, more
than 10-fold more non-sperm cells than sperm cells), the
supernatant from the sample binding may contain sufficient
non-sperm cell DNA for analysis.
[0085] A cell wash buffer located in compartment 2 is then
transferred to compartment 1. The cell wash supernatant from
compartment 1 is then transferred to a waste receptacle, which is
shown as compartment 0. The waste receptacle may or may not be a
contiguous part of the system shown in FIG. 1. In certain
embodiments, when the compartments 1 through 6 of FIG. 1 are
contained in a cartridge, the waste compartment 0 is not part of
the cartridge. In certain embodiments, a cartridge comprises
compartments 0 through 6 of FIG. 1.
[0086] After removal of the cell wash supernatant from compartment
1, a selective sperm lysis buffer is transferred from compartment 3
to compartment 1. The selective sperm lysis buffer lyses the sperm
cells bound to the cell-trapping matrix in compartment 1. Following
lysis of the sperm cells, the lysate supernatant is transferred
from compartment 1 to compartment 4, which contains DNA-binding
particles and a dilution buffer, which, when combined with the
selective sperm lysis buffer, forms a DNA-binding buffer. Following
DNA binding, the supernatant from compartment 4 is transferred to
the waste compartment 0. A DNA wash buffer is then transferred from
compartment 5 to compartment 4. The DNA wash buffer supernatant is
then transferred from compartment 4 to the waste compartment 0.
[0087] In certain embodiments, a second DNA wash buffer is
contained in a compartment 7 (not shown in FIG. 1). In certain such
embodiments, the second DNA wash buffer is then transferred to
compartment 4. The second DNA wash buffer supernatant is then
transferred from compartment 4 to the waste compartment 0.
[0088] Finally, an elution buffer is transferred from compartment 6
to compartment 4. The elution buffer releases sperm cell DNA from
the DNA-binding particles into the elution supernatant. In certain
embodiments, the elution buffer is heated to facilitate elution of
the bound DNA. In certain embodiments, the elution supernatant may
then transferred from compartment 4 to a second collection vessel
(not shown in FIG. 1). The sperm cell DNA can then be subjected to
DNA analysis.
[0089] FIG. 2 shows a second non-limiting exemplary system for
differential extraction of sperm cells and non-sperm cells, e.g.,
epithelial cells, according to certain embodiments. In the system
shown in FIG. 2, the cell-trapping matrix also serves as the
DNA-binding matrix for the non-sperm cell DNA.
[0090] In embodiments depicted in FIG. 2, a sample comprising a
mixture of sperm cells and non-sperm cells is placed in compartment
1. Compartment 1 comprises a cell-trapping matrix, which captures
the sperm cells and the non-sperm cells. The supernatant from
compartment 1 is then transferred to a waste receptacle, which is
shown as compartment 0. The waste receptacle may or may not be a
contiguous part of the system shown in FIG. 2. In certain
embodiments, when the compartments 1 through 7 of FIG. 2 are
contained in a cartridge, the waste compartment 0 is not part of
the cartridge. In certain embodiments, a cartridge comprises
compartments 0 through 7 of FIG. 2.
[0091] A cell wash buffer located in compartment 2 is then
transferred to compartment 1. The cell wash supernatant from
compartment 1 is then transferred to a waste receptacle, which is
shown as compartment 0. After removal of the cell wash supernatant
from compartment 1, a selective sperm lysis buffer is transferred
from compartment 3 to compartment 1. The selective sperm lysis
buffer lyses the sperm cells captured by the cell-trapping matrix
in compartment 1. Following lysis of the sperm cells, the lysate
supernatant is transferred from compartment 1 to compartment 5,
which contains DNA-binding particles and a dilution buffer, which,
when combined with the selective sperm lysis buffer, forms a
DNA-binding buffer. A DNA binding buffer is then transferred from
compartment 4 to compartment 1, which now contains non-sperm cells
captured by the cell-trapping matrix. The DNA-binding buffer is
incubated with the cell-trapping matrix for five minutes with
heating to about 70.degree. C. (heating element not shown in FIG.
2) to lyse the non-sperm cells, e.g., epithelial cells. The heating
element is, in certain embodiments, part of a fluid handling or
magnetic particle handling instrument. Non-sperm cell DNA binds to
the cell-trapping matrix in the DNA binding buffer.
[0092] Following DNA binding in compartments 1 and 5, the
supernatants from compartments 1 and 5 are transferred to the waste
compartment 0. A DNA wash buffer is then transferred from
compartment 6 to each of compartments 1 and 5. The DNA wash buffer
supernatant is then transferred from compartments 1 and 5 to the
waste compartment 0.
[0093] In certain embodiments, a second DNA wash buffer is
contained in a compartment 8 (not shown in FIG. 2). In certain such
embodiments, the second DNA wash buffer is then transferred to
compartments 1 and 5. The second DNA wash buffer supernatant is
then transferred from compartments 1 and 5 to the waste compartment
0.
[0094] Finally, an elution buffer is transferred from compartment 7
to each of compartments 1 and 5. The elution buffer releases sperm
cell DNA from the DNA-binding particles into the elution
supernatant in compartment 5 and releases non-sperm cell DNA from
the DNA-binding particles into the elution supernatant in
compartment 1. In certain embodiments, the elution buffer is heated
to facilitate elution of the bound DNA. In certain embodiments, the
elution supernatant is then transferred from compartment 5 to a
collection vessel (not shown in FIG. 3). The sperm cell DNA can
then be subjected to DNA analysis. In certain embodiments, the
elution supernatant is then transferred from compartment 1 to a
second collection vessel (not shown in FIG. 3). The non-sperm cell
DNA can then be subjected to DNA analysis.
[0095] FIG. 3 shows a third non-limiting exemplary system for
differential extraction of sperm cells and non-sperm cells, e.g.,
epithelial cells, according to certain embodiments. The system
shown in FIG. 3 comprises two separate compartments containing
DNA-binding particles, one of which is used to bind sperm cell DNA
and one of which is used to bind non-sperm cell DNA.
[0096] In embodiments depicted in FIG. 3, a sample comprising a
mixture of sperm cells and non-sperm cells is placed in compartment
1. Compartment 1 comprises a cell-trapping matrix, which captures
the sperm cells and the non-sperm cells. The supernatant from
compartment 1 is then transferred to a waste receptacle, which is
shown as compartment 0. The waste receptacle may or may not be a
contiguous part of the system shown in FIG. 3. In certain
embodiments, when the compartments 1 through 7 of FIG. 3 are
contained in a cartridge, the waste compartment 0 is not part of
the cartridge. In certain embodiments, a cartridge comprises
compartments 0 through 7 of FIG. 3.
[0097] A cell wash buffer located in compartment 2 is then
transferred to compartment 1. The cell wash supernatant from
compartment 1 is then transferred to a waste receptacle, which is
shown as compartment 0. After removal of the cell wash supernatant
from compartment 1, a selective sperm lysis buffer is transferred
from compartment 3 to compartment 1. The selective sperm lysis
buffer lyses the sperm cells captured by the cell-trapping matrix
in compartment 1. Following lysis of the sperm cells, the lysate
supernatant is transferred from compartment 1 to compartment 5,
which contains DNA-binding particles and a dilution buffer, which,
when combined with the selective sperm lysis buffer, forms a
DNA-binding buffer. A DNA binding buffer is then transferred from
compartment 4 to compartment 1, which now contains non-sperm cells
captured by the cell-trapping matrix. While transferring the
DNA-binding buffer from compartment 4, in certain embodiments, the
DNA-binding particles are held in compartment 4 by a magnet on the
fluid handling instrument, to prevent the DNA-binding particles
from being removed with the DNA-binding buffer. The DNA-binding
buffer is incubated with the cell-trapping matrix for five minutes
with heating to about 70.degree. C. (heating element not shown in
FIG. 3) to lyse the non-sperm cells, e.g., epithelial cells. The
heating element is, in certain embodiments, part of a fluid
handling or particle handling instrument. The DNA-binding
buffer/lysate supernatant is then transferred from compartment 1
back to compartment 4, which contains DNA-binding particles.
[0098] Following DNA binding in compartments 4 and 5, the
supernatants from compartments 4 and 5 are transferred to the waste
compartment 0. A DNA wash buffer is then transferred from
compartment 6 to each of compartments 4 and 5. The DNA wash buffer
supernatant is then transferred from compartments 4 and 5 to the
waste compartment 0.
[0099] In certain embodiments, a second DNA wash buffer is
contained in a compartment 8 (not shown in FIG. 3). In certain such
embodiments, the second DNA wash buffer is then transferred to
compartments 4 and 5. The second DNA wash buffer supernatant is
then transferred from compartments 4 and 5 to the waste compartment
0.
[0100] Finally, an elution buffer is transferred from compartment 7
to each of compartments 4 and 5. The elution buffer releases sperm
cell DNA from the DNA-binding particles into the elution
supernatant in compartment 5 and releases non-sperm cell DNA from
the DNA-binding particles into the elution supernatant in
compartment 4. In certain embodiments, the elution buffer is heated
to facilitate elution of the bound DNA. In certain embodiments, the
elution supernatant may then transferred from compartment 5 to a
collection vessel (not shown in FIG. 3). The sperm cell DNA can
then be subjected to DNA analysis. In certain embodiments, the
elution supernatant may then transferred from compartment 4 to a
second collection vessel (not shown in FIG. 3). The non-sperm cell
DNA can then be subjected to DNA analysis.
[0101] In certain embodiments, a dilution buffer and/or a
DNA-binding buffer is kept separate from DNA-binding particles in a
system until the DNA-binding reaction is carried out. Keeping such
components separate may be desirable, in certain embodiments, when
the dilution buffer or DNA-binding buffer comprises alcohol. One
skilled in the art can modify any of the systems of FIGS. 1 to 3 to
keep the DNA-binding particles separate from the dilution and/or
DNA-binding buffer until the DNA-binding reaction is carried
out.
[0102] In certain embodiments, a magnetic force is applied to the
cell-trapping matrix to facilitate cell trapping. The magnetic
force is applied, in certain embodiments, by an instrument portion
of the differential extraction system.
Certain Exemplary Systems and Methods Using Transfer of DNA-Binding
Particles Between Compartments
[0103] FIG. 4 shows a fourth non-limiting exemplary system for
differential extraction of sperm cells and non-sperm cells, e.g.,
epithelial cells, according to certain embodiments. The system
shown in FIG. 4 is designed for a magnetic particle-handling
instrument that is also able to move liquid between compartments.
See, e.g., the BioRobot EZ1 Workstation (Qiagen). The system shown
in FIG. 4 processes sperm cell DNA in compartments 3 to 6 and
non-sperm cell DNA in compartments 7 to 9. The cell-trapping matrix
also serves as the DNA-binding matrix for the non-sperm cell DNA in
the exemplary system shown in FIG. 4. The cell-trapping matrix in
that system is magnetic.
[0104] In embodiments depicted in FIG. 4, a sample comprising a
mixture of sperm cells and non-sperm cells is placed in compartment
1. Compartment 1 comprises a cell-trapping matrix, which captures
the sperm cells and the non-sperm cells. The cell-trapping matrix
is transferred to compartment 2, which contains a cell wash buffer.
The cell-trapping matrix is then transferred to compartment 3,
which contains a selective sperm lysis buffer. The selective sperm
lysis buffer lyses the sperm cells captured by the cell-trapping
matrix. After lysis of the sperm cells, the cell-trapping matrix,
which still has captured non-sperm cells, is transferred to the
DNA-binding buffer in compartment 7. The DNA-binding buffer in
compartment 7 is incubated with the cell-trapping matrix for five
minutes with heating to about 70.degree. C. (heating element not
shown in FIG. 4) to lyse the non-sperm cells, e.g., epithelial
cells. The heating element is, in certain embodiments, part of a
fluid handling or particle handling instrument. The non-sperm cell
DNA binds to the cell-trapping matrix in the DNA binding buffer.
The cell-trapping matrix with the bound non-sperm cell DNA is then
transferred to compartment 8, which contains a DNA wash buffer.
Finally, the cell-trapping matrix with the bound non-sperm cell DNA
is transferred to compartment 9, which contains an elution buffer.
In certain embodiments, the elution buffer is heated to facilitate
elution of the bound DNA. In certain embodiments, the elution
supernatant may then transferred from compartment 9 to a collection
vessel (not shown in FIG. 4). The non-sperm cell DNA can then be
subjected to DNA analysis.
[0105] The selective sperm lysis buffer supernatant in compartment
3 is transferred to compartment 4, which contains DNA-binding
particles and a dilution buffer, which, when combined with the
selective sperm lysis buffer, forms a DNA-binding buffer. The
DNA-binding particles with the bound sperm cell DNA are then
transferred to compartment 5, which contains a DNA wash buffer.
Finally, the DNA-binding particles with the bound sperm cell DNA
are transferred to compartment 6, which contains an elution buffer.
In certain embodiments, the elution buffer is heated to facilitate
elution of the bound DNA. In certain embodiments, the elution
supernatant may then transferred from compartment 6 to a second
collection vessel (not shown in FIG. 4). The sperm cell DNA can
then be subjected to DNA analysis.
[0106] In certain embodiments, a second DNA wash buffer is
contained in a compartment 10 and in a compartment 11 (not shown in
FIG. 4). In certain such embodiments, the cell-trapping matrix with
the bound non-sperm cell DNA is transferred to compartment 10,
which contains a second DNA wash buffer, prior to being transferred
to the elution compartment. In certain such embodiments, the
DNA-binding matrix with the bound sperm cell DNA is transferred to
compartment 11, which contains a second DNA wash buffer, prior to
being transferred to the elution compartment.
[0107] FIG. 5 shows a fifth non-limiting exemplary system for
differential extraction of sperm cells and non-sperm cells, e.g.,
epithelial cells, according to certain embodiments. The system
shown in FIG. 5 is designed for a magnetic particle-handling
instrument that cannot also able to move liquid between
compartments. See, e.g., the Maxwell 16 Instrument (Promega). The
system shown in FIG. 5 processes sperm cell DNA in compartments 3
to 5 and non-sperm cell DNA in compartments 6 to 8. The
cell-trapping matrix also serves as the DNA-binding matrix for the
non-sperm cell DNA in the exemplary system shown in FIG. 5. The
cell-trapping matrix in that system is magnetic.
[0108] In embodiments depicted in FIG. 5, a sample comprising a
mixture of sperm cells and non-sperm cells is placed in compartment
1. Compartment 1 comprises a cell-trapping matrix, which captures
the sperm cells and the non-sperm cells. The cell-trapping matrix
is transferred to compartment 2, which contains a cell wash buffer.
The cell-trapping matrix is then transferred to compartment 3.
Compartment 3 in FIG. 5 comprises two sections that are partitioned
from one another. The partition between the sections is breakable
by the magnetic particle handling instrument, e.g., by physical
force. The top portion of compartment 3 comprises a selective sperm
lysis buffer. The bottom portion of compartment 3, which is
partitioned from the top portion, contains DNA-binding particles
and a dilution buffer. The cell-trapping matrix is transferred into
the top portion initially. The selective sperm lysis buffer lyses
the sperm cells captured by the cell-trapping matrix. After lysis
of the sperm cells, the cell-trapping matrix, which is still has
captured non-sperm cells, is transferred to the DNA-binding buffer
in compartment 6. The DNA-binding buffer is incubated with the
cell-trapping matrix for five minutes with heating to about
70.degree. C. (heating element not shown in FIG. 5) to lyse the
non-sperm cells, e.g., epithelial cells. The heating element is, in
certain embodiments, part of a fluid handling or particle handling
instrument. The non-sperm cell DNA binds to the cell-trapping
matrix in the DNA binding buffer. The cell-trapping matrix with the
bound non-sperm cell DNA is then transferred to compartment 7,
which contains a DNA wash buffer. Finally, the cell-trapping matrix
with the bound non-sperm cell DNA is transferred to compartment 8,
which contains an elution buffer. In certain embodiments, the
elution buffer is heated to facilitate elution of the bound DNA. In
certain embodiments, the elution supernatant may then transferred
from compartment 8 to a collection vessel (not shown in FIG. 5).
The non-sperm cell DNA can then be subjected to DNA analysis.
[0109] After the cell-trapping matrix is removed, the instrument
breaks the breakable partition between the top portion compartment
3 and the bottom portion of compartment 3 to mix the selective
sperm lysis buffer supernatant with the DNA-binding particles and
the dilution buffer, which, when combined with the selective sperm
lysis buffer, forms a DNA-binding buffer. The DNA-binding particles
with the bound sperm cell DNA are then transferred to compartment
4, which contains a DNA wash buffer. Finally, the DNA-binding
particles with the bound sperm cell DNA are transferred to
compartment 5, which contains an elution buffer. In certain
embodiments, the elution buffer is heated to facilitate elution of
the bound DNA. In certain embodiments, the elution supernatant is
then transferred from compartment 5 to a second collection vessel
(not shown in FIG. 5). The sperm cell DNA can then be subjected to
DNA analysis.
[0110] In certain embodiments, a second DNA wash buffer is
contained in a compartment 9 and in a compartment 10 (not shown in
FIG. 5). In certain such embodiments, the cell-trapping matrix with
the bound non-sperm cell DNA is transferred to compartment 9, which
contains a second DNA wash buffer, prior to being transferred to
the elution compartment. In certain such embodiments, the
DNA-binding matrix with the bound sperm cell DNA is transferred to
compartment 10, which contains a second DNA wash buffer, prior to
being transferred to the elution compartment.
[0111] FIG. 6 shows a system similar to the system of FIG. 5,
except the DNA-binding particles used to bind the sperm cell DNA
are kept in a separate compartment (compartment 4) from the
dilution buffer, according to certain embodiments. In embodiments
depicted in FIG. 6, compartment 3 has two separate sections
separated by a breakable seal, similar to compartment 3 of the
system of FIG. 5. In embodiments depicted in FIG. 6, however, the
lower compartment contains only the dilution buffer, so when the
instrument breaks the seal, the selective sperm lysis buffer and
the dilution buffer form a DNA-binding buffer. The instrument then
transfers the DNA-binding particles from compartment 4 to
compartment 3 to bind the sperm cell DNA.
[0112] In certain embodiments, a dilution buffer or a DNA-binding
buffer is kept separate from DNA-binding particles in a system
until the DNA-binding reaction is carried out. Keeping such
components separate may be desirable, in certain embodiments, when
the dilution buffer or DNA-binding buffer comprises alcohol. In
various embodiments, one skilled in the art can modify any of the
systems of FIGS. 3 to 6 to keep the DNA-binding particles separate
from the dilution and/or DNA-binding buffer until the DNA-binding
reaction is carried out.
[0113] In certain embodiments, a system comprises one or more empty
compartments. One or more empty compartments may be used, in
certain embodiments, as a location to dry the DNA-binding
particles, e.g., after a wash and before elution.
* * * * *