U.S. patent application number 11/947869 was filed with the patent office on 2008-05-22 for methods and kits for isolating sperm cells.
This patent application is currently assigned to Promega Corporation. Invention is credited to Laura Flanagan, Paraj Mandrekar, Ryan Olson, Allan Tereba.
Application Number | 20080118909 11/947869 |
Document ID | / |
Family ID | 36034542 |
Filed Date | 2008-05-22 |
United States Patent
Application |
20080118909 |
Kind Code |
A1 |
Tereba; Allan ; et
al. |
May 22, 2008 |
METHODS AND KITS FOR ISOLATING SPERM CELLS
Abstract
Disclosed are methods for isolating sperm cells from an aqueous
sample and kits for isolating sperm cells from an aqueous
sample.
Inventors: |
Tereba; Allan; (Fitchburg,
WI) ; Flanagan; Laura; (Madison, WI) ;
Mandrekar; Paraj; (Oregon, WI) ; Olson; Ryan;
(Madison, WI) |
Correspondence
Address: |
MICHAEL BEST & FRIEDRICH LLP
ONE SOUTH PINCKNEY STREET, P O BOX 1806
MADISON
WI
53701
US
|
Assignee: |
Promega Corporation
Madison
WI
|
Family ID: |
36034542 |
Appl. No.: |
11/947869 |
Filed: |
November 30, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10939105 |
Sep 10, 2004 |
7320891 |
|
|
11947869 |
|
|
|
|
Current U.S.
Class: |
435/2 ;
435/91.1 |
Current CPC
Class: |
C12N 15/1003 20130101;
C12N 5/0612 20130101 |
Class at
Publication: |
435/2 ;
435/91.1 |
International
Class: |
A01N 1/02 20060101
A01N001/02; C12P 21/04 20060101 C12P021/04 |
Claims
1. A method of separating sperm cells from an aqueous sample
comprising: (a) contacting the aqueous sample with a non-aqueous
liquid having a density greater than about 1.00 g/cm.sup.3, wherein
the density of the non-aqueous liquid is sufficiently low to permit
pelleting of at least a portion of the sperm cells in the sample;
(b) applying a force to the contacted sample for a period of time
sufficient to form an aqueous layer, a non-aqueous layer, and a
sperm pellet, thus separating sperm cells from the aqueous
sample.
2. The method of claim 1, wherein the force is applied by
centrifugation.
3. The method of claim 1, wherein the non-aqueous liquid has a
density of at least about 1.01 g/cm.sup.3.
4. The method of claim 1, wherein the non-aqueous liquid has a
density of about 1.29 g/cm.sup.3 or lower.
5. The method of claim 1, wherein the non-aqueous liquid comprises
chloroform.
6. The method of claim 1, wherein the non-aqueous liquid has a
density in the range of from about 1.02 g/cm.sup.3 to about 1.29
g/cm.sup.3.
7. The method of claim 1, wherein the non-aqueous liquid has a
density in the range of from about 1.050 g/cm.sup.3 to about 1.058
g/cm.sup.3.
8. The method of claim 1, wherein the aqueous sample comprises
lysed epithelial cells.
9. The method of claim 1, further comprising the step of: (c)
removing the aqueous layer.
10. The method of claim 9, further comprising the step of: (d)
contacting the non-aqueous layer and sperm pellet with a chaotropic
agent.
11. The method of claim 10, wherein the chaotropic agent comprises
a chaotropic salt.
12. The method of claim 10, wherein the chaotropic agent comprises
a detergent, and further comprising the step of: (e) contacting the
non-aqueous layer and sperm cell pellet of step (d) with
phenol:chloroform.
13. The method of claim 9, further comprising the step of: (d)
removing the non-aqueous layer.
14. The method of claim 13, further comprising the step of: (e)
contacting the cell pellet of step (d) with an aqueous detergent
and phenol:chloroform.
15. A method of isolating DNA from sperm cells in an aqueous sample
comprising lysed epithelial cells comprising: (a) contacting the
aqueous sample with a non-aqueous liquid having a density of
greater than about 1.00 g/cm.sup.3, wherein the density of the
non-aqueous liquid is sufficiently low to permit pelleting of at
least a portion of the sperm cells in the sample; (b) centrifuging
the contacted sample to form an aqueous layer, a non-aqueous layer,
and a sperm cell pellet; (c) removing the aqueous layer; (d)
contacting the non-aqueous layer and sperm cell pellet with lysis
buffer comprising a chaotropic agent and a reducing agent; and (e)
isolating DNA from the contacted sperm cell pellet of step (d).
16. A method of isolating DNA from sperm cells in an aqueous sample
comprising lysed epithelial cells comprising: (a) contacting the
aqueous sample with a non-aqueous liquid having a density of
greater than about 1.00 g/cm.sup.3, wherein the density of the
non-aqueous liquid is sufficiently low to permit pelleting of at
least a portion of the sperm cells in the sample; (b) centrifuging
the contacted sample to form an aqueous layer, a non-aqueous layer,
and a sperm cell pellet; (c) removing the aqueous layer and
non-aqueous layer; (d) contacting the sperm cell pellet with an
aqueous solution comprising a detergent and phenol:chloroform; and
(e) isolating DNA from the contacted sperm cell pellet of step
(d).
17. A kit for isolating sperm cells from a sample comprising sperm
cells, comprising a non-aqueous liquid having a density of greater
than about 1.00 g/cm.sup.3, wherein the density of the non-aqueous
liquid is sufficiently low to permit pelleting of at least a
portion of the sperm cells in the sample.
18. The kit of claim 17, further comprising an aqueous buffer.
19. The kit of claim 17, further comprising Proteinase K.
20. The kit of claim 17, further comprising a chaotropic agent.
21. The kit of claim 17, further comprising a spin basket.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 10/939,105, filed Sep. 10, 2004, which is
incorporated herein by reference in its entirety.
INTRODUCTION
[0002] Genetic material obtained from forensic samples can be used
to identify perpetrators of sexual assaults or to exonerate
innocent suspects. Purified DNA obtained from sperm cells isolated
from forensic samples can be used in subsequent genetic identity
testing. The genetic profile of sperm cell DNA can be compared to
that of a known suspect or to databases containing genetic
information about a large number of convicted felons.
[0003] In sexual assault cases, a vaginal or rectal swab, or
clothing containing a semen stain, is obtained from the victim for
forensic analysis. If sperm cells are present in the sample, DNA
from the sperm cells can be isolated and used in genetic identity
testing. However, a vaginal swab obtained from a sexual assault
victim typically contains relatively few sperm cells and large
numbers of epithelial cells from the victim. As a consequence,
unless the sperm cells are first separated from other cells in the
sample, DNA purified from a forensic sample is susceptible to
overwhelming contamination with epithelial cell DNA. Contamination
with epithelial cell DNA interferes with the ability to establish a
match between the genetic profile of DNA from the sample and that
of the suspect or a member of the database. It is therefore
desirable to isolate sperm cells from other cells in a forensic
sample prior to DNA isolation and analysis.
[0004] Techniques currently used to isolate sperm cells from other
cells in forensic samples are time consuming and labor intensive,
and there is currently a backlog of unprocessed samples. Because of
this backlog, some jurisdictions have a policy against processing
samples unless a suspect has been identified. Consequently, many
unprocessed samples are ultimately discarded, and genetic
information contained in the sample is never compared with or
entered into the national database, which reduces the ability of
law enforcement to identify and apprehend repeat sex offenders.
[0005] Sperm cells are typically isolated from forensic samples
containing epithelial cells by selectively lysing the epithelial
cells by treatment with Proteinase K and a detergent under
nonreducing conditions (Gill et al. 1985). Following epithelial
cell lysis, intact sperm cells are pelleted by centrifugation and
the supernatant, which contains DNA from lysed epithelial cells, is
removed. In order to minimize contamination by soluble epithelial
cell DNA, the sperm pellet is subjected to repeated washing with an
aqueous buffer in an attempt to remove soluble epithelial cell DNA.
This process frequently results in the loss of sperm cells.
[0006] Sperm cells have been isolated from samples containing both
sperm and epithelial cells by selectively binding the sperm cells
to sperm cell specific polyclonal or monoclonal antibodies attached
to a solid support (e.g., paramagnetic particles). After binding
the cells to the immobilized antibodies, the support is washed to
remove unbound cells. This method requires a large amount of
antibodies and is therefore relatively expensive. Furthermore,
sperm cells are lost during the wash steps, resulting in reduced
yield. Because sperm cells undergo structural changes in the
relatively low pH of the vagina, many sperm cell-specific
antibodies do not bind to sperm cells from all semen-containing
samples. In addition, antibodies may not bind efficiently because
of variations or mutations in sperm cell surface antigens in
certain individuals, resulting in poor sperm cell yields.
[0007] Sperm cells may be separated from epithelial cells on the
basis of differences in cell size by filtering the sample through
size selective membranes. This method is problematic because sperm
cells tend to become trapped among the epithelial cells, the sperm
cells form clumps that are too large to pass through the membrane,
and the membrane tends to clog, which ultimately may result in low
yields of sperm cells contaminated with epithelial cells or
epithelial cell DNA.
[0008] In another method, sperm cells are isolated by first
selectively lysing epithelial cells and filtering the lysate to
effect separation of the soluble epithelial cell DNA and intact
sperm cells. However, the method suffers from disadvantages,
including clogging of the membrane, low sperm cell yields and
contamination of the sperm cells with epithelial cell DNA.
[0009] In the field of reproductive medicine, sperm cells have been
isolated from fresh semen using a cell sorter, which although
effective, is not practical in the forensic context because it is
costly, time consuming, and does not address how to effectively
recover sperm cells and epithelial cells from a forensic sample
(e.g., a swab or clothing).
[0010] Thus, there is a need in the art for simplified methods of
separating sperm cells from epithelial cells in forensic
samples.
SUMMARY OF THE INVENTION
[0011] In one aspect, the present invention provides methods for
separating sperm cells from an aqueous sample. The aqueous sample
is contacted with a non-aqueous liquid having a density of greater
than about 1.00 g/cm.sup.3 and having a density sufficiently low to
permit pelleting of at least a portion of the sperm cells in the
sample. A force is applied to the contacted sample for a period of
time sufficient to form an aqueous layer, a non-aqueous layer, and
a sperm cell pellet.
[0012] In another aspect, the present invention provides kits for
isolating sperm cells from an aqueous sample. A kit according to
the present invention includes a non-aqueous liquid having a
density of greater than about 1.00 g/cm.sup.3 and having a density
sufficiently low to permit pelleting of at least a portion of the
sperm cells in the sample. Optionally, the kit may include an
aqueous solvent, a protease, a chaotropic agent, or a protocol for
isolating sperm cells.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 is an electropherogram of amplified DNA isolated from
sperm cells or epithelial cells separated by the method of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0014] Forensic samples obtained from victims of sexual assault
typically contain a large number of epithelial cells and, if
present, relatively few sperm cells. In order to obtain DNA from
the sperm cells for subsequent use in genetic identity testing, it
is necessary to isolate the sperm cells from aqueous soluble
material, especially DNA, that may interfere with or complicate the
interpretation of results. For example, DNA from lysed epithelial
cells is soluble in aqueous solutions.
[0015] Briefly, the method of the present invention involves
contacting an aqueous sample comprising sperm cells and material
soluble in an aqueous solution with a non-aqueous liquid having a
density that is greater than that of water but less than that of
sperm cells, and pelleting the sperm cells. Suitably, the sperm
cells are pelleted by centrifugation. The aqueous soluble material
(e.g., DNA) remains in the aqueous phase and is physically
separated from the pelleted sperm cells by the non-aqueous phase.
As used herein, "sperm cells" may include an intact sperm cell or
essentially intact sperm cell, and a sperm cell that has lost its
flagellum or "tail". Although a sperm cell may have been exposed to
environmental conditions, mechanical sheering, or chemical
treatment (e.g., exposure to Proteinase K or other agents) that
altered the cell, such cells are within the scope of the invention,
especially those cells that retain their nuclei.
[0016] Forensic samples from sexual assault victims are typically
collected on a solid support, such as a swab or cloth (e.g., a
cutting from clothing containing a semen stain). The swab or cloth
may be transferred to a container such as a test tube or other
suitable container and contacted with an aqueous solution such as a
lysis buffer. As described in the Examples, recovery of the aqueous
buffer and sperm cells may be facilitated by transferring the solid
support and aqueous buffer to a second container equipped with a
mechanical barrier that effectively prevents passage of the solid
support while allowing the liquid sample to pass and centrifuging
to recover the aqueous sample. The second container held a
non-aqueous liquid so that removal of the aqueous sample from the
solid support and pelleting of sperm cells through a non-aqueous
liquid was accomplished in a single centrifugation. Alternatively,
the treatment with the non-aqueous liquid could be performed in the
same container in which the solid support was treated with the
lysis buffer by contacting the aqueous sample with the non-aqueous
liquid either before or after removing the solid support. After
adding the non-aqueous liquid, the container may be optionally
fitted with a mechanical barrier, onto which the solid support is
placed prior to centrifugation. In another approach, the sperm
cells in the aqueous sample may be pelleted by centrifugation prior
to contacting the sample with a non-aqueous liquid. Addition of the
non-aqueous liquid will float the aqueous sample off of the pellet
and form a barrier (i.e., a non-aqueous layer) between the sperm
cell pellet and soluble DNA in the aqueous sample. The latter
approach would allow physical separation between pelleted sperm
cells and soluble DNA, but would be less effective separating the
sperm cell pellet from cellular debris than the other approaches in
which the aqueous sample is contacted with the non-aqueous liquid
prior to centrifugation.
[0017] The lysis buffer suitably comprises Proteinase K, and
Sarkosyl or SDS. Optionally, the lysis buffer may comprise any
suitable water soluble dye (e.g., FD & C Yellow) to enhance
visualization of the aqueous phase. The material is treated with a
suitable amount of a protease, such as Proteinase K (270 .mu.g/ml),
under conditions that allow lysis of the epithelial cells, but do
not promote lysis of the sperm cells. Sperm cells are relatively
resistant to proteases because the exposed proteins contain a
relatively large number of disulfide bonds. Therefore, reducing
conditions are not suitable for differential lysis because the
presence of reducing agents would disrupt the disulfide bonds and
increase lysis of sperm cells treated with proteases.
[0018] After treatment with the protease and detergent, the lysate
containing the lysed epithelial cells and intact sperm cells is
contacted with a non-aqueous liquid. A suitable non-aqueous liquid
may include any non-aqueous liquid having a density greater than
that of water but less than that of sperm cells, suitably having a
density of greater than 1.00 g/cm.sup.3 and having a density
sufficiently low to permit at least a portion of the sperm cells to
be pelleted. As demonstrated in the Examples below, a non-aqueous
liquid having a density of less than 1.29 g/cm.sup.3 was found to
allow recovery of sperm cells for subsequent DNA isolation,
amplification, and analysis. However, it is envisioned that
non-aqueous liquids having a density of greater than 1.29
g/cm.sup.3 may be used in the method of the invention, provided
that the density is sufficiently low to permit at least some of the
sperm cells to be pelleted. It is well within the ability of one
skilled in the art to evaluate the suitability of non-aqueous
liquids to isolate sperm cells according to the method of the
invention. The non-aqueous liquid is suitably non-chaotropic so as
to prevent undesired lysis of the sperm cells. The non-aqueous
liquid is preferably one that has a relatively low solubility in
water. Using a non-aqueous liquid having low solubility in water
will typically afford better phase separation and reduced
contamination of the sperm cell pellet with water soluble
materials, such as epithelial cell DNA.
[0019] The non-aqueous liquids diethyl glutarate ("DEG"), dimethyl
glutarate ("DMG"), and 1-chloro-2-methyl-2-propanol have been
evaluated and were found to be useful in the practice of the
invention when used alone or in combination. Optionally, the
density of the non-aqueous liquid may be adjusted by using two or
more non-aqueous liquids having different densities in combination
at ratios effective to obtain the desired density. When two or more
non-aqueous liquids are used, the liquids are suitably
substantially miscible with each other so as to form a liquid
mixture of substantially uniform density.
[0020] Each of DEG, which has a density of approximately 1.02
g/cm.sup.3, 1-chloro-2-methyl-2-propanol, which has a density of
about 1.058 g/cm.sup.3, and DMG, which has a density of 1.09
g/cm.sup.3, afforded acceptable separation of sperm cells from the
soluble epithelial cell DNA when used as the sole non-aqueous
liquid. DEG may be used in combination with DMG at ratios effective
to provide a mixed non-aqueous liquid intermediate between 1.02
g/cm.sup.3 and 1.09 g/cm.sup.3. In the Examples below, a mixed
liquid comprising DEG and DMG in a ratio of about 50:50 was used to
obtain a non-aqueous liquid with a density of about 1.055
g/cm.sup.3, which was found to afford effective separation between
the sperm cell pellet and aqueous soluble epithelial cell DNA. In
addition, mixed liquids prepared from DEG and DMG in ratios of from
about 100:0 to about 0:100 DEG:DMG were found to be effective in
the practice of the invention. We have evaluated the efficacy of
various ratios of DEG and DMG and found ratios of 100:0, 50:50,
40:60, 30:70, 20:80, and 0:100 DEG:DMG to be effective in obtaining
sufficient quantities of sperm cells of acceptable purity using the
methods of the invention.
[0021] In addition, we discovered that DMG can be used in
combination with chloroform to obtain a mixed non-aqueous liquid of
various densities. Mixed liquids of DMG and chloroform are
effective in isolating sperm cells at densities of 1.29 g/cm.sup.3
or lower. Using mixed liquids containing DMG:chloroform in a ratio
of 95:5 and having a density of about 1.108 g/cm.sup.3, sperm cell
yields were comparable to those obtained with DMG alone. A mixed
liquid containing DMG:chloroform in a ratio of 75:25 and having
density of 1.189 g/cm.sup.3 afforded a lower sperm cell yield than
DMG alone. A mixed liquid containing DMG:chloroform in a ratio of
50:50 and having density of 1.29 g/cm.sup.3 gave low but detectable
levels of sperm cells. In other words, the tested combinations of
nonaqueous liquids, having densities of 1.29 g/cm.sup.3 or lower,
were effective in yielding sperm cell DNA in a yield and of a
purity sufficient to permit amplification and genetic identity
testing. It is specifically envisioned that chloroform could be
used in conjunction with DEG or 1-chloro-2-methyl-2-propanol to
isolate sperm cells by selecting appropriate ratios to provide a
mixed non-aqueous liquid having a suitable density.
[0022] As one skilled in the art will appreciate, a mixed
non-aqueous liquid having a relatively low ratio of DEG to DMG will
have a relatively high density, and mixed non-aqueous liquids
having a relatively high ratio of DEG to DMG will have a relatively
low density. It is expected that a non-aqueous liquid having a
relatively low density may afford greater recovery of sperm cells,
whereas a relatively high density may result in the recovery of
fewer sperm cells with less contaminating unlysed epithelial cells
and cell debris. Therefore, depending on the application, ratios of
various non-aqueous liquids may be adjusted to affect greater
recovery of sperm cells or recovery of fewer sperm cells of higher
purity.
[0023] After contacting the aqueous sample comprising lysed
epithelial cells with the non-aqueous liquid, the sample is
subjected to a force for a period of time effective to cause
separation of the aqueous and non-aqueous phases and pelleting of
the sperm cells. Preferably, the force is applied to the sample by
centrifuging the sample. Optionally, a spin basket may be employed
in the centrifugation step to allow removal of the cells and
solution from the solid support.
[0024] Epithelial cell DNA, which is soluble in water, is found in
the aqueous layer, and insoluble epithelial cell debris, which may
also contain trapped epithelial cell DNA, is found at the interface
between the aqueous and non-aqueous layers, depending on the
density of the non-aqueous liquid. The epithelial cell DNA and cell
debris is removed by removing the aqueous layer and interface
region, which can be achieved by any suitable means, including
pipetting. The epithelial cell DNA present in the aqueous phase may
be used as a control in genetic identity testing to confirm the
source of the sample. Following recovery of the aqueous phase, a
small amount of aqueous solution may remain at or near the surface
of the non-aqueous phase and on the container wall. Optionally, to
enhance removal of the aqueous solution and epithelial cell DNA
without disrupting the sperm cell pellet, the residual aqueous
solution may be removed by first layering water or a suitable
aqueous solution onto the non-aqueous phase and then removing the
aqueous phase. Because water does not mix with the non-aqueous
liquid, there is no need to centrifuge the solutions to effect
separation of the aqueous and non-aqueous layers.
[0025] Following removal of the aqueous phase, a lysis buffer
containing a chaotropic agent and a reducing agent may be added to
the non-aqueous liquid and vortexed to form a substantially
homogenous mixture. As shown in Example 2, a lysis buffer
containing guanidine thiocyanate (GTC) and dithiothreitol (DTT) was
combined with the non-aqueous phase and sperm cell pellet to effect
lysis of the sperm cells. Example 2 further demonstrates that a
resin capable of binding DNA can then be added to the sperm cell
lysate to purify the DNA from other cellular material.
[0026] Alternatively, as described in Example 3, both the aqueous
and non-aqueous layers may be removed to leave a sperm cell pellet,
and the sperm cells present may be lysed by contacting the cells
with an aqueous solution comprising a detergent, such as sodium
dodecyl sulfate (SDS) (1% w/v) and DTT, followed by extraction with
phenol:chloroform.
[0027] It is specifically envisioned that, following removal of the
aqueous phase, the sperm cell pellet and the non-aqueous phase
could be extracted with phenol:chloroform and an aqueous solution
containing a detergent (e.g., SDS or Sarkosyl) to effect release of
the sperm cell DNA.
[0028] As described in the Examples, the method of the invention
was found to be effective in isolating sperm cells from epithelial
cells contained within forensic samples such as vaginal or cervical
swabs. It is envisioned that the method will be useful in isolating
sperm cells from other sources, including other solid supports
containing semen, such as cloth. It is reasonably expected that the
method of the invention will be suitable for use with samples
containing sperm cells and contaminating red or white blood cells
or DNA derived from nucleated non-sperm cells.
[0029] The method of the invention is expected to have general
applicability in separating cells on the basis of their
differential densities, or to facilitate isolation of other cell
types from material soluble in an aqueous solvent following
selective lysis. It is also envisioned that the method may be
suitable for isolating various sub-cellular organelles.
[0030] The following non-limiting examples are intended to be
purely illustrative.
EXAMPLE 1
Isolation of Sperm Cells from Samples Containing Epithelial
Cells
[0031] Samples used in evaluating sperm cell isolation included a
fresh buccal swab containing added semen, fresh or four year old
vaginal samples containing added semen, and a four year old 11-hour
post coital vaginal sample. The solid support (e.g., a swab)
containing the samples were placed in a microcentrifuge tube. A 0.5
ml aliquot of a digestion solution containing 50 mM NaCl, 10 mM
Tris, pH 8.0, 10 mM EDTA, 0.2% SDS, FD&C Yellow dye, and 270
.mu.g/ml Proteinase K was added to each sample. The tubes
containing the samples were vortexed for 30 seconds and incubated
at 56.degree. C. for 1 hour. Diethyl glutarate and dimethyl
glutarate were combined at ratios of 100:0, 50:50, 40:60, 30:70,
20:80, and 0:100 DEG:DMG to form mixed non-aqueous liquids. The
ratio of 50:50 formed a liquid with a density of about 1.055
g/cm.sup.3. A 100 .mu.l aliquot of the non-aqueous liquid including
ratios of 100:0, 50:50, 40:60, 30:70, 20:80, or 0:100 DEG:DMG was
transferred to a clean microcentrifuge tube fitted with a spin
basket. Following the incubation, the Proteinase K digestion
reaction and the solid support were transferred to the spin basket.
The spin basket and microcentrifuge tube were placed in a
microcentrifuge and centrifuged for 10 min at 14,000 rpm
(10,000.times.g). The yellow aqueous phase and any cellular debris
near the boundary between the aqueous and non-aqueous phases was
removed by pipetting. A 100 .mu.l aliquot of water was layered onto
the top of the non-aqueous phase, and allowed to stand for about 30
seconds before removing the aqueous phase with a pipette.
[0032] In addition, sperm cells were isolated using ratios of
dimethyl glutarate and chloroform for the non-aqueous solution.
Dimethyl glutarate and chloroform were combined in the ratios of
95:5, 75:25 and 50:50 dimethyl glutarate:chloroform. The density of
the 50:50 mixture was 1.290.
EXAMPLE 2
Isolation of DNA from Sperm Cells or Lysed Epithelial Cells Using
Chaotropic Salt and Reducing Conditions
[0033] The DNA from the sperm cell pellet of Example 1 was isolated
according to DNA isolation methods described in Promega Technical
Bulletin TB296 using components provided in the DNA IQ.TM. system
(Promega Corp., Madison, Wis. Cat. No. DC6701). A 200 .mu.l aliquot
of DNA IQ.TM. lysis buffer containing 4.5 M guanidine thiocyanate
(GTC) and 10 mM dithiothreitol (DTT) was added to the microfuge
tube containing the non-aqueous liquid and sperm cell pellet, and
vortexed briefly to disrupt the sperm cell pellet and to form a
homogenous mixture of the non-aqueous phase and the lysis
buffer.
[0034] A 7 .mu.l aliquot of DNA IQ.TM. Resin was added to the
solution and mixed by vortexing at high speed for 3 seconds and
incubated at room temperature for 5 minutes. The mixture was
vortexed for 2 seconds at high speed, the tube was placed in a
magnetic stand, and after the paramagnetic resin was attracted to
the side of the tube, the homogeneous mixture of lysis buffer and
non-aqueous liquid was removed and discarded. The particles were
washed three times with 100 .mu.l aliquots of DNA IQ.TM. Wash
Buffer. The particles were contacted with 40 .mu.l of DNA IQ.TM.
Elution Buffer, vortexed at high speed for two seconds, and
incubated at 65.degree. C. Immediately following incubation at
65.degree. C., the tube was vortexed for 2 seconds at high speed,
placed in a magnetic stand, and the eluate was transferred to a
clean container.
[0035] Epithelial cell DNA was isolated in the same manner that DNA
from lysed sperm cells was isolated, as described in the preceding
paragraph.
EXAMPLE 3
Isolation of DNA from Sperm Cells Using a Detergent and
Phenol:Chloroform Extraction
[0036] The DNA from the sperm cell pellet of Example 1 was isolated
by first removing the non-aqueous liquid to leave a firm sperm cell
pellet. A 300 .mu.l solution containing 10 mM Tris, pH 8.0, 1 mM
EDTA, 10 mM DTT, and 1% SDS was added to the sperm cell pellet, and
the tube was vortexed to lyse the sperm cells and dissolve the DNA.
An equal volume of phenol:chloroform (1:1, v/v) was added and
vortexed to denature the protein. The aqueous DNA-containing
solution was removed, concentrated in a Microcon.RTM. apparatus,
washed with 200 .mu.l of a buffer containing 10 mM Tris, pH 8.0,
0.1 mM EDTA, and concentrated again in the Microcon.RTM. apparatus
to about 40 .mu.l. The purified DNA was transferred to a clean
container.
EXAMPLE 4
Characterization of Sperm Cell DNA Following Multiplex
Amplification
[0037] Following recovery of DNA from isolated sperm cells or lysed
epithelial cells, according to Example 2 or Example 3, DNA was
amplified using PowerPlex.RTM. 16 (Promega Corp., Madison, Wis.
Cat. # DC6530). The amplified DNA was then analyzed using an ABI
Prism.RTM. 310 Genetic Analyzer.
[0038] Representative results are provided in the electropherogram
shown in FIG. 1. With reference to FIG. 1, DNA was isolated from
sperm cells or epithelial cells as described above in Examples 1
and 2 from a vaginal swab containing sperm cells and stored at room
temperature for 4 years. Following DNA purification from the
epithelial cell lysate or the sperm cell lysate by the DNA IQ.TM.
system, DNA ( 1/400.sup.th of the epithelial cell fraction and
1/80.sup.th of the sperm cell fraction) was amplified using
PowerPlex.RTM. 16 and amplification products were analyzed using
the ABI Prism.RTM. 310 Genetic Analyzer. The electropherogram of
FIG. 1 shows amplification products in the Fluoroscein channel
(FIG. 1A); shows amplification products in the JOE channel (FIG.
1B); and amplification products in the TMR channel (FIG. 1C). The
results are separated according to dye color to permit one to more
easily identify each of the 16 loci, and the results from the sperm
cell DNA are shown just above the results obtained from the
epithelial cell DNA.
[0039] As can be seen from FIG. 1, the method provides separation
of sperm cells from epithelial cells, as is evidenced by the very
low levels of contaminating epithelial cell DNA in the sperm cell
DNA. Only minor sperm cell DNA is seen in the epithelial cell DNA
fraction and is likely due to sperm cell lysis during prolonged
storage at room temperature.
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