U.S. patent application number 14/728862 was filed with the patent office on 2015-09-17 for nucleic acid purification.
The applicant listed for this patent is NETBIO, INC.. Invention is credited to Richard F. Selden, Eugene Tan.
Application Number | 20150259672 14/728862 |
Document ID | / |
Family ID | 42232704 |
Filed Date | 2015-09-17 |
United States Patent
Application |
20150259672 |
Kind Code |
A1 |
Selden; Richard F. ; et
al. |
September 17, 2015 |
Nucleic Acid Purification
Abstract
A self-contained apparatus for isolating nucleic acid, cell
lysates and cell suspensions from unprocessed samples apparatus, to
be used with an instrument, includes at least one input, and: (i) a
macrofluidic component, including a chamber for receiving an
unprocessed sample from a collection device and at least one filled
liquid purification reagent storage reservoir; and (ii) a
microfluidic component in communication with the macrofluidic
component through at least one microfluidic element, the
microfluidic component further comprising at least one nucleic acid
purification matrix; and (iii) at least one interface port to a
drive mechanism on the instrument for driving said liquid
purification reagent, through the microfluidic element and the
nucleic acid purification matrix, wherein the only inputs to the
apparatus are through the chamber and the interface port to the
drive mechanism.
Inventors: |
Selden; Richard F.;
(Lincoln, MA) ; Tan; Eugene; (Arlington,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NETBIO, INC. |
WALTHAM |
MA |
US |
|
|
Family ID: |
42232704 |
Appl. No.: |
14/728862 |
Filed: |
June 2, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13025923 |
Feb 11, 2011 |
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14728862 |
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12699564 |
Feb 3, 2010 |
9012208 |
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13025923 |
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61207017 |
Feb 6, 2009 |
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61206690 |
Feb 3, 2009 |
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Current U.S.
Class: |
536/25.41 ;
422/503; 435/287.2 |
Current CPC
Class: |
B01L 3/502753 20130101;
B01L 2200/027 20130101; B01L 2400/0487 20130101; B01L 2300/087
20130101; B01L 2300/0887 20130101; B01L 2400/043 20130101; B01L
2300/0867 20130101; C12N 15/101 20130101; B01L 3/502715 20130101;
B01L 3/5029 20130101; B01L 2300/0681 20130101; B01L 7/52 20130101;
C12N 15/1003 20130101; B01L 2300/0816 20130101; B01L 3/50825
20130101; B01L 2200/16 20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10; B01L 7/00 20060101 B01L007/00; B01L 3/00 20060101
B01L003/00 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This application was supported by Grant Number
2007-DN-BS-K184 awarded by the National Institutes of Justice,
Office of Justice Programs, US Department of Justice and a contract
from MIT Lincoln Laboratory entitled "Field-Deployable Accelerated
Nuclear DNA Equipment" awarded on Sep. 30, 2009.
Claims
1. A self-contained apparatus for isolating nucleic acid from at
least one unprocessed sample, said apparatus to be used with an
instrument, said apparatus consisting of a macrofluidic component,
a microfluidic component, at least one drive line, and two inputs,
a first input for receiving unprocessed sample or samples from a
collection device, and a second input for interfacing at least one
drive mechanism on said instrument at, at least one port: (i) said
macrofluidic component consisting of: (a) at least one sample
chamber, each of said at least one sample chamber composed of said
first input for receiving said at least one unprocessed sample from
a collection device; (b) at least one, but no less than all reagent
storage chambers necessary for isolating nucleic acid from said at
least one unprocessed sample, each of said reagent storage chambers
pre-filled with at least one necessary liquid reagent, including,
at least two lysis reagent storage chambers pre-filled with lysis
reagent; a wash reagent storage chamber pre-filled with wash
reagent; and an elution reagent storage chamber pre-filled with
elution reagent and (c) at least one empty holding chamber; (ii)
said microfluidic component composed of at least one microfluidic
element and at least one nucleic acid purification matrix, said
microfluidic component in communication with said macrofluidic
component via said at least one microfluidic element, and said at
least one drive line; (iii) said second input composed of at least
one drive mechanism interface port for connection to said drive
mechanism on said instrument configured to drive said first and
second lysis reagents, said wash reagent, and said elution reagent
and combinations thereof sequentially through said microfluidic
element and said nucleic acid purification matrix, and back into
said holding chamber (iv) said at least one drive line in
communication with said second input for interfacing with said at
least one drive mechanism on said instrument and with said at least
one microfluidic element to supply controlled flow or controlled
pressure or a controlled volumetric displacement of gas or liquid
to the apparatus. whereby in use, spent lysis, wash and elution
reagents and combinations thereof are driven through said
microfluidic element and back into said at least one sample
chamber, now serving as a waste chamber for spent process
reagents.
2. The apparatus of claim 1 wherein said collection device and/or
chamber is labeled with a bar code or RFID.
3. The apparatus of claim 1 wherein said drive mechanism is
pneumatic, mechanical, magnetic, or fluidic.
4. The apparatus of claim 1 wherein the unprocessed sample is
selected from the group consisting of: i) a nasal swab,
nasopharyngeal swab, buccal swab, oral fluid swab, stool swab,
tonsil swab, vaginal swab, cervical swab, blood swab, wound swab,
or tube containing blood, sputum, purulent material, or aspirates;
(ii) a forensic swab, cutting, adhesive tape lift, or card; or
(iii) an environmental air filter, water filter, and swab.
5. The apparatus of claim 1 wherein the nucleic acid purification
matrix is selected from the group consisting of comprises silica
membranes, silica beads, silica magnetic beads, ion exchange
resins, and ion exchange beads.
6. The apparatus of claim 1 wherein said at least one microfluidic
element is selected from the group consisting of: channels,
reservoirs, active valves, passive valves, pneumatically actuated
valves, reaction chambers, mixing chambers, venting elements,
access holes, pumps, metering elements, mixing elements, heating
elements, magnetic elements, reaction chambers, filtration
elements, purification elements, drive lines, and actuation
lines.
7. The apparatus of claim 1, wherein the apparatus can be placed
into or interfaces with another instrument that performs at least
one of thermal cycling, capillary electrophoresis, microfluidic
electrophoresis, nucleic acid fragment sizing, short tandem repeat
(STR), Y-STR, and mini-STR, single nucleotide polymorphism, PCR,
highly multiplexed PCR, Real-time-PCR, Reverse Transcription PCR,
sequencing, hybridization, microarray, VNTR, immunoassays, mass
spectroscopy and RFLP analyses.
8. The apparatus of claim 1, wherein said first lysis reagent
storage chamber of said at least two pre-filled lysis reagent
storage chambers is pre-filled with guanidinum and said second
lysis reagent storage chamber of said at least two pre-filled lysis
reagent storage chambers is pre-filled with ethyl alcohol and said
pre-filled wash reagent storage chamber is pre-filled with an
ethanol based wash reagent.
9. The apparatus of claim 1, wherein an aggregate fluid volume of
the at least one sample chamber, the at least two pre-filled lysis
reagent storage chambers, the pre-filled was reagent storage
chamber, and the pre-filled elution reagent storage chamber is
between about 1 and 1000 mL.
10. A method for purifying nucleic acids from an unprocessed sample
comprising, providing the apparatus of claim 1; providing said at
least one unprocessed sample comprising nucleic acids to at least
one sample chamber of said apparatus; driving at least a portion of
a first lysis reagent from a first of said at least two lysis
reagent storage chambers into said at least one sample chamber to
provide a first mixture; bubbling a gas through the first mixture
to provide a stirred first mixture; driving at least a portion of
said second lysis reagent from said second lysis reagent chamber
into said at least one sample chamber to provide a second mixture;
and driving at least a portion of the stirred first mixture through
said at least one nucleic acid purification matrix to provide a
filtrate and a retentate, wherein the retentate comprises at least
a portion of the nucleic acids; driving at least a portion of said
wash reagent from said wash reagent storage chamber and through
said at least one purification matrix to provide a washed retentate
and a waste; optionally drying the washed retentate; driving at
least a portion of said elution reagent from said elution reagent
storage chamber through said at least one purification matrix to
provide an eluted nucleic acid solution; and bubbling a gas through
the eluted nucleic acid solution to provide a homogenized eluted
nucleic acid solution driving said homogenized eluted nucleic acid
solution through said particulate filter to and into said at least
one holding chamber to provide a lysate and ethanol mixture;
driving said lysate and ethanol mixture through said at least one
nucleic acid purification matrix and back into a sample chamber,
serving as a waste chamber.
11. The method of claim 10, wherein the at least one unprocessed
sample is selected from the group consisting of: nasal swab,
nasopharyngeal swab, buccal swab, oral fluid swab, stool swab,
tonsil swab, vaginal swab, cervical swab, blood swab, wound swab,
tube containing blood, sputum, purulent material, or aspirates,
forensic swab, cutting, adhesive tape lift, adhesive tape card,
environmental air filter, water filter and an environmental swab.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/025,923, filed Feb. 11, 2011, which is a
continuation of U.S. patent application Ser. No. 12/699,564, filed
Feb. 3, 2010, and claims the benefit of the filing dates of U.S.
Provisional Application Ser. No. 61/206,690, filed Feb. 3, 2009;
and No. 61/207,017, filed Feb. 6, 2009. Each of the preceding are
hereby incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
A. The Unmet Need--Unprocessed Clinical and Forensic Samples
[0003] From the first isolation of nucleic acids by Miescher and
Altmann in the second half of the nineteenth century (Miescher,
Friedrich (1871) "Ueber die chemische Zusammensetzung der
Eiterzellen," in F. Miescher. Die Histochemischen and
physiologischen Arbeiten Vol. 2:3-23) to the most sophisticated
molecular biological techniques available today, the process of DNA
extraction has been streamlined substantially. Nevertheless, there
is a pressing need in the clinical, biothreat detection, and
forensics communities for sensitive, robust, and reliable
integrated methods of DNA purification that are rapid,
cost-effective, and neither labor- nor space-intensive. In
particular, there is an unmet need for methods and devices that can
rapidly purify nucleic acids from unprocessed clinical or forensic
field samples without any manual handling or processing.
[0004] Ideally, novel methods for nucleic acid purification are
needed to address the numerous and varied existing and emerging
markets for delivering genomic information, particularly the
delivery of genomic information in the field, and for point-of-care
and near point-of-care applications. For example, in the field of
human identification, there is an unmet need in the forensic
community to be able to generate a DNA fingerprint rapidly, whether
in the laboratory or in the field (e.g. at borders, ports of entry,
the battlefield, and military checkpoints).
[0005] Similarly, in order to protect civilian and military
populations, it is critical to improve the identification of
environmental biothreats. More rapid, more sensitive, more
specific, and more detailed identification will allow improved
strategic and tactical responses by civilian and military
authorities, and more effective remediation activities. The rapid
application of nucleic acid analysis technologies including nucleic
acid amplification, hybridization, and sequencing can provide
critical information in this regard.
[0006] Furthermore, the ability to rapidly diagnose clinical
infections (whether caused by biothreats or conventional pathogens)
would have a profound impact on society. For example, drawing a
blood sample from a septic patient and determining both the
identity of the pathogen or pathogens as well as their antibiotic
resistance profiles based on nucleic acid analyses within an hour
or less would allow specific antimicrobial therapy to begin
immediately (the analogous situation for viral diagnostics and drug
resistance profiles is also critically important). The ability to
rapidly generate nucleic acid analytic information from clinical
samples would also have substantial impact on the diagnosis and
treatment of a wide range of diseases ranging from cancers to
immune system disorders; essentially every category of diseases
would be impacted. The same approach could also be applied to
pharmacogenomics, the use of genetic information to predict the
suitability of a given pharmacologic intervention.
B. Prior Art Approaches to DNA Purification
[0007] The basic approach to extraction and purification of nuclear
DNA from mammalian cells was developed over three decades ago (N.
Blin, D. W. Stafford (1976). A general method for isolation of high
molecular weight DNA from eukaryotes. Nucleic Acids Res. 3(9):
2303-8) and has two major steps: the lysis of the cell types of
interest and the purification of DNA from other cellular components
in solution (particularly proteins) and cellular and tissue debris.
Cell lysis and (when appropriate) DNA solubilization can be
accomplished by mechanical (reviewed in J. Brent (1998). Breaking
Up Isn't Hard To Do: A cacophony of sonicators, cell bombs and
grinders" The Scientist 12(22):23) and non-mechanical techniques.
Simple mechanical approaches include the use of a blenders and
homogenization by forcing cells through restrictive openings.
Sonication is based on the exposure of cells to high-frequency
sound waves, and bead approaches are based on exposing cells to
violent mixing in the presence of various beads.
[0008] Chemical disruption of cells is an alternative to mechanical
disruption. Detergents are important chemical lytic agents that act
by disrupting lipid bilayers. Additional properties of detergents
may allow protein structure to be maintained (e.g. zwitterionic and
nonionic detergents) or disrupted (ionic detergents). Sodium
dodecyl sulfate (SDS), an ionic detergent, is commonly used in
forensic DNA extraction protocols due in part to its ability to
solubilize macromolecules and denature proteins within the cell (J.
L. Haines et al (2005) Current Protocols in Human Genetics Vol. 2,
(2005 John Wiley and Sons, Inc. Pub.). Proteinase K is often used
in tandem with detergent-based (e.g. SDS, Tween-20, Triton X-100)
lysis protocols. Another form of detergent lysis is based on FTA
paper (L. A. Burgoyne (1997) Convenient DNA Collection and
Processing: Disposable Toothbrushes and FTA Paper as a
Non-threatening Buccal-Cell Collection Kit Compatible with
Automatable DNA Processing, 8th International Symposium on Human
Identification, Sep. 17-20, 1997 Orlando, Fla.; G. M. Fomovskaia et
al., U.S. Pat. No. 6,958,392). This is a cellulose filter
impregnated with a weak base, an anionic detergent, a chelating
agent, and preservatives.
[0009] In the case of a clinical or environmental sample, a
critical first step towards nucleic acid analysis is the isolation
or purification of some or all of the nucleic acid present in the
sample. The biological material in the sample may be lysed and
nucleic acids within the lysate may be purified prior to further
analysis. Alternatively, nucleic acids contained within the lysate
may be analyzed directly (e.g. Phusion Blood Direct PCR kit
(Finnzymes, Espoo, FN) and Daniel et al., U.S. Pat. No.
7,547,510).
[0010] As those skilled in the art will recognize, purifying
nucleic acids from unprocessed clinical, environmental, or forensic
samples requires the automation of pre-processing steps suited to
the particular field sample under investigation. The diversity of
sample types, sample volumes, sampling technologies, sample
collection devices, sample processing requirements, and the
complexities inherent in resolving field samples has created an
unmet need for robust methods and devices for purifying nucleic
acids from such diverse samples.
C. Microfluidic Approaches to Purification from Clinical and
Environmental Samples
[0011] The field of microfluidics offers a potential solution to
the unmet need for methods and devices capable of isolating nucleic
acids from unprocessed clinical, environmental, and forensic
samples. Microfluidics is based on the manipulation of small fluid
volumes of microliters or less and emerged as a hybrid of molecular
biology and microelectronics in the early 1990's (See Manz et al.
Sens. Actuators B1:244-248 (1990)). A major focus in microfluidics
is to integrate multiple components to develop a system with
sample-in, results-out functionality (reviewed in Erickson et al.,
Anal. Chimica Acta 507: 11-26 (2004)).
[0012] Some progress using this approach has been made with regard
to environmental detection of biothreats. The automated pathogen
detection system (Hindon et al., Anal. Chem. 77:284-289 (2005))
collects air samples and performs microfluidic DNA extraction and
real-time PCR capable of detecting B. anthracis and Y. pestis
(detection limits were between 10.sup.3-10.sup.7 organisms per mL
of concentrated sample). The Cepheid (Sunnyvale, Calif.) GeneXpert
system also collects air samples and performs integrated B.
anthracis spore lysis (by microsonication), DNA extraction, and
real-time PCR (detection limits were 68 cfu (equivalent to 148
spores) per mL concentrated sample for Ames spores and
10.sup.2-10.sup.3 cfu per mL concentrated sample for Sterne
spores). Despite these advances, there is no available system or
device capable of purifying unprocessed nucleic acids from clinical
or environmental samples (or from environmental samples collected
manually) without human intervention. Indeed, all of the available
technologies rely on manual processing of some or all of the
steps.
D. DNA Purification from Forensic Samples
[0013] One of the earliest DNA purification methods for forensic
samples was the use of phenol/chloroform extraction (D. M. Wallace
(1987) Large and small scale phenol extractions. Methods Enzymol.
152:33-41; Maniatis, T. et al., "Purification of Nucleic Acids" in
Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.). In this method, most protein
moves to the organic phase or the organic-aqueous interface, and
solubilized DNA remains in the aqueous phase. The DNA-containing
phase can be subjected to ethanol precipitation, and DNA isolated
following a series of centrifugation and wash steps. In forensic
practice, DNA is often recovered from the aqueous phase with
centrifugal dialysis devices, such as the Microcon columns
(Millipore Corporation, Billerica, Mass.). The advantage of the
organic extraction approach is that it yields high quality DNA
preparations (with relatively low amounts of protein and relatively
low degradation) and remains one of the most reliable methods
available today. The major disadvantages are that the procedure is
time- and labor-intensive, requires cumbersome equipment, and is
relatively difficult to adapt to high-throughput settings.
[0014] Accordingly, the forensic community has moved to a series of
purification technologies that are simpler to use, many of which
serve as the basis of commercially available kits. There are an
enormous number of approaches to nucleic acid purification, several
of which are summarized as follows:
[0015] Silica Matrices/Chaotropic Agents.
[0016] The use of silica beads for DNA isolation has been a
standard technique for over a quarter century, with the initial
protocols based on the binding of DNA to silica in the presence of
chaotropic agents such as sodium iodide (B. Vogelstein et al.,
(1979) "Preparative and analytical purification of DNA from
agarose," Proc Nat Acad Sci USA 76(2):615-9). Many years earlier,
guanidinium salts had been found to be potent destabilizers of
macromolecules (von Hippel P. H. et al., (1964) "Neutral Salts: The
Generality of Their Effects on the Stability of Macromolecular
Conformations." Science 145:577-580). Certain guanidinium salts
also have the advantage of deactivating nucleases (Chirgwin J. M.
et al., (1979) "Isolation of biologically active ribonucleic acid
from sources enriched in ribonuclease," Biochemistry
18(24):5294-9). These observations were synthesized by Boom (Boom,
R. et al., (1990) "Rapid and Simple method for purification of
nucleic acids," J Clin Microbiol. 28(3):495-503), who, in effect,
used two related properties of guanidinium salts. The first, the
ability of the salts to lyse cells, and the second, the ability of
the salts to enhance DNA binding to silica particles, have led to a
number of lysis/purification approaches widely utilized in
forensics laboratories today (e.g. DNAIQ Systems, Promega, Madison,
Wis.). An alternative to silica beads is the use of silica
membranes (QIAamp, Qiagen Hilden, DE). In addition, the silica
beads themselves may be modified to further enhance DNA
binding.
[0017] Silica Matrices/Non-Chaotropic Agents.
[0018] Silica matrices can also be utilized in the absence of
chaotropes. One approach is to modify silica beads such that they
have a net positive charge at a given pH and are capable of binding
DNA (Baker, M. J., U.S. Pat. No. 6,914,137). The modification
contains an ionizable group, such that the DNA binding is reversed
at a higher pH (when the ionizable group is neutral or negatively
charged), sometimes at elevated temperature. As wide swings in pH
can damage DNA, a critical feature of this approach is to choose a
modification that allows reversible binding of DNA within a
relatively narrow pH range. A widely used approach of this type is
based on the ChargeSwitch bead (Life Technologies, Inc. Carlsbad,
Calif.).
[0019] Magnetic Beads.
[0020] Although DNA binding properties are determined primarily by
the surface structure of a given bead, the use of magnetic beads
has become increasingly important in DNA purification protocols.
These particles are generally paramagnetic; they are not themselves
magnetic but form dipoles when exposed to a magnetic field. The
utility of these beads relates to their ease of handling and
adaptation to automated systems. For example, beads can be readily
removed from a suspension in the presence of a magnet, allowing
them to be washed and transported efficiently. Two commonly used
magnetic beads are the ChargeSwitch and DNAIQ beads described
above.
[0021] Ion Exchange.
[0022] Ion exchange allows DNA molecules to reversibly bind to an
immobile bead. The bead generally consists of a porous organic or
inorganic polymer with charged sites that allow one ion to be
replaced by another at a given ionic strength. In practice, a
solution containing DNA and other macromolecules is exposed to the
ion exchange resin. The negatively charged DNA (due to its
phosphate backbone) binds relatively strongly to the resin at a
given salt concentration or pH. Protein, carbohydrate, and other
impurities bind relatively weakly (if at all) and are washed from
the beads (e.g. in a column format or by centrifugation). Purified
DNA can then be eluted in a high ionic strength buffer. A
commercially available anion exchange resin used today is based on
DEAE-modified silica beads (Genomic-tip, Qiagen).
[0023] Chelex.
[0024] Chelex-100 (Bio-Rad, Hercules, Calif.). is a modified resin
that efficiently binds multivalent metal cations. As such cations
are required for enzymes that degrade DNA and themselves inhibit
PCR enzymes, this method is representative of those that
essentially avoid a DNA purification step (Walsh P. S. et al.,
Chelex 100 as a medium for simple extraction of DNA for PCR-based
typing from forensic material. Biotechniques 10(4):506-13).
[0025] When using cotton swabs to collect material, there can be
problems removing biological material from the cotton matrix; as
the cotton swab dries after collection, the biological material can
adhere to the swab. For example, due to the saccharic composition
of the spermatocyte membrane, spermatocytes stick to solid
supports, especially cotton (Lazzarino, M. F. et al, (2008) DNA
Recovery from Semen Swabs with the DNA IQ System. Forensic Science
Communications 10(1)). In order to release the maximum amount of
material from the swabs, a variety of buffers have been tested and
compared to the standard differential extraction buffer. Use of
detergents such as 1-2% sodium dodecyl sulfate (SDS) has shown to
increase sperm cell recovery (Norris, J. V. et al., (2007)
"Expedited, chemically enhanced sperm cell recovery from cotton
swabs for rape kit analysis." J Forensic Sci 52(4): 800-5). Also,
the addition of low amounts of cellulase has shown to release more
epithelial and sperm cells from the cotton swab matrix than buffer
elution alone (Voorhees, J. C. et al., (2006). "Enhanced elution of
sperm from cotton swabs via enzymatic digestion for rape kit
analysis." J Forensic Sci 51(3): 574-9).
[0026] There can be many challenges to obtaining forensic short
tandem repeat (STR) profiles from biological materials including
low quantity or quality of DNA. Low copy number samples (containing
less than 50-100 picograms of DNA) as well as low quality, degraded
samples require highly efficient collection, extraction, and
amplification procedures. These samples are seen in a variety of
forensic evidence including touch evidence and aged samples.
Amplification kits such as the Life Technologies Minifiler.TM. have
smaller amplicon sizes which have shown to increase the ability to
obtain STR profiles from these difficult samples.
[0027] PCR inhibitors are another challenge and must be eliminated
before downstream applications can be performed. Common inhibitors
are indigo dyes from denim, heme from blood, humic acid found in
plants and soil, and collagen found in various tissues. The
majority of these inhibitors are effectively eliminated using
silica based DNA extraction methods or additional purification with
charge or size exclusion columns. The presence of inhibitors can be
detected by performing PCR with internal positive controls. If
present, some inhibitors can be neutralized by various treatments
including sodium hydroxide washes or further purification with
Millipore Microcon YM.RTM. columns.
[0028] The need to reconcile the "real world" requirements of
sample collection with the microfluidic requirements of a fully
integrated microfluidic DNA processing biochip can be referred to
as the "macro-to-micro interface" or the "world-to-chip interface"
(Fredrickson, C. and Fan, Z. (2004) "Macro-to-micro interfaces for
microfluidic devices," Lab Chip 4(6): 526-33). Much of the reported
research on addressing this interface is focused on resolving the
mismatch between the macrofluidic and microfluidic volumetric
requirements, but little or no research concerning the reconciling
of specific forensic sampling requirements and formats with
microfluidic devices has been reported.
[0029] The (non-forensic) volumetric mismatch has been commercially
addressed by Agilent (Santa Clara, Calif.) in the Bioanalyzer 2100
by the use of a capillary to aspirate samples from a microtiter
plate to a chip for enzyme assays (Lin 2003). Similarly, Gyros
(Uppsala, SE) has developed a capillary dispenser for a LabCD
system where samples are aspirated from a well plate into a
dispensing nozzle and then directed upwards onto a rotating device
(Jesson 2003). These devices, however, do not address the format
incompatibility of collected forensic samples--particularly on the
commonly used collection devices based on swabs.
E. Partially Automated DNA Purification
[0030] A variety of laboratory instruments have been developed for
the partially automated purification of nucleic acids. For example,
the Maxwell 16 instrument (Promega) is designed to purify nucleic
acids from forensic samples. To purify DNA from a buccal swab, the
operator performs a number of steps including cutting the cotton
collection portion in half, placing it into a 1.5 mL centrifuge
tube, preparing and adding lysis reagents, incubating the sample in
a heat block, vortexing the tube, transferring the reagents and
swab sample to a spin basket, and centrifuging the basket. Next, a
plunger is placed into the Maxwell cartridge, the sample is
pipetted into the cartridge, and the cartridge is placed into the
instrument for nucleic acid purification.
[0031] The iPrep instrument (Life Technologies) is also used for
the processing of forensic and clinical samples to purify nucleic
acids. For example, the tip of a buccal swab is placed into a 1.5
mL centrifuge tube and subjected to a series of manual steps
similar to those required for the Maxwell 16. After manual sample
preparation, the crude lysate is transferred to a 1 mL elution tube
for processing within the instrument. The Qiagen EZ1, BioRobot M48,
and Qiacube systems (Qiagen) partially automate nucleic acid
purification. Buccal swabs are collected, allowed to dry for two
hours, and manually processed essentially as with the instruments
described above. Innuprep (analytikJena, Itzehoe, DE), LabTurbo
(Taigen, Taipei, T W), Xiril 150 (Xiril AG, Hombrechtikon, CH), and
Quickgene (FujiFilm Corp., Tokyo, JP) systems are partially
automated instruments requiring substantial user manipulation and
intervention. U.S. Patent App. Pub. No. 20080003564 (Chen et al)
describes a macrofluidic sample processing tube that accepts a swab
and transports reagents mechanically using macrofluidic features
and flexible tubing. US Patent App. Pub. No. 20070092901 (Ligler,
F. et al.) have described a system that accepts liquid biological
samples for semi-automated nucleic acid purification.
[0032] Several groups including those of Landers (Wolfe, K. A. et
al., (2002) Toward a microchip-based solid phase extraction method
for isolation of nucleic acids. Electrophoresis 23 (5):727-33; Wen,
J. et al., (2006) DNA extraction using a tetramethyl
orthosilicate-grafted photopolymerized monolithic solid phase. Anal
Chem. 78(5):1673-81; Easley, C. J. et al., (2006) A fully
integrated microfluidic genetic analysis system with
sample-in-answer-out capability. Proc Natl Acad Sci USA
103(51):19272-7); Hagan K. A. et al. (2008) Microchip-based
solid-phase purification of RNA from biological samples, Anal Chem
80:8453-60), Locascio (Becker, H. et al., (2002) Polymer
microfluidic devices Talanta 56(2):267-287; Martynova, L. et al.,
(1997) Fabrication of plastic microfluid channels by imprinting
methods. Anal Chem. 69(23):4783-9), Mathies (Lagally, E. T. et al.,
(2001) Fully integrated PCR-capillary electrophoresis microsystem
for DNA analysis. Lab Chip 1(2):102-7: Yeung, S. H., et al., (2006)
Rapid and high-throughput forensic short tandem repeat typing using
a 96-lane microfabricated capillary array electrophoresis
microdevice. J Forensics Sci. 51(4):740-7), and others (Liu R. H.
et al., (2004) "Self-contained, fully integrated biochip for sample
preparation, polymerase chain reaction, amplification, and DNA
microarray detection Anal Chem 76(7):1824-31) have been working on
microfluidics for DNA purification and analysis (reviewed in Liu,
P. and Mathies, R. A., (2009), "Integrated microfluidic systems for
high-performance genetic analysis." Trends in Biotechnology
27(10):572-81). Easley has demonstrated DNA isolation from 750
nanoliters of whole blood and 1 microliter of nasal aspirate using
a guanidinium lysis/silica bead purification protocol (Easley, C.
J. et al., Proc Natl Acad Sci supra). The whole blood sample
contained approximately 2.5 million bacteria (Bacillus anthracis)
per mL 1500-2000 cfu in the 750 nL sample), a concentration too
high to be relevant for clinical diagnostics. U.S. Patent App.
US2008/0014576 A1 describes nucleic acid purification modules that
accept samples for purification in solutions, beads, colloids, or
multiple-phase solutions and may be integrated with downstream
preparation devices such as thermal cyclers and separation
instruments.
SUMMARY OF THE INVENTION
[0033] The inventions of this disclosure comprise apparatus,
methods and instruments for isolating nucleic acid, cell lysates
and cell suspensions from unprocessed samples. In one invention,
the apparatus comprises a self-contained apparatus for isolating
nucleic acid from an unprocessed sample, said apparatus to be used
with an instrument, said apparatus comprising, at least one input,
and:
[0034] (i) a macrofluidic component, comprising a chamber for
receiving said unprocessed sample from a collection device and at
least one filled liquid purification reagent storage reservoir;
and
[0035] (ii) a microfluidic component in communication with said
macrofluidic component via at least one microfluidic element, said
microfluidic component further comprising; at least one nucleic
acid purification matrix
[0036] (iii) a drive mechanism on said instrument for driving said
liquid purification reagent, through said microfluidic element and
said nucleic acid purification matrix, wherein the only inputs to
said apparatus are via said chamber and said drive mechanism.
[0037] In another invention, the apparatus comprises a
self-contained apparatus for isolating nucleic acid from an
unprocessed sample, said apparatus to be used with an instrument,
said apparatus comprising, at least one input, and:
[0038] (i) a macrofluidic component comprising: [0039] a chamber
for receiving said unprocessed sample from a collection device;
[0040] at least two pre-filled lysis reagent storage reservoirs;
[0041] a pre-filled wash reagent storage reservoir; and a
pre-filled elution reagent storage reservoir; and
[0042] (ii) a microfluidic component in communication with said
macrofluidic component via at least one microfluidic element, said
microfluidic component further comprising; at least one nucleic
acid purification matrix;
[0043] (iii) a drive mechanism on said instrument for driving said
first and second lysis reagents, said wash reagent, and said
elution reagent and sequentially through said microfluidic element
and said nucleic acid purification matrix, wherein the only inputs
to said apparatus are via said chamber and said drive
mechanism.
[0044] In related inventions, the claimed apparatus may have
collection devices and/or chambers are labeled, said labels
comprising and a bar code or RFID. In other related inventions, the
drive mechanisms may be pneumatic, mechanical, magnetic, or
fluidic. In still other related inventions the unprocessed sample
comprises: (i) a nasal swab, nasopharyngeal swab, buccal swab, oral
fluid swab, stool swab, tonsil swab, vaginal swab, cervical swab,
blood swab, wound swab, or tube containing blood, sputum, purulent
material, or aspirates; (ii) a forensic swab, cutting, adhesive
tape lift, or card; or (iii) an environmental air filter, water
filter, or swab.
[0045] In other related inventions the purification matrix of the
claimed apparatus comprises silica membranes, silica beads, silica
magnetic beads, ion exchange resins, or ion exchange beads. In
still other related inventions the microfluidic component of the
claimed apparatus comprises channels, reservoirs, active valves,
passive valves, pneumatically actuated valves, reaction chambers,
mixing chambers, venting elements, access holes, pumps, metering
elements, mixing elements, heating elements, magnetic elements,
reaction chambers, filtration elements, purification elements,
drive lines, and actuation lines.
[0046] Another invention of this disclosure is a method for
purifying nucleic acids from an unprocessed sample comprising,
[0047] providing a sample comprising nucleic acids to the chamber
of a claimed apparatus;
[0048] driving at least a portion of a first lysis reagent from
said first lysis reagent chamber into the chamber to provide a
first mixture;
[0049] driving at least a portion of a second lysis reagent from
said second lysis reagent chamber into the chamber to provide a
second mixture;
[0050] driving at least a portion of the said second mixture
through the purification membrane to provide a filtrate and a
retentate, wherein the retentate comprises at least a portion of
the nucleic acids;
[0051] driving at least a portion of the wash reagent through the
purification membrane to provide a washed retentate and a
waste;
[0052] optionally drying the washed retentate; and
[0053] collecting at least a portion of the nucleic acids from the
washed retentate by driving at least a portion of an elution
reagent from the elution reagent chamber through the purification
matrix.
[0054] Yet another invention of this disclosure is a method for
purifying nucleic acids from an unprocessed sample comprising,
[0055] providing a sample comprising nucleic acids to the chamber
of a claimed apparatus;
[0056] driving at least a portion of a first lysis reagent from
said first lysis reagent chamber into the chamber to provide a
first mixture;
[0057] bubbling a gas through the first mixture to provide a
stirred first mixture
[0058] driving at least a portion of a second lysis reagent from
said second lysis reagent chamber into the chamber to provide a
second mixture; and
[0059] driving at least a portion of the stirred first mixture
through the purification matrix to provide a filtrate and a
retentate, wherein the retentate comprises at least a portion of
the nucleic acids;
[0060] driving at least a portion of the wash reagent through the
purification matrix to provide a washed retentate and a waste;
[0061] optionally drying the washed retentate;
[0062] driving at least a portion of an elution reagent from the
elution reagent chamber through the purification matrix to provide
an eluted nucleic acid solution; and bubbling a gas through the
eluted nucleic acid solution to provide a homogenized eluted
nucleic acid solution.
[0063] Still another invention of this disclosure is a method for
purifying nucleic acids from pathogens in whole blood
comprising,
[0064] providing a sample comprising anticoagulated whole blood and
pathogens in a blood collection tube to the sample collection
chamber of a claimed apparatus;
[0065] driving at least a portion of the blood through a leukocyte
retention filter to provide a reduced-leukocyte filtrate;
[0066] driving at least a portion of a leukocyte wash reagent
through the leukocyte retention filter to provide a washed
reduced-leukocyte filtrate;
[0067] driving at least a portion of the a reduced-leukocyte
filtrate through a pathogen capture membrane
[0068] driving at least a portion of the pathogen resuspension
solution across the capture membrane to provide a concentrated
pathogen suspension
[0069] driving at least a portion of the concentrated pathogen
suspension into a first lysis reagent chamber containing said first
lysis reagent to provide a first mixture;
[0070] driving at least a portion of a second lysis reagent from
said second lysis reagent chamber into the first lysate reagent
chamber to provide a second mixture; driving at least a portion of
the said second mixture through the purification membrane to
provide a filtrate and a retentate, wherein the retentate comprises
at least a portion of the nucleic acids; [0071] driving at least a
portion of the wash reagent through the purification membrane to
provide a washed retentate and a waste;
[0072] optionally drying the washed retentate; and
[0073] collecting at least a portion of the nucleic acids from the
washed retentate by driving at least a portion of an elution
reagent from the elution reagent chamber through the purification
matrix.
[0074] Another invention of this disclosure is a method for
purifying nucleic acids from pathogens in whole blood
comprising,
[0075] providing a sample comprising anticoagulated whole blood and
pathogens in a blood collection tube to the sample collection
chamber of a claimed apparatus;
[0076] driving at least a portion of the blood through a leukocyte
retention filter to provide a reduced-leukocyte filtrate;
[0077] driving at least a portion of a leukocyte wash reagent
through the leukocyte retention filter to provide a washed
reduced-leukocyte filtrate;
[0078] driving at least a portion of the leukocyte resuspension
solution across the retention filter to provide a concentrated
leukocyte suspension;
[0079] driving at least a portion of the concentrated leukocyte
suspension into a first lysis reagent chamber containing said first
lysis reagent to provide a first mixture;
[0080] driving at least a portion of a second lysis reagent from
said second lysis reagent chamber into the first lysate reagent
chamber to provide a second mixture; driving at least a portion of
the said second mixture through the purification membrane to
provide a filtrate and a retentate, wherein the retentate comprises
at least a portion of the nucleic acids;
[0081] driving at least a portion of the wash reagent through the
purification membrane to provide a washed retentate;
[0082] optionally drying the washed retentate; and
[0083] collecting at least a portion of the nucleic acids from the
washed retentate by driving at least a portion of an elution
reagent from the elution reagent chamber through the purification
matrix.
[0084] In a related invention the method additionally
comprises,
[0085] driving a leukocyte lysis solution into the concentrated
leukocyte suspension to provide a differentially lysed suspension;
driving at least a portion of the differentially lysed suspension
through a pathogen retention filter; driving at least a portion of
the retention filter wash reagent through the pathogen retention
filter to provide a washed pathogen retentate; and resuspending,
lysing, and purifying nucleic acids from the pathogen
retentate.
[0086] Another invention of this disclosure is a self-contained
apparatus for generating cell lysate from an unprocessed sample,
said apparatus to be used with an instrument, said apparatus
comprising at least one input, and:
[0087] (i) a macrofluidic component, comprising: a chamber for
receiving said unprocessed sample from a collection device, and at
least one filled reagent storage reservoir; and
[0088] (ii) a microfluidic component in communication with said
macrofluidic component via at least one microfluidic element;
and
[0089] (iii) a drive mechanism on said instrument for driving said
reagent, through said microfluidic element, wherein the only inputs
to said apparatus are via said chamber and said drive
mechanism.
[0090] Yet another invention is a self-contained apparatus for
lysing cells from an unprocessed sample, said apparatus to be used
with an instrument, said apparatus comprising at least one input,
and:
[0091] (i) a macrofluidic component, comprising a chamber for
receiving said unprocessed sample from a collection device and at
least one pre-filled lysis storage reservoir; and
[0092] (ii) a microfluidic component in communication with said
macrofluidic component via at least one microfluidic element;
and
[0093] (iii) a drive mechanism on said instrument for driving
reagent in said storage reservoir, through said microfluidic
element,
[0094] wherein the only inputs to said apparatus are via said
chamber and said drive mechanism.
[0095] In a related inventive method for lysing cells from a sample
comprising using the apparatus, comprising at least one input, and:
providing a sample comprising cells to a chamber; introducing said
lysis reagent into the chamber to provide a mixture; bubbling a gas
through the mixture to provide a stirred mixture; wherein the
stirred mixture comprises lysed cells.
[0096] Still another invention is a self-contained apparatus for
generating a suspension of cells from an unprocessed sample, said
apparatus to be used with an instrument, said apparatus comprising
at least one input, and:
[0097] (i) a macrofluidic component, comprising: a chamber for
receiving said unprocessed sample from a collection device, and at
least one filled reagent storage reservoir storing a substantially
isotonic reagent; and
[0098] (ii) a microfluidic component in communication with said
macrofluidic component via at least one microfluidic element;
and
[0099] (iii) a drive mechanism on said instrument for driving said
reagent, through said microfluidic element, wherein the only inputs
to said apparatus are via said chamber and said drive
mechanism.
[0100] In another invention, the instruments which comprise the
claimed inventive apparatus also perform at least one of thermal
cycling, capillary electrophoresis, microfluidic electrophoresis,
nucleic acid fragment sizing, short tandem repeat (STR), Y-STR, and
mini-STR, single nucleotide polymorphism, PCR, highly multiplexed
PCR, Real-time-PCR, Reverse Transcription PCR, sequencing,
hybridization, microarray, VNTR, immunoassays, mass spectroscopy
and RFLP analyses.
[0101] In still another invention, the apparatus of the invention
can be placed into or interface with another instrument that
performs at least one of thermal cycling, capillary
electrophoresis, microfluidic electrophoresis, nucleic acid
fragment sizing, short tandem repeat (STR), Y-STR, and mini-STR,
single nucleotide polymorphism, PCR, highly multiplexed PCR,
Real-time-PCR, Reverse Transcription PCR, sequencing,
hybridization, microarray, VNTR, immunoassays, mass spectroscopy
and RFLP analyses.
[0102] It also is an invention of this disclosure that the claimed
apparatus and instruments are ruggedized to withstand transport and
extremes of at least one of temperature, humidity, and airborne
particulates.
BRIEF DESCRIPTION OF THE DRAWINGS
[0103] FIG. 1 depicts an apparatus suitable for biothreat detection
from a blood sample.
[0104] FIG. 2 is an electropherogram showing near-quantitative
recovery of B. subtilis using a purification cartridge.
[0105] FIG. 3 is a side view of a purification cartridge for blood
samples. The blood collection tube is labeled 1, the cover is
labeled 2; the macrofluidic component is labeled 3; the
microfluidic component is labeled 4; a pneumatic interface port is
labeled 5.
[0106] FIG. 4 is a side view of the macrofluidic component of a
purification cartridge for blood samples. The macrofluidic
component is labeled 3; the first wash reservoir is labeled 6; the
eluate homogenization chamber is labeled 7; the waste chamber is
labeled 8; the eluate reservoir is labeled 9; the resuspension
solution reservoir is labeled 10; the lysis chamber is labeled 11;
the ethanol reservoir is labeled 12; the lysis reservoir is labeled
13; the holding chamber is labeled 14; the second wash reservoir is
labeled 15 and the blood collection tube cavity is labeled 16.
[0107] FIG. 5 is a top view of the pneumatic layer of a
purification cartridge for blood samples. Pneumatic channels are
labeled 17; through holes to reagent reservoirs and chambers of the
macrofluidic component are labeled 18 and pneumatic interface ports
are labeled 19.
[0108] FIG. 6 is a top view of the microfluidic layer of a
purification cartridge for blood samples. Fluidic channels are
labeled 20; the track etch membrane is labeled 21; the Leukosorb
filter is labeled 22 and the purification filter is labeled 23.
[0109] FIG. 7 is a side view of a forensics cartridge. The swab cap
is labeled 24; the cover is labeled 25; the macrofluidic component
is labeled 26; the microfluidic component is labeled 27; the
pneumatic interface ports are labeled 28A, 28B, and 28C; the
instrument is labeled 1000 and the drive mechanism is labeled
1005.
[0110] FIG. 8 is a side view of the macrofluidic component of a
forensics cartridge. The macrofluidic component is labeled 26; the
wash reservoir is labeled 29; the eluate homogenization chamber is
labeled 30; the eluate reservoir is labeled 31; the swab chamber is
labeled 32; the ethanol reservoir is labeled 33; the lysis
reservoir is labeled 34 and the holding chamber is labeled 35.
[0111] FIG. 9 is a top view of the pneumatic layer of a forensics
cartridge. Pneumatic channels are labeled 36; through holes to
reagent reservoirs are labeled 37 and pneumatic interface ports are
labeled 38.
[0112] FIG. 10 is a top view of the microfluidic component of a
forensics cartridge. Fluidic channels are labeled 39; the
particulate filter is labeled 40 and the purification filter is
labeled 41.
[0113] FIG. 11 is an STR profile obtained from DNA purified from a
buccal swab.
[0114] FIG. 12 is a STR profile obtained from DNA purified from a
dried bloodstain sample.
[0115] FIG. 13 is an STR profile obtained from DNA purified from
saliva isolated from saliva.
[0116] FIG. 14 is an STR profile obtained from DNA purified from a
touch sample.
[0117] FIG. 15 is a side view of a cervical swab cartridge. The
swab cap is labeled 42; the cover is labeled 43; the macrofluidic
component is labeled 44; the microfluidic component is labeled 45
and a pneumatic interface port are labeled 46.
[0118] FIG. 16 is a side view of the macrofluidic portion of a
cervical swab cartridge. The wash reservoir is labeled 47; the
eluate homogenization chamber is labeled 48; the eluate reservoir
is labeled 49; the swab chamber is labeled 50; the ethanol
reservoir is labeled 51; the lysis reservoir is labeled 52; the
holding chamber is labeled 53.
DETAILED DESCRIPTION OF THE INVENTION
[0119] The invention provides a series of apparatus,
instrumentation, and methods that can be used to provide rapid,
efficient purification of nucleic acids from a variety of
biological sample types. As illustrated in the examples herein,
nucleic acid can be purified based on devices comprising both
macrofluidic and microfluidic features and accompanying
instrumentation. In general, the macrofluidic component of the
apparatus of this invention comprises chambers (including sample,
reagent storage reservoir, reaction, holding, homogenization, and
waste chambers) with aggregate volume of 1-1000 mL or greater and
individual volumes of 1 mL and greater. Particularly preferred are
aggregate volumes in the range of 1-250 mL. The macrofluidic
component may also optionally comprise chambers with volumes of
20-1000 .mu.L. The microfluidic component comprises microfluidic
elements with microliter and nanoliter volumes. It is preferred
that the microfluidic elements have individual volumes in the range
of 0.1-1000 .mu.L and particularly preferred that the individual
elements have volumes of 0.1 to 100 .mu.L.
[0120] The teachings of the invention can be applied to nucleic
acid purification such that the nucleic acid product can be removed
and analyzed separately or the nucleic acid can be transferred
directly to analytic modules in an integrated instrument. Types of
analysis and approaches to such integration include those described
in Tan et al., Integrated Nucleic Acid Analysis, PCT/US08/04462,
which is herein fully incorporated by reference.
[0121] The apparatus and instrumentation of the invention allow
nucleic acid to be purified from unprocessed biological samples.
Unprocessed biological samples are those that are collected by an
individual and then inserted into the sample receiving chamber of
the apparatus with no intermediate processing steps (although the
sample collection device may be labeled and/or stored prior to
processing). The operator need only collect or otherwise obtain the
sample, insert the sample into the apparatus, insert the apparatus
into the instrument (not necessary if the apparatus was previously
placed in the instrument), and press a start button. No processing,
manipulation, or modification of the sample is required prior to
insertion in the apparatus--the operator does not have to cut a
swab, open a blood tube, collect a tissues or biologic fluid,
transfer a sample to another holder, or expose the sample to a
reagent or a condition (e.g. heat, cold, vibration). Accordingly,
the operator need not have extensive training in the biological
sciences or laboratory techniques.
[0122] The apparatus of the invention are self-contained in that
the only inputs to the apparatus are via the sample receiving
chamber and the drive mechanism. As all required reagents are
present within the apparatus in pre-filled reagent storage
reservoirs, the operator is not required to add process reagents to
the apparatus. The fact that the apparatus contains all reagents
on-board is an important factor in ease of operation. Similarly, as
the instrument contains no purification process reagents, the
operator need not add reagents to the instrument. The
self-contained nature of the apparatus minimizes operating
procedures, maintenance procedures, and operator requirements.
Taken together, the self-contained apparatus and the use of
unprocessed samples dramatically simplifies the process of nucleic
acid purification. Another advantage of the self-contained
apparatus of the invention is that this format reduces both the
possibility of sample contamination as well as operator exposure to
sample, reagents, and process waste.
[0123] Furthermore, the apparatus and instrumentation of the
invention are designed to be operable outside of conventional
laboratory environments. Depending upon the application, they can
be ruggedized to withstand transport and extremes of temperature,
humidity, and airborne particulates. Use of the invention by
non-technical operators in offices, out of doors, in the
battlefield, in airports, at borders and ports, and at the
point-of-care will allow much broader application of genetic
technology in society. The use of unprocessed samples in a
self-contained apparatus further supports the broad application of
the methods of the invention.
[0124] In practice, biological samples are collected using a myriad
of collection devices, all of which can be used with the apparatus
of the invention. The collection devices will generally be
commercially available but can also be specifically designed and
manufactured for a given application. For clinical samples, a
variety of commercial swab types are available including nasal,
nasopharyngeal, buccal, oral fluid, stool, tonsil, vaginal,
cervical, and wound swabs. The dimensions and materials of the
sample collection devices vary, and the devices may contain
specialized handles, caps, scores to facilitate and direct
breakage, and collection matrices. Blood samples are collected in a
wide variety of commercially available tubes of varying volumes,
some of which contain additives (including anticoagulants such as
heparin, citrate, and EDTA), a vacuum to facilitate sample entry, a
stopper to facilitate needle insertion, and coverings to protect
the operator from exposure to the sample. Tissue and bodily fluids
(e.g. sputum, purulent material, aspirates) are also collected in
tubes, generally distinct from blood tubes. These clinical sample
collection devices are generally sent to sophisticated hospital or
commercial clinical laboratories for testing (although certain
testing such as the evaluation of throat/tonsillar swabs for rapid
streptococcal tests can be performed at the point of care).
Environmental samples may be present as filters or filter
cartridges (e.g. from air breathers, aerosols or water filtration
devices), swabs, powders, or fluids.
[0125] Collection of biological evidence from crime scenes is a
process that gathers a number of cells from a variety of surfaces,
preserves the collected cells to minimize molecular degradation,
and allows release of the collected material for downstream
processing. Blood, semen, epithelial cells, urine, saliva, stool,
various tissues, and bone can be associated with the crime scene
and require careful and effective collection (Lee, H. C. et al.,
(1998) "Forensic applications of DNA typing: part 2: collection and
preservation of DNA evidence." Am J Forensic Med Pathol 19(1):
10-8.
[0126] A common collection technique for forensic evidence is
performed using a cotton swab. A single swab is taken from an area
or a wet-dry double swab technique can be used. The double swab
technique may be the most prevalent and a number of different
fluids including water, buffered saline, or lysis buffers can be
used to moisten the first swab (Leemans, P. 2006. "Evaluation and
methodology for the isolation and analysis of LCN-DNA before and
after dactyloscopic enhancement of fingerprints." Int Congress Ser
1288: 583-5). This technique allows for dried samples to become
re-hydrated, with the majority of material collected on the first
swab and the dry second swab collecting the remainder of the
sample. In addition to cotton, the swab collection matrix can be
comprised of various materials such as natural fiber (cotton) and
synthetic matrices (modified cellulose, foam, Nylon, Polyester and
Rayon). Swabs are commercially available from Bode (Lorton Va.),
Puritan (Guilford, Me.), Fitzco (Spring Park, Minn.), Boca (Coral
Springs, Fla.), Copan (Murrieta, Calif.) and Starplex (Etobicoke,
ON, Canada). Swabbing can also be performed using gauze-like
materials, disposable brushes, or commercially available biological
sampling kits (Lauk, C. and Schaaf, J. 2007. "A new approach for
the extraction of DNA from postage stamps" Forensic Science
Communications 9(1)).
[0127] Another forensic collection technique involves taking
cuttings of the area of interest such as a biological fluid from
clothing; however this destroys the integrity of the evidence.
Adhesive tape lifts are also used on a variety of surfaces to
collect trace evidence that may contain human DNA. Cards such as
FTA cards (Whatman plc, Kent, UK) are also used to collect
samples.
[0128] Biological evidence from an individual that is present in
person is often collected using buccal swabs. A widely used
commercial buccal swab is the SecurSwab (The Bode Technology Group,
Lorton, Va.). Buccal samples are collected by instructing the
subject or operator to place the swab into the mouth on the inner
cheek surface and to move the swab up and down one or more
times.
[0129] After the unprocessed samples of the invention are
collected, if they are not processed immediately they are sometimes
allowed to dry to prevent fungal or bacterial growth. Evidentiary
samples are generally not immediately sealed in plastic, which can
result in microbial growth and cause degradation of the DNA.
Typically, swabs or cuttings are placed in breathable containers
made of paper or cardboard. Storing collected evidence in cool, dry
environments also minimizes sample deterioration (Lee, H. C. and
Ladd, C. (2001) "Preservation and Collection of Biological
Evidence" Croat Med J 42(3): 225-8). To be truly useful to the
forensic community, nucleic acid purification apparatus,
instrumentation, and methods should be able to obtain highly
purified nucleic acids from commercially available collection
devices and be compatible with accepted forensic collection and
analysis protocols.
[0130] Regardless of the type of sample, the sample receiving
chamber of the apparatus and the cover (if present) are designed to
accept and fit snugly with the sample collection device. In the
case of samples such as cloth or adhesive tape (e.g. sample
collection devices that have no handle or cap), following their
placement into the chamber, a snug-fitting cap is placed on the
cover to close the chamber. Depending on application, the sample
collection device can be locked (reversibly or irreversibly) into
the apparatus. Furthermore, the device and apparatus can form a
seal (air- and water-tight); in this case, a vent or vent membrane
may be placed to allow fluid flow into the chamber. Unless
otherwise specified, the chambers of the apparatus that receive
fluid from elsewhere on the apparatus must contain vents or vent
membranes to allow for air to escape during chamber filling.
[0131] The apparatus of the invention comprise a macrofluidic
component and a microfluidic component in communication with one
another. The macrofluidic component comprises a sample chamber for
receiving a biological sample from a sample collection device, and
other chambers that may include reservoirs for purification
reagents, holding chambers, homogenization chambers, metering
chambers, reaction chambers, mixing chambers, and waste chambers.
The microfluidic component comprises a chamber comprising a nucleic
acid purification media and at least one microfluidic feature and
one pneumatic drive-line. The macrofluidic chambers are in
communication with microfluidic features and the macrofluidic
chambers are in communication with each other via the microfluidic
component. Fluids pass from one macrofluidic chamber through the
microfluidic component back to another macrofluidic chamber. The
volumes of the chambers are determined by the use of the purified
nucleic acids. For example, elution reservoir volume is chosen to
allow the final concentration of the purified nucleic acid to be
optimal for subsequent reactions.
[0132] Following the purification processes in the apparatus of the
present invention, the nucleic acid solution provided may be
transferred for additional analytic steps. The nucleic acid
solution may be automatically transferred to other analytic modules
within the same instrument, or the apparatus itself may be
transferred to a compatible instrument. Alternatively, the chamber
that collects the purified nucleic acid sample may contain a
removable nucleic acid storage tube. Samples processed according to
this invention may be a precursor to a wide variety of analytical
methods, including without limitation nucleic acid fragment sizing,
short tandem repeat (STR), Y-STR, and mini-STR, single nucleotide
polymorphism, PCR, highly multiplexed PCR, Real-time-PCR, Reverse
Transcription PCR, sequencing, hybridization, microarray, VNTR, and
RFLP analyses. Similarly, the apparatus, methods, and instruments
of the invention can also be applied to immunoassays and protein
and mass spectroscopy assays in general and other analytical
methods well known to those skilled in the art.
[0133] The apparatus of the invention may also have an optional
cover to route channels between the drive mechanism of the
instrument and each of the individual chambers. In addition, the
cover also provides optional functions of venting gases within the
chambers to the environment and locking sample collection device
following insertion. The cover comprises at least one layer,
preferably fabricated of plastic. Additional layers can be added as
the number of pneumatic channels or the complexity of routing and
other features increases. Layer features can be macrofluidic or
microfluidic, and features can be fabricated by CNC machining, hot
embossing patterns, die cutting, or laser cutting of plastic
sheets, or injection molding of thermoplastic resin. In addition
the incorporation of vent membranes into the layer can be achieved
by welding and bonding. When two or more layers are required, the
individual layers are bonded together to form a single part.
Bonding methods for fabrication of the cover include thermal
bonding, solvent bonding, ultrasonic bonding, adhesive and laser
bonding.
[0134] The microfluidic component of the apparatus may contain a
variety of fine features or microfluidic elements, including
channels (which may be independent, connected, or networked),
reservoirs, valves, reaction chambers, liquid and lyophilized
reagent storage chambers, mixing chambers, mixing elements, venting
elements, access holes, pumps, metering elements, heating elements,
magnetic elements, reaction chambers, filtration elements,
purification elements, drive lines, actuation lines, optical
excitation and detection regions, optical windows. The microfluidic
component of the apparatus may use valves for flow control to halt
or allow flow of fluids within channels. Valves can be passive or,
most preferably, active, and valving approaches for microfluidic
devices are well known in the art (reviewed in Zhang, C., et al.
(2007) "Micropumps, microvalves, and micromixers within PCR
microfluidic chips: Advances and trends." Biotechnol Adv 25(5):
483-514). Active valve structures include mechanical
(thermopneumatic and shape memory alloy), non-mechanical (hydrogel,
sol-gel, paraffin, and ice), and external (modular built-in,
pneumatic, and non-pneumatic) microvalves. The pneumatic and
mechanical microvalve structures can also apply either elastomeric
or non-elastomeric membranes. Passive valves include in-line
polymerized gel, passive plug, and hydrophobic valves.
[0135] The fluids required for the methods of the invention are
nucleic acid purification reagents and gasses (e.g. air, nitrogen,
or oxygen). The purification reagents and media can be based on any
of the well-characterized methods of the literature, including
silica matrices/chaotropic agents (Boom, R. et al., (1990), supra),
silica matrices/non-chaotropic agents, ion exchange, and many
others as well known in the art. Many such methods are summarized
in Current Protocols in Molecular Biology (Edited by Ausubel et al,
John Wiley and Sons, 2010). Similarly, many types of purification
media can be used in the apparatus. Silica nucleic acid binding
membranes, for example, vary in size, pore size, flow rate,
retention volume, reagent compatibility, and binding capacity;
appropriate membranes are chosen based on a given application. Cell
separation media are also selected based on the physical and
chemical properties of the cellular material to be separated.
Finally, in some cases, particle removal filters may be used,
preferably to remove particulates that may inhibit, slow down, or
otherwise interfere with a downstream separation or purification
process. In many forensic embodiments, particle removal filters are
preferred.
[0136] The drive mechanism to allow fluid transport throughout the
apparatus can be pneumatic, mechanical, magnetic, fluidic, or any
other means that allows precise control of the fluid movement. A
pneumatic drive allows controlled flow or a controlled pressure or
a controlled volumetric displacement of air (or other gasses) to
the apparatus via one or more drive lines, and are particularly
preferred. The pneumatic drive lines can be utilized to move
liquids, create bubbles, burst foils, actuate mechanical features,
and perform any other movements required for a given nucleic acid
purification method. The drive of the instrument must interface
with the drive lines of the apparatus. For a pneumatic drive, the
interface may be located at one or more macrofluidic or
microfluidic regions of the apparatus. The drive is contained
within the instrument, which may also contain a power supply, a
housing to accept the apparatus, features that allow ruggedization
and protection from environmental exposure, an on-board computer, a
process controller, a monitor, and other features based on the
nucleic acid analysis to be conducted. The pneumatic drive system
may contain the following components: pumps, electromechanical
valves, pressure regulators, pressure tanks, tubing, pneumatic
manifolds, and flow and pressure sensors. The pneumatic drive
system allows the generation and deliver of a defined flow,
pressure, or volume to each of the pneumatic lines of the
apparatus. A process controller can execute a programmed script
following insertion of the unprocessed sample into the apparatus.
More than one class of drive mechanism can be utilized with the
apparatus. However, the use of a single drive mechanism, preferably
pneumatic, reduces the complexity of both the instrument and the
apparatus.
[0137] Once in the sample chamber, the biological sample may be
lysed by a number of methods. Chaotic bubbling is caused by the
flow of fluid, preferably air, into a chamber of the macrofluidic
component. The flow may be turbulent, which may contribute shearing
forces that would contribute to cell lysis and which may be
appropriate for mixing or homogenization of reagents. Other
approaches to potentiating lysis include mechanical actuation by
vibration, ultrasonic actuation, and heat.
[0138] In one embodiment of the invention, the nucleic acid to be
purified is DNA. Other embodiments are based on the purification of
RNA and total nucleic acids. The reagents required to purify DNA,
RNA, and total nucleic acids are well-known in the art. See, e.g.,
Gjerde, D. T. et al., RNA Purification & Analysis: Sample
Preparation, Extraction, Chromatography (2009 Wiley-VCH Pub.);
Ausubel, F. M. et al., (Eds)., Current Protocols in Molecular
Biology (2008 John Wiley Pub.). In another embodiment of the
invention, the apparatus contains macrofluidic and microfluidic
elements that allow cell separation. These elements may allow a
variety of cell separations including white blood cells (WBC) to be
separated from red blood cells, bacteria or viruses to be separated
from host cells, sperm cells to be separated from vaginal
epithelial cells, and intracellular viruses and bacteria to be
separated from their mammalian hosts.
[0139] The apparatus of the invention can be fabricated in several
ways. Based on the time and cost allotted for fabrication and the
number of apparatus to be fabricated a variety of methods are
available. The apparatus may be fabricated out of glass or more
preferably, out of thermoplastic polymers such as polyethylene,
polypropylene, polycarbonate, polystyrene, cyclic olefin polymer,
and cyclic olefin copolymer. The apparatus may be fabricated in one
or more parts, macrofluidic and microfluidic. If the apparatus is
made of plastic parts, the components may be bonded together using
clamping, thermal bonding, ultrasonic bonding, solvent bonding,
laser bonding, or adhesive bonding (bonding methods are reviewed in
Tsao and DeVoe, Microfluid Nanofluid (2009) 6:1-16). A rapid and
straightforward method of fabrication is by
computer-numerical-controlled machining. Other methods include blow
molding, extrusion, and embossing.
[0140] A preferred method of fabrication is by injection molding.
The macrofluidic portion of the apparatus of the invention
comprises of a set of chambers of tubular structure that may be
injection molded together to form a single part. The top surfaces
of each chamber are preferably coupled pneumatically to a cover to
provide pneumatic drive to each of the individual chambers. The
bottom surfaces of each chamber are preferably coupled
pneumatically and fluidically to the microfluidic component.
Tube-like structures that have been injection molded as a single
part include microtiter plates with 96, 384 and 1536 wells.
Microcentrifuge plates with 96 wells in a 12.times.8 configuration
8.4 mm in diameter and 16 mm deep is described by Turner (U.S. Pat.
No. 6,340,589). While these plates have a high density of tubular
structures in high packing density, the depth of these tubes are
not more 16 mm. PCR tubes-tube strips with 8 or 12 tubes have been
fabricated in-line by injection molding, with each tube being 8.65
mm in diameter and 30 mm deep with a capability to hold 0.2 mL.
These are all coupled versions of the plastic reaction vessel
described by Gerken (U.S. Pat. No. 4,713,219). These strip tubes
have a low packing density, and are oriented in-line. Finally,
injection molding of two long coupled tubes are described by Spehar
(U.S. Pat. No. 4,753,536).
[0141] The tubular structures of the macrofluidic portion have thin
walls. When injection molded, the macrofluidic portion is
essentially a series of thin walled tubes held together as opposed
to a solid block with tubes drilled out. The tubes have wall
thicknesses of 0.1-5.0 mm, preferably 0.3 to 3.0 mm, more
preferably 0.5 to 1.5 mm, still more preferably 0.7 to 1.3 mm, and
most preferably 0.9 to 1.2 mm.
[0142] The injection molded tubular structures of the macrofluidic
portion preferably have a tube length in excess of 16 mm, 18 mm, 20
mm, 25 mm, 30 mm, 40 mm, 50 mm, 60 mm, 70 mm, 80 mm, 90 mm, or 100
mm. The tubular structures are oriented in a two dimensional
configuration. The tops of the tubular structures must be flat to
achieve strong pneumatic coupling to optional cover, and the
bottoms of the tubular structures must be flat to achieve strong
pneumatic and fluidic coupling to the microfluidic portion. Spacing
of the tubular structures must be maintained to accurately match
the footprint of the microfluidic portion. The tubular structures
may taper from top to bottom to facilitate precise interfacing with
the microfluidic component. Similarly, the form of the tubular
structure may be adapted for a particular purpose, such as a
narrowed lower portion to facilitate chaotic bubbling and mixing or
a narrowed central or upper portion to maintain the position of a
sample collection device.
[0143] The tubular structures of the macrofluidic component are
packed densely, with one tube present per approximately 200
mm.sup.2 surface area at the top of the component, more preferably
approximately 150 mm.sup.2. For other applications, one tube is
present, preferably, per approximately 100 mm.sup.2 surface area at
the top of the component, and most preferably one tube is present
per approximately 50 mm.sup.2 surface area at the top of the
component. Similarly, the surface area occupied by the tubular
structures as compared to the total area at the top of the
macrofluidic component is greater than 30%, more preferably greater
than 40%. For other applications, greater than 50%, for other
applications still more preferably greater than 60%, and most
preferably greater than 90%.
[0144] The total volume of the apparatus is based in part on the
number and volume of chambers and the number of samples to be
processed simultaneously. The volume will be at least 2 mL, and may
be 45 mL, 65 mL, 100 mL, 500 mL, 1000 mL, 1500 mL or more, and
preferably in the range of 45-1500 mL, and even more preferred in
the range of 65-1500 mL.
[0145] The apparatus of the invention can accept and process one or
more samples. For some embodiments the apparatus may be configured
to accept 2, 4, 8, 16, 24, 36, 48, 96, 192, or 384 samples. As the
number of units increases, the approach to manufacturing may
change. For example, a single sample unit fabricated by injection
molding may be used as the basis for a 5-sample apparatus. Sets of
5-sample apparatus can be bonded, generating 10-sample, 15-sample
apparatus. Alternatively, a 15-sample apparatus can be manufactured
as a single large unit. The apparatus, instruments, and methods of
the invention allow the rapid purification of nucleic acids. From
the time the process is initiated following insertion of the
unprocessed sample to the time purified nucleic acids from the
sample are generated is preferably less than 30 minutes, more
preferably less than 20 minutes, even more preferably less than 10
minutes, and most preferably less than 5 minutes.
Examples
Example I
Extracellular Bacteria Present in Blood
[0146] Pathogens such as staphylococci, streptococci, and Yersinia
enterocolitica may be present in the blood. In some cases, it is
advantageous to isolate extracellular pathogens from the cellular
elements of human (or other animal host) blood. For example, in
order to make best use of the advantages of a microfluidic device,
an ideal volume for the purified DNA that is the end product of the
DNA extraction/purification module is 25 .mu.L or less. This volume
can be quickly transferred and manipulated on a microfluidic chip.
By limiting this volume, however, an analogous limit is also placed
on the maximum amount of DNA that can be present within that
volume. In 3 mL of whole blood, assume a total of 15 million white
blood cells and 150 bacteria (50 per mL). The total DNA in this
sample is approximately 90 .mu.g, with essentially all of this due
to leukocyte DNA. If this DNA were purified and recovered with 100%
efficiency in a solution of 25 .mu.L, the DNA concentration would
be 3.6 mg/mL, almost certainly inhibitory for PCR (F. B. Cogswell,
C. E. Bantar, T. G. Hughes, Y. Gu, and M. T. Philipp (1996) "Host
DNA can interfere with detection of Borrelia burgdorferi in skin
biopsy specimens by PCR" J Clin Microbiology 34:980-982) and too
viscous for microfluidic manipulation. In contrast, the small
amount of bacterial DNA--only 250 genomes of approximately 5
Mbp/genome--present in 25 .mu.L would be approximately 1 pg. If
only one-tenth of the total DNA were to be used for the
microfluidic reaction, the limit of detection would by definition
decrease ten-fold. As blood volume increases, the number of
leukocytes per unit blood volume increases, the microfluidic
solution volume decreases, and the number of organisms per sample
decrease, this problem becomes even more severe. The conclusion
from this analysis is that in certain applications (particularly
those in which bacterial load is low early in an infection), most
of the leukocyte DNA should be removed before the final
microfluidic volume is reached. This would allow most or all of the
pathogen DNA to be analyzed following purification. Similarly,
background environmental DNA such as that which accumulates on the
filters of air breathers can interfere with the sensitivity and
specificity of pathogen identification.
[0147] Removal of leukocytes in 3 mL of fresh human whole blood was
achieved by stacking 13 layers of binding media with nominal pore
size of 8 .mu.m (Leukosorb B media, Pall Corporation, Port
Washington, N.Y.) and filtered using initial vacuum pressure of
0.25 psi and then was increased to 25 psi for final collection of
filtrate. Useful pore sizes of binding media can range from less
than 1 micron to over 100 microns, depending on the type of cells,
virions, bacteria, fungi, and particulates to be separated.
Recovered volume was approximately 1.5 mL with filtration completed
in 1 minute. WBC counting of filtrates indicated that greater than
99% of leukocytes were retained by the filter.
[0148] 3 mL of fresh human whole blood samples were spiked with 100
.mu.L of B. subtilis (ATCC.RTM. 7003.TM.), with each 100 .mu.L
containing varying concentrations of B. subtilis per sample. In
this experiment, B. subtilis was used as a model for pathogenic
organisms and biothreat agents (e.g. B. anthracis). These
blood-bacteria samples were passed through the stacked media using
the apparatus of FIG. 1. Sample application was followed by wash
with 3 mL TSB (Tryptic Soy Broth media), allowing retrieval of
bacteria that did not initially pass through the binding matrix.
Collected flow-through of approximately 4.5 mL was passed through a
single layer of 0.2 .mu.m polycarbonate track-etch membrane
(SPI-Pore.TM. Track-Etch Membrane, Structure Probe, Inc., West
Chester, Pa.) to concentrate the bacteria through capture on the
membrane. This concentration method reduced reagent volumes, sizes
of the purification cartridge reservoir chambers, and process time.
The captured organisms were collected from the surface of the
membrane by resuspending in 100 .mu.L PBS (Phosphate-buffered
saline).
[0149] In this embodiment, the lysis of bacterial cells was based
on chaotropic salt extraction method for DNA and RNA. In
particular, the preferred lysis buffer solution contained 4M
guanidinium hydrochloride, 80 mM Tris-HCL (pH 7.5), 20 mM EDTA and
5% Triton X-100 (other useful lysis buffers are described in
Ausubel et al., supra). To 100 .mu.L of resuspended organisms, 450
.mu.L of lysis buffer with 1 mg/mL final concentration of
proteinase K was transferred and mixed thoroughly in the
purification cartridge through the chaotic bubbling method defined
in the pneumatic script. To this, 550 .mu.L of absolute (200 proof)
ethanol was added and again mixed by bubbling. The lysate was
passed microfluidically through a silica-based membrane for DNA
binding in the microfluidic portion of the purification apparatus.
After the entire lysate was filtered, the membrane is washed with 2
mL of 1.times. wash solution prepared by mixing 1 unit volume of
200 mM NaCL solution, 0.5 unit volume of 200 proof ethanol and 0.5
unit volume of >99% isopropanol. Following wash step, the
membrane was then allowed to dry for 1 minute by exposure to air
from the pneumatic system. DNA was finally eluted in 20 .mu.L TE
buffer, pH 8.0. Pressures for lysate filtration, membrane washing
and drying, and elution were approximately 5 psi. Fast PCR
amplification (Giese, H. et al., (2009), "Fast multiplexed
polymerase chain reaction for conventional and microfluidic short
tandem repeat analysis" J Forensic Sci 54(6): 1287-97) using glnA
(glutamine synthetase) primers in a microfluidic biochip and
separated and detected microfluidically results in the expected
343-bp fragment characteristic of the B. subtilis glnA gene with
signal intensity proportional to the input copies in blood samples.
PCR was performed using biochips and a fast thermal cycler as
described in "Methods for Rapid Multiplexed Amplification of Target
Nucleotides," PCT/US08/04487, which is hereby incorporated by
reference. Separation and detection were performed on Genebench as
described in "Plastic Microfluidic Separation and Detection
Platforms," PCT/US08/04405, and "Integrated Nucleic Acid Analysis,"
PCT/US08/04462, both of which are hereby incorporated by reference.
FIG. 2 shows an electropherogram showing approximately 2 genome
equivalents of B. subtilis; this represents the amplification of
only .about.6% of the total material recovered from a 33 cfu/mL
blood sample and using 40% of the PCR product for electrophoretic
analysis. Bacterial recovery is near-quantitative.
[0150] An alternative method for quantitation was to use a
Petroff-Hausser chamber. Collected flow-through was plated on
TSB-agar plates to determine the effect of filtration through
stacked media on the recovery of bacteria. Recovered bacteria are
normalized using plating efficiency based on colonies recovered in
unfiltered control samples. At clinical relevant concentrations of
bacteria in blood, approximately 100% of the bacteria were
recovered.
TABLE-US-00001 Expected Bacteria # of Colonies Recovered by % %
Recovery in 3 mL of Blood Plating Filtered Samples Recovery
Normalized ~1000 860 .+-. 239 85 .+-. 18 104 .+-. 10 ~100 88 .+-.
16 88 .+-. 11 97 .+-. 17 ~10 9 .+-. 2 96 .+-. 19 103 .+-. 26
[0151] FIG. 3 shows an integrated purification cartridge for blood
samples. FIGS. 4, 5, and 6 show the macrofluidic component,
pneumatic layer of the microfluidic component, microfluidic layer
of the microfluidic component, respectively, of the integrated
purification cartridge. The macrofluidic portion [3] of the
apparatus is composed of 11 chambers, [6] to [16], that hold
preloaded reagent solutions or serve as holding/reaction chambers
during the DNA purification process. One chamber is used to accept
the blood collection tube; six chambers are pre-filled with 3 mL of
wash buffer, 100 .mu.L of resuspension solution, 450 .mu.L of lysis
solution, 550 .mu.L of absolute ethanol, 2 mL of wash buffer and 20
.mu.L of TE (pH 8) elution buffer.
[0152] The apparatus accepts a standard 3 cc vacutainer tube (for
separation experiments, blood should be collected in tubes
containing appropriate anticoagulants. The blood collection tube
[1] is inserted into the cartridge with the rubber stoppered end
down. The purification process is initiated when the user presses a
start button. The apparatus together with the instrument execute an
automated script and generate purified DNA. Within the instrument,
the blood collection tube is pushed onto two hollow pins located at
the base of blood collection tube cavity [16]. The hollow pins
pierce through the rubber stopper to fluidically and pneumatically
couple the blood collection tube to the apparatus. The blood
collection tube [16] is pressurized pneumatically to 5 psi to drive
the blood from the blood collection tube [16] through the leukosorb
filter [22] and track-etch membrane [21] to the waste chamber [8].
The filtrate that passes through the leukosorb filter contains the
biological material (e.g. bacterial, viral, or fungal pathogens)
for analysis, a leukocyte-reduced filtrate. This filtrate is then
driven through a track-etch membrane, and the pathogens of interest
are retained by the membrane (the pore size of the pathogen capture
membrane is elected based on the dimensions of the pathogens to be
analyzed). Wash solution from wash reservoir 2 [15] is
pneumatically driven through the leukosorb filter [22] and
track-etch membrane [21] to the waste chamber [8]. Resuspension
solution from the resuspension solution reservoir [4-10] is applied
to the surface of the track-etch membrane [6-21]. This solution
will resuspend the pathogens retained on the track etch membrane,
generating a concentrated pathogen suspension (which may also
include residual leukocytes). This suspension is pneumatically
driven into the lysis/waste chamber [11]. Lysis reagent is
pneumatically driven into the lysis chamber [11]. Air is
pneumatically driven into the lysis/waste chamber [11] to effect
chaotic bubbling of the lysate within the lysis/waste chamber [11].
This bubbling creates flow of the lysate to mediate cell lysis.
Ethanol from the ethanol reservoir [12] is driven into the
lysis/waste chamber [11]. Continued application of pneumatic drive
through the ethanol reservoir [12] after all the ethanol has been
dispensed forces air through the lysate and ethanol solution to
effect mixing by chaotic bubbling. All the lysate and ethanol
mixture is pneumatically driven into the holding chamber [11]. From
the holding chamber [11] the lysate and ethanol mixture is
pneumatically driven through the purification membrane [23] and
into the lysis/waste chamber [11]. Wash solution from wash
reservoir 1 [6] is pneumatically driven through the purification
membrane [23] and into the lysis/waste chamber [11]. This wash
removes unbound material and residual lysis solution. Continued
application of pneumatic drive through the wash chamber [6] after
all the wash solution has been dispensed will force air through the
purification filter and dry the filter. Elution solution is
pneumatically driven from the eluate reservoir [9] through the
purification membrane [6-23] to the eluate homogenization chamber
[7]. Continued application of pneumatic drive through the eluate
reservoir [9] after all elution solution has been dispensed will
force air through eluate homogenization chamber [7] to effect
mixing by chaotic bubbling. Homogenized purified DNA solution in
the eluate homogenization chamber [7] is ready for subsequent
analysis.
Example II
Intracellular Bacteria Present in Blood
[0153] Certain bacteria such as Francisella tularenis and Chlamydia
trachomastis spend a significant portion of their life cycles
within mammalian cells. Some are obligate intracellular organisms
and others are optionally intracellular. A comprehensive summary of
known human pathogens is provided by Gorbach, S. L. (et al. Eds.)
Infectious Disease (3rd Ed), (2004 Lippincott Williams &
Wilkins Pub). The DNA purification process for such intracellular
bacteria in blood is similar to that of extracellular bacteria with
a major exception. Following application of whole blood on and
through the cell separation filter, the leukocytes trapped by the
filter contain the DNA of interest. The filter is washed,
resuspended in 100 .mu.L, and subjected to guanidinium-based
purification as described in Example I with corresponding reduction
is reagent volume.
[0154] If desired, the apparatus can be design to initially lyse
the leukocytes (osmotically, for example), taking advantage of the
relative ease of lysis of mammalian cells as compared to bacteria.
In this setting, the intact intracellular bacteria are released,
and the cell extract is based through a bacterial capture filter
and washed. Bacterial DNA is then purified as described in Example
I. Similarly, whole blood can be lysed in the absence of cell
separation, allowing extracellular or intracellular bacterial or
viral DNA to be purified.
Example III
Purification of DNA from Biological Sample(s) Collected by a
Validated Forensic Collection Swab
[0155] Forensic samples can be broadly divided into two types;
casework samples are those that are collected at a crime scene or
in connection with an investigation, and reference samples are
collected directly from an individual. Several collection methods
are available based on the specific type of sample to be analyzed
and are designed to obtain and protect biological evidence from the
crime scene. Swabbing is a well-established forensic sample
collection method, and commercially available swabs have collection
matrices consisting of various materials such as cotton, modified
cellulose, foam, nylon, polyester and rayon.
[0156] FIG. 7 shows a purification cartridge for forensic swab
samples. FIGS. 8-10 show the macrofluidic component, pneumatic
layer of the microfluidic component, and the microfluidic layer of
the microfluidic component of the purification cartridge. The
microfluidic portion [27] of the purification cartridge contains
valves to control the flow of the solutions to and from the
macrofluidic portion [26], a particulate filter [40], and a
purification filter [41].
[0157] To purify DNA from a forensic swab sample, a BodeSecur swab
was manually inserted into the sample collection chamber of the
purification cartridge and was locked into place for sample
processing. The chamber that accepts the swab was designed to allow
the swab cap to fit snugly. The cartridge was designed to allow the
operator to insert the swab into the chamber and initiate DNA
purification without further user manipulation.
[0158] The macrofluidic portion of the purification cartridge was
composed of 7 chambers that hold preloaded reagent solutions or
serve as holding/reaction chambers during the DNA purification
process. One chamber was used to hold the cotton swab with the DNA
sample; four chambers were pre-filled with 550 .mu.L of lysis
solution, 550 .mu.L of absolute ethanol, 2 mL of wash buffer and
100 .mu.L of TE (pH 8) elution buffer. The microfluidic portion of
the purification cartridge contained valves to control the flow of
the solutions to and from the macrofluidic portion, a particulate
filter, and a purification filter.
[0159] The sample collection swab (Bode SecurSwab) [24] is
comprised of a cap, cotton swab head, and a shaft connecting the
two; the total length of this sample collection device is
approximately 9.1 cm. The swab head has a nominal dimension of 5 mm
to 5.1 mm in diameter and is approximately 12 mm long. When the
SecurSwab is inserted into the apparatus, the swab head enters a
tubular section of the sample chamber and is positioned between 0
mm to 1.5 mm from the bottom of the sample chamber. The tubular
section is 5.85 mm in diameter and 24 mm in length. An air inlet
port that is 1 mm in diameter is located at the bottom of the
tubular section. The diameter of the inlet port (between 0.1 mm and
2.5 mm and preferably between 0.7 mm and 1.3 mm) and the dimensions
of the tubular section of the sample chamber can be modified to
optimize fluid flow and chaotic bubbling.
[0160] The purification process was initiated by simply pressing a
button that starts the automated script that controls the pneumatic
drive [1005] on the instrument [1000]. The pneumatic drive [1005]
applies the required pressures and vacuums for the required times
to enable all process steps to be conducted automatically, without
user intervention. Lysis solution was pneumatically driven through
interface port [28B] from the lysis reagent reservoir [34] into the
swab chamber [32] and brought in contact with the swab. Continued
application of pneumatic drive through interface port [28B] through
the lysis reservoir [34] after all the lysis reagent had been
dispensed forced air through swab chamber effect "chaotic
bubbling". This was carried out, by the application of 5.7 psi
pressure for 60 seconds. This bubbling created turbulent flow
around the swab head, mediating cell lysis and the removal of
cellular material from the swab head. Ethanol from the ethanol
reservoir [33] was driven into the swab chamber [32]. Continued
application of pneumatic drive through the ethanol reservoir [32]
after all the ethanol had been dispensed forced air through the
lysate and ethanol solution to effect mixing by chaotic bubbling
for 30 seconds. All of the lysate and ethanol mixture was
pneumatically driven in through interface port [280] through a
particulate filter [40] into the holding chamber [35]. From the
holding chamber [35] the lysate and ethanol mixture was
pneumatically driven in through interface port [28A] through the
purification membrane [41] and into the swab chamber [32]. The swab
chamber now served as a waste chamber for spent process reagents.
Wash solution from wash reservoir [29] was pneumatically driven
through the purification membrane [41] and into the swab chamber
[32]. Washing of the purification membrane with wash buffer was
conducted to remove unbound material (including protein) and
residual lysis solution. Continued application of pneumatic drive
via interface port [28A] through the wash reservoir [29] after all
the wash solution had been dispensed forced air through the
purification filter [41] (i.e., purification filter comprising
nucleic acid purification matrix) and dried the filter for 105
seconds. Elution solution was pneumatically driven from the eluate
reservoir [31] through the purification membrane [41] to the eluate
homogenization chamber [30]. Continued application of pneumatic
drive through the eluate reservoir [31] after all elution solution
had been dispensed forced air through eluate homogenization chamber
[30] to effect mixing by chaotic bubbling. Homogenized purified DNA
solution in the eluate homogenization chamber [30] was ready for
subsequent analysis.
[0161] To evaluate the DNA generated by the purification cartridge,
rapid multiplex PCR reactions were performed as described in Geise
et al. 2009 (supra) using AmpFlSTR.RTM. Identifiler.RTM. primers
(Life Technologies) in a volume of 7 .mu.L in approximately 17
minutes. Amplified products were separated and detected using
NetBio's Genebench. To 2.7 .mu.L of each amplified product 10.2
.mu.L Hi-Di formamide and 0.1 .mu.L of Genescan 500 LIZ internal
lane standard (both Life Technologies) were added. After
denaturation at 95.degree. C. for 3 min and snap cooling on ice,
samples were loaded into the wells of the separation biochip and
electrophoretically moved into the separation channels by applying
a 350 V/cm electric field for 90 seconds. This was followed by the
application of a 150 V/cm electric field along the separation
channel to separate the DNA fragments. All separations were carried
out at 50.degree. C. Raw data were analyzed with the
GeneMarker.RTM. HID STR Human Identification Software, Version 1.51
(SoftGenetics LLC, State College, Pa.).
[0162] Full allelic profiles from various swab samples (buccal
swabs, dried and wet whole blood in swabs, saliva and cellular
touch) were generated. Buccal cell samples (FIG. 11) are obtained
by lightly scraping the swabs on the inside cheek of a human
subject. Dried blood sample are prepared by swabbing dried
bloodstains (FIG. 12). Saliva samples (FIG. 13) are collected by
swabbing saliva present on a ceramic tile. Touch samples (FIG. 14)
are prepared by swabbing a ceramic tile that was handled by a
single donor. The swab head was pre-wet with sterile DI water.
Example IV
Bacterial DNA from a Vaginal Swab
[0163] A vaginal swab is inserted into the purification cartridge
sample chamber through a clamping port to hold the swab in place.
The purification is essentially the same as that described for
forensic swabs in Example III; the main difference is that the
sample chamber is modified to accept and secure the vaginal swab.
The geometry of the swab chamber can be modified to accommodate
essentially any swab type, regardless of the dimensions of the swab
handle or collection region of the swab. The sample chamber is
designed such that the swab can be directly inserted into the
purification cartridge for processing. The cap of the swab may be
modified to lock irreversibly to minimize the possibility of
sample-to-sample contamination, and the swab assembly may be
modified to allow sample identification (e.g. by bar-code or RFID
chip).
[0164] FIG. 15 shows a purification cartridge for vaginal or
cervical swab samples and FIG. 16 shows the macrofluidic portion of
that cartridge. The microfluidic layers are essentially the same as
those of FIGS. 9 and 10.
[0165] The macrofluidic portion of the purification cartridge is
composed of 7 chambers that hold preloaded reagent solutions or
serve as holding/reaction chambers during the DNA purification
process. One chamber is used to hold the cotton swab with the DNA
sample; four chambers are pre-filled with 550 .mu.L of lysis
solution, 550 .mu.L of absolute ethanol, 2 mL of wash buffer and
100 .mu.L of TE (pH 8) elution buffer.
[0166] The purification process is initiated by simply pressing a
button that starts the automated script that controls the pneumatic
drive. The pneumatic drive applies the required pressures and
vacuums for the required times to enable all process steps to be
conducted automatically, without user intervention. Lysis solution
is pneumatically driven from the lysis reagent reservoir [52] into
the swab chamber [50] and brought in contact with the swab.
Continued application of pneumatic drive through the lysis
reservoir [52] after all lysis reagent has been dispensed will
force air through swab chamber effect "chaotic bubbling" at 5 psi
for 60 seconds. This bubbling creates turbulent flow around the
swab head, mediating cell lysis and the removal of cellular
material from the swab head. Ethanol from the ethanol reservoir
[51] is driven into the swab chamber [50]. Continued application of
pneumatic drive through the ethanol reservoir [51] after all the
ethanol has been dispensed will force air through the lysate and
ethanol solution to effect mixing by chaotic bubbling for 30
seconds. All the lysate and ethanol mixture is pneumatically driven
through a particulate filter [40] into the holding chamber [35].
From the holding chamber [35] the lysate and ethanol mixture is
pneumatically driven through the purification membrane and into the
swab chamber [50]. The swab chamber now serves as a waste chamber
for spent process reagents. Wash solution from wash reservoir [47]
is pneumatically driven through the purification membrane and into
the swab chamber [50]. Washing of the purification membrane with
wash buffer is conducted to remove unbound material (including
protein) and residual lysis solution. Continued application of
pneumatic drive through the wash reservoir [47] after all the wash
solution has been dispensed will force air through the purification
filter and dry the filter for 105 seconds. Elution solution is
pneumatically driven from the eluate reservoir [49] through the
purification membrane to the eluate homogenization chamber [48].
Continued application of pneumatic drive through the eluate
reservoir [49] after all elution solution has been dispensed will
force air through eluate homogenization chamber [48] to effect
mixing by chaotic bubbling. Homogenized purified DNA solution in
the eluate homogenization chamber [48] is ready for subsequent
analysis.
[0167] Total nucleic acid concentration is quantified by absorbance
at 260 nm. Fast PCR amplification in biochip using
fluorescently-labeled primer sets specific for sexually transmitted
diseases (including Chylamdia trachomatis, human immunodeficiency
virus, Trichomonas vaginalis, Neisseria gonorrhoeae) and
electrophoretic separation and detection in Genebench generate
bands characteristic of the pathogen causing either symptomatic or
asymptomatic infection.
[0168] While these inventions have been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
the form and details may be made therein without departing from the
spirit and scope of the inventions, as described in the appended
claims.
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