U.S. patent application number 13/414592 was filed with the patent office on 2012-09-13 for methods and compositions for isolating nucleic acid.
This patent application is currently assigned to NORTHWESTERN UNIVERSITY. Invention is credited to Sujit Jangam, David M. Kelso, Sally McFall, Douglas Yamada.
Application Number | 20120231466 13/414592 |
Document ID | / |
Family ID | 42074177 |
Filed Date | 2012-09-13 |
United States Patent
Application |
20120231466 |
Kind Code |
A1 |
Kelso; David M. ; et
al. |
September 13, 2012 |
METHODS AND COMPOSITIONS FOR ISOLATING NUCLEIC ACID
Abstract
The present invention relates to compositions and methods for
isolating and purifying nucleic acid. In particular, the present
invention relates to methods of isolating nucleic acid from cells
for use in further analysis.
Inventors: |
Kelso; David M.; (Wilmette,
IL) ; Jangam; Sujit; (Chicago, CA) ; Yamada;
Douglas; (Los Angeles, CA) ; McFall; Sally;
(Evanston, IL) |
Assignee: |
NORTHWESTERN UNIVERSITY
EVANSTON
IL
|
Family ID: |
42074177 |
Appl. No.: |
13/414592 |
Filed: |
March 7, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12570542 |
Sep 30, 2009 |
8148071 |
|
|
13414592 |
|
|
|
|
61101236 |
Sep 30, 2008 |
|
|
|
Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12N 15/1017
20130101 |
Class at
Publication: |
435/6.12 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method for processing a blood sample, comprising: contacting a
blood sample with a device comprising a sample collection membrane
with a pore size configured to retain nucleic acid and allow lysed
red blood cells to flow through; treating the membrane with a
buffer that causes lyses of red blood cells in the blood sample;
drying the membrane to provide a dried, treated membrane.
2. The method of claim 1, wherein the treating comprises treating
with a buffer that lyses red blood cells and leaves purified white
blood cells or components thereof on the membrane.
3. The method of claim 1, wherein said contacting comprises
contacting a blood sample with a device comprising a sample
collection membrane comprising a lying agent.
4. The method of claim 1, further comprising mailing the dried
membrane for further processing.
5. The method of claim 1, wherein said treating retains nucleic
acid from white blood cells on the membrane.
6. The method of claim 5, wherein the nucleic acid is an RNA.
7. The method of claim 1, wherein said contacting comprising
contacting a device comprising the sample collection membrane that
is in contact with a blotter material.
8. A method for analysis of a blood sample, comprising: receiving a
sample collection membrane with a pore size configured to retain
nucleic acid and allow lysed red blood cells to flow through on
which a blood sample was placed, treated with a buffer to cause
lysis of red blood cells, and dried to form a dried sample; and
processing the dried sample on the sample collection membrane to
detect the presence of a particular nucleic acid.
9. The method of claim 8, wherein the receiving comprising
receiving a sample collection membrane treated with buffer that
lyses red blood cells and leaves purified white blood cells or
components thereof on the membrane.
10. The method of claim 9, wherein the receiving comprising
receiving a sample collection membrane treated with buffer that
lyses red blood cells and leaves nucleic acid from white blood
cells on the membrane.
11. The method of claim 10, wherein the nucleic acid is genomic DNA
or an RNA.
12. The method of claim 11, wherein the RNA is a viral RNA.
13. The method of claim 10, wherein the processing comprises
amplifying the nucleic acid.
14. The method of claim 13, wherein the processing comprising
placing at least a portion of the sample collection membrane with
the dried sample in an amplification process.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/570,542, filed Sep. 30, 2009, now allowed, which claims
priority to U.S. Provisional Application Ser. No. 61/101,236, filed
Sep. 30, 2008. Both documents are herein incorporated by reference
in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions and methods
for isolating and purifying nucleic acid. In particular, the
present invention relates to methods of isolating nucleic acid from
cells for use in further analysis.
BACKGROUND OF THE INVENTION
[0003] It is estimated that up to 5% of infants born to HIV
seropositive mothers are HIV infected due to
mother-to-child-transmission (MTCT). In 2005 alone, about 2.3
million infants were infected with HIV and about 2 million of these
were in sub-Saharan Africa. Mortality rates are very high in
infants infected with HIV. In HIV infected infants, mortality rate
is as high as 26-45% by the first year of birth and 35-59% by the
second, stressing the urgent need for early and accurate diagnosis
and therapy. Early diagnosis of HIV could result in timely
treatment of HIV infected infants and may result in lower mortality
rates.
[0004] HIV DNA PCR detects close to 100% of all infections at
6-weeks of age. Roche AMPLICOR.RTM. HIV-1 DNA test version 1.5 is
the most widely used commercial DNA PCR assay that is capable of
detecting all subtypes of HIV and has been used for diagnosis of
HIV in infants. Nucleic Acid Sequence Based Amplification (NASBA)
has been shown to be as sensitive and specific as DNA PCR.
Recently, dried blood spots (DBS) based DNA PCR has been developed
for early diagnosis of HIV in infants. It does not involve
venipuncture and only requires a sterile lancet for a heel prick.
Briefly, a blood sample is collected on 903 filter paper (Whatman
Inc., Kent, UK) and dried. Blood can be stored and transported
easily in the form of DBS. Furthermore, it has been demonstrated
that there is a 100% correlation between diagnosis from heel prick
blood and venous blood by DNA PCR. Many groups have reported
successful PCR with high sensitivity and specificity from DNA
extracted from DBS. DBS have resulted in improved accessibility to
rural areas. A DBS sample is collected from infants at the POC when
they are brought in for their first immunization at the age of 6
weeks. The samples are shipped to a central laboratory for testing
and the results are then shipped back to the rural clinic in time
for the second immunization at 10 weeks post-birth.
[0005] However, very often, the results do not get back in time for
the second immunization resulting in high lost-to-lab and lost to
follow-up rates up to 50%. Furthermore, the extraction of DNA in a
central lab requires expensive equipment and trained personnel
making the test inaccessible to developing nations due to cost at
about 50 USD/test. In contrast, results from early POC testing can
be provided to the infant's caregiver at the same appointment, and
appropriate treatment can be initiated significantly reducing
lost-to-follow up rates.
[0006] Many challenges must be overcome when conducting HIV DNA
tests both in centralized laboratories and out in the field. Large
laboratories use automated or semi-automated robotic systems for
high-volume HIV viral load assays. However, sample processing is
typically the most troublesome part of these tests. Currently,
sample-processing procedures involve many steps, often requiring
centrifugation and extraction steps. Also, these methods often do
not adequately purify the target nucleic acid. They often leave
inhibitory or interfering substances in the reaction mixture that
can cause inhibition of the amplification reaction and result in
false-negative results. The manual nature of current
sample-processing techniques also can lead to specimen
cross-contamination, which can cause false-positive results.
[0007] Considerable effort has been made in trying to automate the
sample preparation process, since this would allow for the more
widespread use of PCR or other nucleic analysis techniques.
However, existing automated high-throughput systems perform
multiple extraction and purification steps, and still require
certain manual preparations, including sample and reagent loading,
and waste removal. Hence, highly trained technicians are required
to conduct the assay and maintain the instrument. The automated
systems are very expensive because they use complex robotic arms to
move solutions or magnetic particles and precision instruments to
pipette liquids. The cost of an automated system is often difficult
to justify for smaller laboratories, especially those in resource
limited settings. Cross-contamination is also a problem since they
employ amplification technologies. Clinical laboratories often use
separate rooms for reagent preparation, sample preparation,
amplification, and post-amplification analysis. For these reasons,
despite the automation, DNA proviral testing is considered
high-complexity tests under the Clinical Laboratory Improvement
Amendments (CLIA). To date, no Nucleic Acid Test (NAT) system has
qualified for CLIA-waived status, largely because of the
difficulties in automating sample preparation and reagent
handling.
[0008] Performing field-use or near-patient NATs involves even more
challenges, especially since they will inevitably be conducted by
less-experienced users in non-laboratory environments.
[0009] What is needed are fast, inexpensive, and efficient methods
of preparing samples for nucleic acid analysis.
SUMMARY OF THE INVENTION
[0010] The present invention relates to compositions and methods
for isolating and purifying nucleic acid. In particular, the
present invention relates to methods of isolating nucleic acid from
cells for use in further analysis.
[0011] For example, in some embodiments, the present invention
provides a biological sample processing kit, comprising: at least
one sample processing device comprising a sample collection
membrane with a pore size configured to retain nucleic acid (e.g.,
in white blood cells or white blood cell nuclei) and allow lysed
red blood cells to flow through; and a buffer configured to lyse
red blood cells retained on said membrane. In some embodiments, the
kit further comprises reagents for performing a nucleic acid (e.g.,
genomic DNA or pathogen) detection reaction. In some embodiments,
the nucleic acid detection reaction is an amplification reaction
(e.g., including but not limited to, polymerase chain reaction
(e.g., real time PCR), loop mediated isothermal amplification,
ligase chain reaction, rolling circle replication, nucleic acid
sequence based amplification and self-sustained sequence
replication). In some embodiments, the membrane holds up to 200
.mu.l or more (e.g., greater than 1 .mu.l, 5 .mu.l, 10 .mu.l, 50
.mu.l, greater than 100 .mu.l, 50 .mu.l-200 .mu.l, 100 .mu.l-200
.mu.l, or 150 .mu.l-200 .mu.l) of blood. In some embodiments, the
membrane has a length of less than 10 cm (e.g., less than 5 cm, 3
cm, 1, etc.) and a width of less than 3 cm (e.g., less than 2 cm, 1
c, 0.5 cm, 0.3 cm, etc.). In some embodiments, the sample
processing device further comprises a blotter material in physical
contact with the membrane. In some embodiments, the physical
contact permits horizontal lateral or vertical flow of fluid from
said membrane to said blotter. In some embodiments, the kit further
comprises reagents for detecting amplified nucleic acid. In some
embodiments, the amplified nucleic acid is pathogen (e.g., viral)
nucleic acid. In some embodiments, the kit further comprises
reagents for lysing white (e.g., comprising the nucleic acid of
interest) and red blood cells.
[0012] In some embodiments, the present invention provides a
system, comprising at least one sample processing device comprising
a sample collection membrane with a pore size configured to retain
nucleic acid (e.g., in white blood cells or white blood cell
nuclei) and allow lysed red blood cells to flow through; and a
buffer configured to lyse red blood cells retained on said
membrane. In some embodiments, the system further comprises a
detection apparatus. In some embodiments, the detection apparatus
is configured to directly receive the membrane (e.g., in a membrane
holder). In some embodiments, the detection apparatus is an
apparatus for performing an amplification reaction (e.g.,
polymerase chain reaction (e.g., real time PCR), loop mediated
isothermal amplification, ligase chain reaction, rolling circle
replication, nucleic acid sequence based amplification and
self-sustained sequence replication.
[0013] In further embodiments, the present invention provides
method of processing biological samples, comprising: contacting at
least one sample processing device comprising a sample collection
membrane with a pore size configured to retain nucleic acid (e.g.,
in white blood cells or white blood cell nuclei) and allow red
blood cell components to flow through with a blood sample; and
contacting the membrane with a buffer under conditions such that
the buffer lyses red blood cells, removes debris, and leaves
purified white blood cells or components thereof on the membrane.
In some embodiments, the method is a multiplex method (e.g., uses
multiple membranes simultaneously). In some embodiments, the method
further comprises the step of performing a nucleic acid (e.g.,
genomic DNA or pathogen nucleic acid) detection reaction on the
purified white blood cells. In some embodiments, the nucleic acid
detection reaction is an amplification reaction (e.g., including
but not limited to, polymerase chain reaction (e.g., real time
PCR), loop mediated isothermal amplification, ligase chain
reaction, rolling circle replication, nucleic acid sequence based
amplification and self-sustained sequence replication). In some
embodiments, the method purifies up to 200 .mu.l of blood. In some
embodiments, the method further comprises the step of detecting the
amplified nucleic acid. In some embodiments, the amplified nucleic
acid is pathogen (e.g., viral) nucleic acid. In some embodiments,
the method further comprises the step of lysing white (e.g.,
containing nucleic acid) and red blood cells prior to contacting
the sample with the sample processing device.
DESCRIPTION OF THE FIGURES
[0014] FIG. 1 shows a schematic of a lateral flow technique for
cell separation.
[0015] FIG. 2 shows a picture of the setup used in embodiments of
the present invention (bottom right). A picture of the tube with
the glass fibers broken up after PCR is shown on the bottom
left.
[0016] FIG. 3A shows melt curves of specific product formation when
.beta..sub.2-microglobulin was tested from whole blood using SYBR
green detection. FIG. 3B shows amplification curves of PCR in
combination with 8964 procedure to detect
.beta..sub.2-microglobulin from whole blood using SYBR green
detection.
[0017] FIG. 4A shows amplification plots indicating that up to 4
cells/.mu.L can be detected using the 8964 procedure in conjunction
with the Abbott RealTime.RTM. HIV-1 assay. FIG. 4B shows standard
curves indicating that PCR efficiency is slightly below 100%.
[0018] FIG. 5A shows a schematic of the vertical flow technique for
cell separation. FIG. 5B shows a picture of the setup. FIG. 5C
shows a picture of disk in the reaction tube. FIG. 5D shows a
picture of an alternative setup.
[0019] FIG. 6A shows amplification curves indicating that PCR in
combination with Fusion 5 procedure can be used for detection of
.beta..sub.2-microglobulin from whole blood using SYBR green
detection. FIG. 6B shows melt curves that show specific product
formation of .beta..sub.2-microglobulin from whole blood using SYBR
green detection.
[0020] FIG. 7A shows amplification plots indicating that up to 4
cells/.mu.L can be detected using the Fusion 5 procedure in
conjunction with an Abbott RealTime.RTM. HIV-1 assay. FIG. 7B shows
standard curves indicating that PCR efficiency is very close to
100%.
[0021] FIGS. 8A-8B show that the number of copies detected did not
change significantly after storage for 5 weeks.
[0022] FIG. 9 shows agarose gel electrophoresis showing the
formation of ladder pattern expected of LAMP products.
[0023] FIG. 10 shows an exemplary device of the present
invention.
[0024] FIG. 11 shows a schematic of an exemplary protocol for rapid
isolation of nucleic acids.
[0025] FIG. 12 shows amplification plots obtained with blood
reconstituted with 8E5 cells to obtain a total of 20,000-40 HIV-1
copies. HIV-1 DNA is detected in this real-time PCR assay.
[0026] FIG. 13 shows that a standard curve obtained with modified
method shows approximately 100% PCR efficiency.
[0027] FIG. 14 shows amplification plots obtained with blood
containing different total count of White Blood Cells (WBCs) in the
range of 62500-20000000 cells.
[0028] FIG. 15 shows standard curves obtained with blood containing
different total count of White Blood Cells (WBCs) in the range of
62500-20000000 cells.
[0029] FIG. 16 shows that HIV-1 DNA is stable after storage up to
one month. Blood samples used for this study were spiked with 40
copies of HIV-1 (8E5 cells)
[0030] FIG. 17 shows blood samples containing HIV-1 (10,000-40
copies) lysed by adding to a microtube containing a pellet of
freeze dried lysing reagent and tested for HIV-1 using real-time
PCR.
[0031] FIG. 18 shows blood samples containing HIV-1 (20,000-40
copies) lysed by adding to a filter membrane containing dried
lysing reagent and tested for HIV-1 using real-time PCR.
[0032] FIG. 19 shows blood samples containing HIV-1 (10,000-40
copies) were collected using an Analox lysing capillary. The sample
gets lysed during collection and nucleic acid isolation is done by
adding to a filter membrane and washing as described before.
[0033] FIG. 20 shows capillary devices for blood collection.
[0034] FIG. 21 shows a composite device consisting of capillary and
microtube.
[0035] FIG. 22 shows usage of plastic transfer pipette like device
for blood collection.
[0036] FIG. 23 shows a SIM with a separation membrane.
[0037] FIG. 24 shows that a SIM could possibly have energy
directors for ultrasonic welding of membrane to plastic.
[0038] FIG. 25 shows a separation device with ring to aid in blood
collection.
[0039] FIG. 26 shows a separation device with extended neck to aid
in blood collection directly from the heel without the need for a
collection device.
[0040] FIG. 27 shows a separation module with spring fixture for
improved contact of blotter material with separation membrane.
[0041] FIG. 28 shows a composite device of separation module and
reaction vessel.
DEFINITIONS
[0042] To facilitate an understanding of this disclosure, terms are
defined below:
[0043] "Purified polypeptide" or "purified protein" or "purified
nucleic acid" means a polypeptide or nucleic acid of interest or
fragment thereof which is essentially free of, e.g., contains less
than about 50%, preferably less than about 70%, and more preferably
less than about 90%, cellular components with which the polypeptide
or polynucleocide of interest is naturally associated.
[0044] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or DNA or polypeptide, which
is separated from some or all of the coexisting materials in the
natural system, is isolated. Such polynucleotide could be part of a
vector and/or such polynucleotide or polypeptide could be part of a
composition, and still be isolated in that the vector or
composition is not part of its natural environment.
[0045] "Purified product" refers to a preparation of the product
which has been isolated from the cellular constituents that the
product is normally associated and from other types of cells which
may be present in the sample of interest.
[0046] A "capture reagent," as used herein, refers to an unlabeled
specific binding member that is specific either for the analyte as
in a sandwich assay, for the indicator reagent or analyte as in a
competitive assay, or for an ancillary specific binding member,
which itself is specific for the analyte, as in an indirect assay.
The capture reagent can be directly or indirectly bound to a solid
phase material before the performance of the assay or during the
performance of the assay, thereby enabling the separation of
immobilized complexes from the test sample.
[0047] "Solid phases" ("solid supports") are known to those in the
art and include the walls of wells of a reaction tray, test tubes,
polystyrene beads, magnetic or non-magnetic beads, nitrocellulose
strips, membranes, microparticles such as latex particles, and
others. The "solid phase" is not critical and can be selected by
one skilled in the art. Thus, latex particles, microparticles,
magnetic or non-magnetic beads, membranes, plastic tubes, walls of
microtiter wells, glass or silicon chips, are all suitable
examples. It is contemplated and within the scope of the present
invention that the solid phase also can comprise any suitable
porous material.
[0048] As used herein, the terms "detect", "detecting", or
"detection" may describe either the general act of discovering or
discerning or the specific observation of a detectably labeled
composition.
[0049] The term "polynucleotide" refers to a polymer of ribonucleic
acid (RNA), deoxyribonucleic acid (DNA), modified RNA or DNA, or
RNA or DNA mimetics. This term, therefore, includes polynucleotides
composed of naturally-occurring nucleobases, sugars and covalent
internucleoside (backbone) linkages as well as polynucleotides
having non-naturally-occurring portions which function similarly.
Such modified or substituted polynucleotides are well-known in the
art and for the purposes of the present invention, are referred to
as "analogues."
[0050] As used herein, the term "nucleic acid molecule" refers to
any nucleic acid containing molecule, including but not limited to,
DNA or RNA. The term encompasses sequences that include any of the
known base analogs of DNA and RNA including, but not limited to,
4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,
pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil,
5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
[0051] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence that comprises coding sequences necessary for the
production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA).
The polypeptide can be encoded by a full length coding sequence or
by any portion of the coding sequence so long as the desired
activity or functional properties (e.g., enzymatic activity, ligand
binding, signal transduction, immunogenicity, etc.) of the
full-length or fragment are retained. The term also encompasses the
coding region of a structural gene and the sequences located
adjacent to the coding region on both the 5' and 3' ends for a
distance of about 1 kb or more on either end such that the gene
corresponds to the length of the full-length mRNA. Sequences
located 5' of the coding region and present on the mRNA are
referred to as 5' non-translated sequences. Sequences located 3' or
downstream of the coding region and present on the mRNA are
referred to as 3' non-translated sequences. The term "gene"
encompasses both cDNA and genomic forms of a gene. A genomic form
or clone of a gene contains the coding region interrupted with
non-coding sequences termed "introns" or "intervening regions" or
"intervening sequences." Introns are segments of a gene that are
transcribed into nuclear RNA (hnRNA); introns may contain
regulatory elements such as enhancers. Introns are removed or
"spliced out" from the nuclear or primary transcript; introns
therefore are absent in the messenger RNA (mRNA) transcript. The
mRNA functions during translation to specify the sequence or order
of amino acids in a nascent polypeptide.
[0052] The term "nucleic acid amplification reagents" includes
conventional reagents employed in amplification reactions and
includes, but is not limited to, one or more enzymes having
polymerase activity, enzyme cofactors (such as magnesium or
nicotinamide adenine dinucleotide (NAD)), salts, buffers,
deoxynucleotide triphosphates (dNTPs; for example, deoxyadenosine
triphosphate, deoxyguanosine triphosphate, deoxycytidine
triphosphate and deoxythymidine triphosphate) and other reagents
that modulate the activity of the polymerase enzyme or the
specificity of the primers.
[0053] As used herein, the terms "complementary" or
"complementarity" are used in reference to polynucleotides (i.e., a
sequence of nucleotides such as an oligonucleotide or a target
nucleic acid) related by the base-pairing rules. Complementarity
may be "partial," in which only some of the nucleic acids' bases
are matched according to the base pairing rules. Or, there may be
"complete" or "total" complementarity between the nucleic acids.
The degree of complementarity between nucleic acid strands has
significant effects on the efficiency and strength of hybridization
between nucleic acid strands. This is of particular importance in
amplification reactions, as well as detection methods which depend
upon binding between nucleic acids.
[0054] The term "homology" refers to a degree of identity. There
may be partial homology or complete homology. A partially identical
sequence is one that is less than 100% identical to another
sequence.
[0055] As used herein, the term "hybridization" is used in
reference to the pairing of complementary nucleic acids.
Hybridization and the strength of hybridization (i.e., the strength
of the association between the nucleic acids) is impacted by such
factors as the degree of complementary between the nucleic acids,
stringency of the conditions involved, the Tm of the formed hybrid,
and the G:C ratio within the nucleic acids.
[0056] As used herein, the term "Tm" is used in reference to the
"melting temperature." The melting temperature is the temperature
at which a population of double-stranded nucleic acid molecules
becomes half dissociated into single strands. The equation for
calculating the Tm of nucleic acids is well known in the art. As
indicated by standard references, a simple estimate of the Tm value
may be calculated by the equation: Tm=81.5+0.41(% G+C), when a
nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson
and Young, Quantitative Filter Hybridization, in Nucleic Acid
Hybridization (1985). Other references include more sophisticated
computations which take structural as well as sequence
characteristics into account for the calculation of Tm.
[0057] As used herein the term "stringency" is used in reference to
the conditions of temperature, ionic strength, and the presence of
other compounds, under which nucleic acid hybridizations are
conducted. With "high stringency" conditions, nucleic acid base
pairing will occur only between nucleic acid fragments that have a
high frequency of complementary base sequences. Thus, conditions of
"weak" or "low" stringency are often required when it is desired
that nucleic acids which are not completely complementary to one
another be hybridized or annealed together.
[0058] The term "wild-type" refers to a gene or gene product which
has the characteristics of that gene or gene product when isolated
from a naturally occurring source. A wild-type gene is that which
is most frequently observed in a population and is thus arbitrarily
designed the "normal" or "wild-type" form of the gene. In contrast,
the term "modified" or "mutant" refers to a gene or gene product
which displays modifications in sequence and or functional
properties (i.e., altered characteristics) when compared to the
wild-type gene or gene product. It is noted that
naturally-occurring mutants can be isolated; these are identified
by the fact that they have altered characteristics when compared to
the wild-type gene or gene product.
[0059] The term "oligonucleotide" as used herein is defined as a
molecule comprised of two or more deoxyribonucleotides or
ribonucleotides, preferably at least 5 nucleotides, more preferably
at least about 10-15 nucleotides and more preferably at least about
15 to 30 nucleotides, or longer. The exact size will depend on many
factors, which in turn depends on the ultimate function or use of
the oligonucleotide. The oligonucleotide may be generated in any
manner, including chemical synthesis, DNA replication, reverse
transcription, or a combination thereof.
[0060] Because mononucleotides are reacted to make oligonucleotides
in a manner such that the 5' phosphate of one mononucleotide
pentose ring is attached to the 3' oxygen of its neighbor in one
direction via a phosphodiester linkage, an end of an
oligonucleotide is referred to as the "5' end" if its 5' phosphate
is not linked to the 3' oxygen of a mononucleotide pentose ring and
as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of
a subsequent mononucleotide pentose ring. As used herein, a nucleic
acid sequence, even if internal to a larger oligonucleotide, also
may be said to have 5' and 3' ends. A first region along a nucleic
acid strand is said to be upstream of another region if the 3' end
of the first region is before the 5' end of the second region when
moving along a strand of nucleic acid in a 5' to 3' direction.
[0061] When two different, non-overlapping oligonucleotides anneal
to different regions of the same linear complementary nucleic acid
sequence, and the 3' end of one oligonucleotide points towards the
5' end of the other, the former may be called the "upstream"
oligonucleotide and the latter the "downstream"
oligonucleotide.
[0062] The term "primer" refers to an oligonucleotide which is
capable of acting as a point of initiation of synthesis when placed
under conditions in which primer extension is initiated. An
oligonucleotide "primer" may occur naturally, as in a purified
restriction digest or may be produced synthetically.
DETAILED DESCRIPTION OF THE INVENTION
[0063] The present invention relates to compositions and methods
for isolating and purifying nucleic acids. In particular, the
present invention relates to methods of isolating nucleic acids
from human cells for use in further analysis.
[0064] Cell separation using filtration membranes has been studied
in great detail. Nucleic acid isolation using lysing agents on
membranes has been successfully performed as well. However, most
such methods require a pressure drop in the form of a syringe-type
system and/or require multiple washes to get rid of contaminating
proteins and other cellular components. Some methods also require a
digestion step involving proteinase K digestion. There are reagents
that are available that enable PCR from whole blood. However, these
reagents can only process small volumes of blood that do not
generate sufficient nucleic acids for amplification reactions
involving low copy number targets such as viral nucleic acids.
There are devices that can collect blood and separate cellular
components but do not clean them enough for PCR.
[0065] Experiments conducted during the course of development of
embodiments of the present invention resulted in the development of
a method that enables the usage of up to 200 .mu.L (e.g., from
approximately 1 .mu.l to greater than 100 .mu.l) whole blood and
also enables real-time optical detection. Many methods also require
agitation in order to separate contaminants. The methods of
embodiments of the present invention do not require a pressure drop
for operation and can use, for example, only a one chase step with
RBC lysis buffer to isolate the WBCs from other contaminating
components of blood, eliminating the need for agitation as
well.
[0066] An exemplary buffer that finds use in the kits, compositions
and methods of the present invention is 0.8M NH.sub.4Cl, nuclease
free H.sub.2O and 10-20 mM NaOH. Good results were obtained with 10
mM NaOH because of improved S/N, although any number of buffers can
be used.
[0067] In some embodiments, the invention provides a membrane
configured to separate blood components. In some embodiments, the
cell separation step takes place by capillary action by placing a
blotter pad below the membrane while RBC lysis buffer is added on
the top (FIG. 5). In other embodiments, the assay is performed
using lateral flow, where a blotter pad is placed on the side of
the membrane resulting in lateral flow (FIG. 1). The end result is
the entrapment of cells on the surface of the membrane which enter
PCR or other nucleic acid analysis methods.
I. Devices
[0068] As described above, embodiments of the present invention
provide devices and systems for performing separation and analysis
reactions. In some embodiments, devices comprise a membrane for
capturing white blood cells. In some embodiments, the membrane has
a pore size of approximately 1 to 50 .mu.M (e.g., 5-20 .mu.M). In
some embodiments, methods are lateral flow methods (e.g., as
described in FIG. 1). In other embodiments, vertical flow methods
are utilized (e.g., as described in FIG. 5). The present invention
is not limited to a particular membrane. In some embodiments, the
membrane is in contact with blotter paper (e.g., to adsorb waste
material). In some embodiments, the membrane has a pore size of
approximately 1 to 50 .mu.M (e.g., 5-20 .mu.M). As described in the
Experimental section below, a number of membranes were tested and
found to be suitable in the methods of embodiments of the present
invention.
[0069] One exemplary device and method of use is depicted in FIG.
10. In some embodiments, the device has an opening for inserting
blood or other fluids. In some embodiments (Step 1 of FIG. 10), the
filtration module contains a cell separation membrane and an
absorbent pad that separates the leukocytes. In some embodiments,
blood is added to the module that results in separation of cellular
components from the plasma by capillary action.
[0070] In some embodiments, the next step (Step 2 of FIG. 10) is a
wash step with 500-1000 .mu.L wash buffer to separate the
erythrocytes, platelets and other blood components from the
leukocytes. In some embodiments, the whole procedure takes about
2-3 min.
[0071] In some embodiments (Step 3 of FIG. 10), the membrane holder
with the disk (with entrapped leukocytes or nucleic acids) is then
removed from the module and used as PCR template by inserting into
a reaction vessel specifically designed for real-time PCR in the
presence of a membrane holder.
[0072] In some embodiments (Step 4 of FIG. 10), a real-time PCR
instrument that has been adapted to work with the reaction vessel
is utilized. The reaction vessel has a large surface to volume
ratio to aid in efficient thermal transfer for fast thermal
cycling. It also has a polished side wall for fluorescence
detection. The circular cap ensures good sealing. The thickness of
the sidewall was chosen based on the thickness of the membrane
holder so as to allow 1-1.5 mm window for fluorescence
detection.
[0073] In some embodiments, vertical flow methods utilize a
membrane sandwiched between two pieces of blotter paper. In other
embodiments, parafilm is used as the top layer instead of blotter
paper.
[0074] In some embodiments, the method involves the following
steps: 1) dispense a volume of whole blood on a filtration
membrane/absorbent pad sandwich, 2) rinse membrane with a buffer,
3) transfer the membrane to an analysis solution. This removes RBC
cell debris, platelets and serum contaminants without a pressure
drop and without further purification of the nucleic acid. The
membrane pore size is chosen such that lymphocytes cannot pass
through the membrane. This method is also suitable for use on dried
blood spots on membranes.
[0075] In some embodiments, systems and/or devices of the present
invention are shipped containing all components necessary to
perform purification and analysis (e.g., amplification reagents).
In some embodiments, additional reaction components are supplied in
separate vessels packaged together into a kit.
[0076] Any of these compositions, alone or in combination with
other compositions disclosed herein or well known in the art, may
be provided in the form of a kit. Kits may further comprise
appropriate controls and/or detection reagents. Any one or more
reagents that find use in any of the methods described herein may
be provided in the kit.
[0077] In some embodiments, systems that automate one or more steps
of the process may be used and system employing multiple parallel
membranes for higher throughput may be used. For example, a system
may comprise automated or multiple sample delivery to a membrane or
membranes (e.g., via a multi-pipette or other dispensing device).
One or more membranes may be positioned in a sample processing
component with the ability to insert, remove, cut, or otherwise
process membranes by hand or via robotics or other automated
processes. Fluid may be supplied or removed from the system via
tubing, channels, or other fluidic components to facilitate sample
processing, washing, or other desired functions. In some
embodiments, purified samples are transferred manually of in an
automated fashion to a reaction chamber or chambers that cause the
isolated nucleic acid to be chemical process (e.g., amplified,
reverse transcribed, labeled, etc.). In some embodiments, the
system comprises detection hardware and/or software to permit
detection of the nucleic acid or a label associated with the
nucleic acid and/or to process data associated with a detection
event or events. The system may be self-contained in a single
apparatus or may be provided as two or more separate
components.
II. Methods
[0078] As described above, the present invention provides sample
preparation devices and methods of using the devices. In some
embodiments, the present invention provides a quick and simple
means of preparing nucleic acid (e.g., genomic DNA) from whole
blood for further analysis (e.g., PCR or other amplification
method). This method finds use in many different diagnostic and
genotyping systems. For example, in experiments conducted during
the course of development of embodiments of the present invention,
DNA purified using the methods was used for detection of HIV-1
proviral DNA in whole blood using real-time PCR and detection of
human genes (.beta.2-microglobulin and .beta.-Globin) with both
real-time PCR and LAMP. These experiments demonstrated that the
nucleic acid was of sufficient purity (e.g., free from interfering
contaminants and inhibitors) and quantity to function with diverse
nucleic acid amplification techniques, without further isolation or
purification. Additional experiments resulted in the development of
a device for implementing such isolation/amplification
processes.
[0079] The compositions and methods described herein find use in
diagnosis of HIV and other diseases at a point-of-care location.
The compositions and methods of the present invention further find
use as a processing method for dried blood spots. In some
embodiments, dried blood spots are collected at a point of care and
then mailed to a central lab for further processing.
[0080] In some embodiments, the methods and compositions described
herein are quantitative and can be used to determine viral
reservoirs (e.g., HIV) in infected subjects. In other embodiments,
the methods are qualitative and distinguish positive from negative
samples.
[0081] In further embodiments, the methods and compositions
described herein are used for detection of cellular mRNA and in
subsequent analyses (e.g., gene expression studies).
[0082] In yet other embodiments, the compositions and methods
described herein are used as an inexpensive alternative to amplify
genomic DNA from cell culture suspensions without having to purify
using other extensive protocols. It does not incorporate any
pressure drop and hence is free from the requirement of complex
filtration modules or centrifuges. The amplified DNA is free in
solution and thus can be used for downstream cloning and other
applications. In some embodiments, lysing agents are included on
the membrane. In some embodiments, cells (e.g., white and red blood
cells) are lysed prior to contacting them with the sample
preparation devices of embodiments of the present invention.
Exemplary lysing methods are described, for example, in Example 2
below.
[0083] In still further embodiments, the compositions and methods
described herein are used for loop mediated isothermal
amplification (LAMP) applications.
[0084] A. Sample
[0085] Any sample suspected of containing the desired material for
purification and/or analysis may be used according to the disclosed
methods. In some embodiments, the sample is biological sample. Such
a sample may be cells (e.g. cells suspected of being infected with
a virus), tissue (e.g., biopsy samples), blood, urine, semen, or a
fraction thereof (e.g., plasma, serum, urine supernatant, urine
cell pellet or prostate cells), which may be obtained from a
patient or other source of biological material, e.g., autopsy
sample or forensic material.
[0086] B. Modification/Analysis/Detection
[0087] The purified sample may be detected using any suitable
methods, including, but not limited to, those disclosed herein. The
description below provides exemplary techniques for biological
molecules such as nucleic acids and proteins. Other techniques may
be applied for biological molecules or non-biological molecules, as
desired or needed.
[0088] Examples of nucleic modification/analysis/detection methods
include, but are not limited to, nucleic acid sequencing, nucleic
acid hybridization, and nucleic acid amplification. Illustrative
non-limiting examples of nucleic acid sequencing techniques
include, but are not limited to, chain terminator (Sanger)
sequencing and dye terminator sequencing. Those of ordinary skill
in the art will recognize that because RNA is less stable in the
cell and more prone to nuclease attack experimentally RNA is
usually reverse transcribed to DNA before sequencing. Illustrative
non-limiting examples of nucleic acid hybridization techniques
include, but are not limited to, in situ hybridization (ISH),
microarray, and Southern or Northern blot. Nucleic acids may be
amplified prior to or simultaneous with detection.
[0089] Illustrative non-limiting examples of nucleic acid
amplification techniques include, but are not limited to,
polymerase chain reaction (PCR), reverse transcription polymerase
chain reaction (RT-PCR), transcription-mediated amplification
(TMA), ligase chain reaction (LCR), strand displacement
amplification (SDA), and nucleic acid sequence based amplification
(NASBA). Those of ordinary skill in the art will recognize that
certain amplification techniques (e.g., PCR) require that RNA be
reversed transcribed to DNA prior to amplification (e.g., RT-PCR),
whereas other amplification techniques directly amplify RNA (e.g.,
TMA and NASBA).
[0090] The polymerase chain reaction (U.S. Pat. Nos. 4,683,195,
4,683,202, 4,800,159 and 4,965,188, each of which is herein
incorporated by reference in its entirety), commonly referred to as
PCR, uses multiple cycles of denaturation, annealing of primer
pairs to opposite strands, and primer extension to exponentially
increase copy numbers of a target nucleic acid sequence. In a
variation called RT-PCR, reverse transcriptase (RT) is used to make
a complementary DNA (cDNA) from mRNA, and the cDNA is then
amplified by PCR to produce multiple copies of DNA. For other
various permutations of PCR see, e.g., U.S. Pat. Nos. 4,683,195,
4,683,202 and 4,800,159; Mullis et al., Meth. Enzymol. 155: 335
(1987); and, Murakawa et al., DNA 7: 287 (1988), each of which is
herein incorporated by reference in its entirety.
[0091] Transcription mediated amplification (U.S. Pat. Nos.
5,480,784 and 5,399,491, each of which is herein incorporated by
reference in its entirety), commonly referred to as TMA,
synthesizes multiple copies of a target nucleic acid sequence
autocatalytically under conditions of substantially constant
temperature, ionic strength, and pH in which multiple RNA copies of
the target sequence autocatalytically generate additional copies.
See, e.g., U.S. Pat. Nos. 5,399,491 and 5,824,518, each of which is
herein incorporated by reference in its entirety. In a variation
described in U.S. Publ. No. 20060046265 (herein incorporated by
reference in its entirety), TMA optionally incorporates the use of
blocking moieties, terminating moieties, and other modifying
moieties to improve TMA process sensitivity and accuracy.
[0092] The ligase chain reaction (Weiss, R., Science 254: 1292
(1991), herein incorporated by reference in its entirety), commonly
referred to as LCR, uses two sets of complementary DNA
oligonucleotides that hybridize to adjacent regions of the target
nucleic acid. The DNA oligonucleotides are covalently linked by a
DNA ligase in repeated cycles of thermal denaturation,
hybridization and ligation to produce a detectable double-stranded
ligated oligonucleotide product.
[0093] Strand displacement amplification (Walker, G. et al., Proc.
Nall. Acad. Sci. USA 89: 392-396 (1992); U.S. Pat. Nos. 5,270,184
and 5,455,166, each of which is herein incorporated by reference in
its entirety), commonly referred to as SDA, uses cycles of
annealing pairs of primer sequences to opposite strands of a target
sequence, primer extension in the presence of a dNTPaS to produce a
duplex hemiphosphorothioated primer extension product,
endonuclease-mediated nicking of a hemimodified restriction
endonuclease recognition site, and polymerase-mediated primer
extension from the 3' end of the nick to displace an existing
strand and produce a strand for the next round of primer annealing,
nicking and strand displacement, resulting in geometric
amplification of product. Thermophilic SDA (tSDA) uses thermophilic
endonucleases and polymerases at higher temperatures in essentially
the same method (EP Pat. No. 0 684 315).
[0094] Other amplification methods include, for example: nucleic
acid sequence based amplification (U.S. Pat. No. 5,130,238, herein
incorporated by reference in its entirety), commonly referred to as
NASBA; one that uses an RNA replicase to amplify the probe molecule
itself (Lizardi et al., BioTechnol. 6: 1197 (1988), herein
incorporated by reference in its entirety), commonly referred to as
Q.beta. replicase; a transcription based amplification method (Kwoh
et al., Proc. Natl. Acad. Sci. USA 86:1173 (1989)); and,
self-sustained sequence replication (Guatelli et al., Proc. Natl.
Acad. Sci. USA 87: 1874 (1990), each of which is herein
incorporated by reference in its entirety). For further discussion
of known amplification methods see Persing, David H., "In Vitro
Nucleic Acid Amplification Techniques" in Diagnostic Medical
Microbiology: Principles and Applications (Persing et al., Eds.),
pp. 51-87 (American Society for Microbiology, Washington, D.C.
(1993)).
[0095] Non-amplified or amplified target nucleic acids can be
detected by any conventional means. For example, target mRNA can be
detected by hybridization with a detectably labeled probe and
measurement of the resulting hybrids. Illustrative non-limiting
examples of detection methods are described below.
[0096] One illustrative detection method, the Hybridization
Protection Assay (HPA) involves hybridizing a chemiluminescent
oligonucleotide probe (e.g., an acridinium ester-labeled (AE)
probe) to the target sequence, selectively hydrolyzing the
chemiluminescent label present on unhybridized probe, and measuring
the chemiluminescence produced from the remaining probe in a
luminometer. See, e.g., U.S. Pat. No. 5,283,174 and Norman C.
Nelson et al., Nonisotopic Probing, Blotting, and Sequencing, ch.
17 (Larry J. Kricka ed., 2d ed. 1995, each of which is herein
incorporated by reference in its entirety).
[0097] Another illustrative detection method provides for
quantitative evaluation of the amplification process in real-time.
Evaluation of an amplification process in "real-time" involves
determining the amount of amplicon in the reaction mixture either
continuously or periodically during the amplification reaction, and
using the determined values to calculate the amount of target
sequence initially present in the sample. A variety of methods for
determining the amount of initial target sequence present in a
sample based on real-time amplification are well known in the art.
These include methods disclosed in U.S. Pat. Nos. 6,303,305 and
6,541,205, each of which is herein incorporated by reference in its
entirety. Another method for determining the quantity of target
sequence initially present in a sample, but which is not based on a
real-time amplification, is disclosed in U.S. Pat. No. 5,710,029,
herein incorporated by reference in its entirety. In some
embodiments, the Abbott RealTime.RTM. Assay (See e.g., US
2005/0227257, herein incorporated by reference in its entirety) is
utilized.
[0098] Amplification products may be detected in real-time through
the use of various self-hybridizing probes, most of which have a
stem-loop structure. Such self-hybridizing probes are labeled so
that they emit differently detectable signals, depending on whether
the probes are in a self-hybridized state or an altered state
through hybridization to a target sequence. By way of non-limiting
example, "molecular torches" are a type of self-hybridizing probe
that includes distinct regions of self-complementarity (referred to
as "the target binding domain" and "the target closing domain")
which are connected by a joining region (e.g., non-nucleotide
linker) and which hybridize to each other under predetermined
hybridization assay conditions. In a preferred embodiment,
molecular torches contain single-stranded base regions in the
target binding domain that are from 1 to about 20 bases in length
and are accessible for hybridization to a target sequence present
in an amplification reaction under strand displacement conditions.
Under strand displacement conditions, hybridization of the two
complementary regions, which may be fully or partially
complementary, of the molecular torch is favored, except in the
presence of the target sequence, which will bind to the
single-stranded region present in the target binding domain and
displace all or a portion of the target closing domain. The target
binding domain and the target closing domain of a molecular torch
include a detectable label or a pair of interacting labels (e.g.,
luminescent/quencher) positioned so that a different signal is
produced when the molecular torch is self-hybridized than when the
molecular torch is hybridized to the target sequence, thereby
permitting detection of probe:target duplexes in a test sample in
the presence of unhybridized molecular torches. Molecular torches
and many types of interacting label pairs are known (e.g., U.S.
Pat. No. 6,534,274, herein incorporated by reference in its
entirety).
[0099] Another example of a detection probe having
self-complementarity is a "molecular beacon" (see U.S. Pat. Nos.
5,925,517 and 6,150,097, herein incorporated by reference in
entirety). Molecular beacons include nucleic acid molecules having
a target complementary sequence, an affinity pair (or nucleic acid
arms) holding the probe in a closed conformation in the absence of
a target sequence present in an amplification reaction, and a label
pair that interacts when the probe is in a closed conformation.
Hybridization of the target sequence and the target complementary
sequence separates the members of the affinity pair, thereby
shifting the probe to an open conformation. The shift to the open
conformation is detectable due to reduced interaction of the label
pair, which may be, for example, a fluorophore and a quencher
(e.g., DABCYL and EDANS).
[0100] Other self-hybridizing probes are well known to those of
ordinary skill in the art. By way of non-limiting example, probe
binding pairs having interacting labels (e.g., see U.S. Pat. No.
5,928,862, herein incorporated by reference in its entirety) may be
adapted for use in the compositions and methods disclosed herein.
Probe systems used to detect single nucleotide polymorphisms (SNPs)
might also be used. Additional detection systems include "molecular
switches," (e.g., see U.S. Publ. No. 20050042638, herein
incorporated by reference in its entirety). Other probes, such as
those comprising intercalating dyes and/or fluorochromes, are also
useful for detection of amplification products in the methods
disclosed herein (e.g., see U.S. Pat. No. 5,814,447, herein
incorporated by reference in its entirety).
[0101] In some embodiments, detection methods are qualitative
(e.g., presence or absence of a particular nucleic acid). In other
embodiments, they are quantitative (e.g., viral load).
[0102] C. Data Analysis
[0103] In some embodiments, following purification and detection, a
computer-based analysis program is used to translate the raw data
generated by the detection assay (e.g., the presence, absence, or
amount of a given target molecule) into data of predictive value
for a clinician or researcher. In some embodiments, the software
program is integrated into an automated device. In other
embodiments, it is remotely located. The clinician can access the
data using any suitable means. Thus, in some preferred embodiments,
the present invention provides the further benefit that the
clinician, who is not likely to be trained in genetics or molecular
biology, need not understand the raw data. The data is presented
directly to the clinician in its most useful form. The clinician is
then able to immediately utilize the information in order to
optimize the care of the subject.
[0104] Any method may be used that is capable of receiving,
processing, and transmitting the information to and from
laboratories conducting the assays, information provides, medical
personal, and subjects. For example, in some embodiments of the
present invention, a sample (e.g., a biopsy or a serum or urine
sample) is obtained from a subject and submitted to a service
(e.g., clinical lab at a medical facility, genomic profiling
business, etc.), located in any part of the world (e.g., in a
country different than the country where the subject resides or
where the information is ultimately used) to generate raw data.
Where the sample comprises a tissue or other biological sample, the
subject may visit a medical center to have the sample obtained and
sent to the profiling center, or subjects may collect the sample
themselves (e.g., a urine sample) and directly send it to a
profiling center. Where the sample comprises previously determined
biological information, the information may be directly sent to the
profiling service by the subject (e.g., an information card
containing the information may be scanned by a computer and the
data transmitted to a computer of the profiling center using an
electronic communication systems). Once received by the profiling
service, the sample is processed and a profile is produced (i.e.,
expression data), specific for the diagnostic or prognostic
information desired for the subject.
[0105] The profile data is then prepared in a format suitable for
interpretation by a treating clinician. For example, rather than
providing raw data, the prepared format may represent a diagnosis
or risk assessment (e.g., HIV infection status) for the subject,
along with recommendations for particular treatment options. The
data may be displayed to the clinician by any suitable method. For
example, in some embodiments, the profiling service generates a
report that can be printed for the clinician (e.g., at the point of
care) or displayed to the clinician on a computer monitor.
[0106] In some embodiments, the information is first analyzed at
the point of care or at a regional facility. The raw data is then
sent to a central processing facility for further analysis and/or
to convert the raw data to information useful for a clinician or
patient. The central processing facility provides the advantage of
privacy (all data is stored in a central facility with uniform
security protocols), speed, and uniformity of data analysis. The
central processing facility can then control the fate of the data
following treatment of the subject. For example, using an
electronic communication system, the central facility can provide
data to the clinician, the subject, or researchers.
[0107] In some embodiments, the subject is able to directly access
the data using the electronic communication system. The subject may
chose further intervention or counseling based on the results. In
some embodiments, the data is used for research use. For example,
the data may be used to further optimize the inclusion or
elimination of markers as useful indicators of a particular
condition or stage of disease.
EXPERIMENTAL
[0108] The following examples are provided to demonstrate and
illustrate certain preferred embodiments and aspects of the
compositions and methods disclosed herein, but are not to be
construed as limiting the scope of the claimed invention.
Example 1
[0109] This Example describes two methods of blood collection and
purification to separate white blood cells from the other
components of blood. The method removes PCR inhibitors and PCR can
be directly performed and the sample collection membrane.
[0110] Lateral Flow Based Separation:
[0111] In this method size-based separation of cells is performed
laterally by using a chase buffer as shown in FIGS. 1 and 2.
[0112] The chase buffer lyses the RBCs and leaves the genetic
material in the WBCs intact. The WBCs get lysed in the subsequent
PCR reaction in the presence of detergents and due to thermal
cycling. The wicking action of the membrane results in the movement
of RBC debris and other proteinaceous material due to the small
pore size of the membrane. The movement of the WBCs is impeded by
the pore size. Various membranes such as HF90 (Millipore), HF75
(Millipore), HF135 (Millipore), 8964 (Ahlstrom) and AE99 (Whatman)
were tried that yielded successful results for detection of genomic
DNA from normal blood. Least variability was observed with 8964
glass fibers. This membrane allows the collection of up to 100
.mu.L of blood in a 6 mm disk. The volume of chase buffer required
to clear the RBCs and other contaminants was about 0.5-0.7 mL. The
total processing time was 4-5 min.
[0113] Using the 8964 fibers, .beta.2-microglobulin from human
blood was easily detected as shown in FIG. 3a and FIG. 3b. All
amplification reactions were done with the following polymerases:
Taq, Tth and HemoKlenTaq. Furthermore, HIV proviral DNA was
successfully detected from whole blood from an HIV-1 seronegative
donor spiked with cultured 8E5 cells that harbor a single copy of
the HIV-1 provirus in the range of 4-400 cells/.mu.L (FIG. 4).
Another parameter that was evaluated was the slope of the standard
curve generated using various dilutions of the 8E5 cells in blood
(FIG. 4b). The slope was slightly more than the -3.3 required for
100% efficiency indicating a lower PCR efficiency than 100%.
However, 4 cells/.mu.L was reliably detected using this
technique.
[0114] Vertical Flow Based Separation:
[0115] In this method size-based separation of cells is performed
vertically by using a chase buffer. The chase buffer specifically
lyses the RBCs and leaves the WBCs intact. The wicking action of
the membrane results in the movement of RBC debris and other
proteinaceous material due to the small pore size of the membrane.
The movement of the WBCs is impeded by the pore size. Most of the
wicking action is vertical but some lateral flow based separation
is also observed. Various membranes such as VF1 (Whatman), VF2
(Whatman), MF1 (Whatman), LF1 (Whatman) and Fusion 5 (Whatman) were
tried that yielded successful results for detection of genomic DNA
from normal blood via PCR. All amplification reactions were done
with the following polymerases: Taq, Tth and HemoKlenTaq. Least
variability was observed with Fusion 5 membrane. This membrane
allows the collection of up to 2004 of blood in a 7 mm disk. The
volume of chase buffer required to clear the RBCs and other
contaminants was about 1 mL. The procedure is a shown in FIG.
5.
[0116] The total procedure takes 4-5 min. Using the Fusion 5
membrane, .beta.2-microglobulin from human blood was easily
detected as shown in FIG. 6a and FIG. 6b. Furthermore, HIV proviral
DNA was successfully detected from whole blood from an HIV-1
seronegative donor spiked with cultured 8E5 cells that harbor a
single copy of the HIV-1 provirus in the range of 0.4-400
cells/.mu.L (FIG. 7). Another parameter that was evaluated was the
slope of the standard curve generated using various dilutions of
the 8E5 cells in blood (FIG. 7b). The slope was very close to the
-3.3 required for 100% efficiency indicating PCR efficiency close
to 100%. Also LOD measurement indicated that 0.2 cells/.mu.L was
reliably detected using this technique.
[0117] The above vertical flow method was also tested after drying
blood samples on the membrane for a 0-5 weeks at 37.degree. C. in
presence of desiccant and then chasing it with RBC lysis buffer.
The results obtained are shown in FIG. 8. FIG. 8 indicated that the
number of copies detected did not change significantly even after
storage for 5 weeks. Similar results were obtained with blood spots
that were chased after blood collection and then dried before PCR.
These experiments showed that this method can be used for
collection of dried blood spots which can then be mailed to a
central lab where the rest of the process is completed.
[0118] LAMP (loop mediated isothermal amplification) was also
successfully performed with the lateral flow method. The data in
FIG. 9 show that LAMP was successfully performed to detect
.beta..sub.2-microglobulin from blood processed by the lateral flow
method. The membrane used was Nitrocellulose AE99 (Whatman).
[0119] In addition to the above examples, lysing agents can be
immobilized on the surface of the membrane and can be washed away
using capillary action. This was demonstrated by using FTA
(Whatman) paper. A blotter pad was placed below the FTA paper and
the method described above was performed. The data is shown in
table 1.
TABLE-US-00001 TABLE 1 Method type Ct FTA normal 26.26 FTA normal
26.11 FTA lateral 24.67 FTA lateral 25.75
Example 2
Blood Genomic DNA Isolation
[0120] This Example describes additional methods for isolation of
genomic DNA. In some embodiments, order to increase the amount of
genomic DNA isolated from blood, a pre-lysing step was added to the
original method. While the additional step improves the yield of
nucleic acid, it may or may not be necessary depending on the
requirements of the PCR assay and sensitivity required. For
applications that require whole cells to be assayed, this step is
not needed. Pre-lysing extends the realm of samples to bacterial
samples and viral samples where cell size or virion size is too
small for effective cell capture. With those sample matrices,
pre-lysing will help release nucleic acids that can be captured by
the separation membrane. Thus, bacterial and/or viral DNA can be
detected. Pre-lysing can also be used with tissue samples such as
material obtained from swabs or biopsies that can be pre-lysed and
the lysate can then be used for nucleic acid isolation via the
following the protocol. Furthermore, by using the appropriate
detergent for pre-lysis, nuclei can be captured specifically
without releasing nucleic acids. Other modifications include, 1) A
larger disk size (9 mm diameter) can be used instead of the 7 mm
disk used in the original method to shorten processing time. 2) To
minimize interference of disk with the optics of the real-time
thermal cycler, an adhesive transfer tape such as the 3M
Double-coated polyester diagnostic tape (product #9965) can be used
to immobilize the disk on the wall of the reaction tube away from
the path of excitation and emission optics.
Modified Protocol for Nucleic Acid Isolation:
[0121] A schematic of the original process is shown in FIG. 1 and
FIG. 5 of the provisional patent application. A schematic of the
modified process is shown in FIG. 11.
[0122] In the modified method, a blood sample is treated with a
lysing agent to lyse all the cellular components of blood including
RBCs and WBCs (step 1). This releases genomic DNA in the lysate
which is then added to the filtration membrane (step 2) that
physically entraps the released genomic DNA. This is followed by
adding 600-1000 .mu.l of 10 mM NaOH to the disk that then washes
away ghosts and hemoglobin (step 3). The disk containing template
genomic DNA is then added to a PCR reaction (step 4). The blood
samples used for this study were reconstituted with 8E5 cells that
harbor a single copy of HIV-1 DNA to obtain 400-0.4 HIV-1
copies/.mu.l blood. The reconstituted blood sample was then used
for nucleic acid isolation. The blood samples are tested for HIV-1
detection and the detection of an endogenous gene namely, human
.beta.-globin. A multiplexed real-time PCR assay is used for
detection.
[0123] Using the modified method as little as 10 copies of HIV-1
can be detected consistently in 100 .mu.l blood. Furthermore,
isolated DNA can be stored after step 2 or step 3 for at least 1
month (FIG. 16).
Pre-Lysing Methods
[0124] Step 1 in which blood samples are pre-lysed can be done in
various ways: [0125] 1. In the method described above, 10 .mu.l of
lysing solution (10% Triton-X-100 OR 0.5% SDS) was added to 100
.mu.l blood in a microcentrifuge tube and finger-tapped a few times
until the sample becomes translucent. The lysate was then added to
the filter membrane. [0126] 2. In another method, 100 .mu.l of
lysing solution (1% Triton-X-100, 1% Trehalose OR 0.5% SDS) is
lyophilized to obtain a pellet of lysing agent. For cell lysis,
blood was added to this pellet which resuspends instantly and forms
a translucent lysate on finger-tapping a few times. Washing was
done as described above and real-time PCR was performed (FIG. 17)
[0127] 3. In another method, lysing solution (1% Triton-X-100 or
0.5% SDS) was added to the filter membrane which was then dried.
Blood was then added directly to the membrane impregnated with
lysing material. This trapped the genomic DNA and washing was done
as described before with 10 mM NaOH followed by real-time PCR (FIG.
18). [0128] 4. In another method, capillaries containing dried
lysing agents (Saponin) obtained from Analox Instruments USA Inc.
were used to collect blood which were then lysed in the capillaries
and added to the filter membrane followed by washing and real-time
PCR (FIG. 19).
Devices Needed to Adapt Aforementioned Methods to Point-of-Care
Testing
[0129] The above mentioned protocol simplifies the process of
nucleic acid extraction and cell capture. It also makes the process
rapid and inexpensive to perform in a laboratory. This method can
also be used for point-of-care diagnostic testing. In order to
adapt the method to point-of-care testing, the following
embodiments can be used namely, 1) Sample collection/lysing device
2) Separation module 3) Wash dropper/container.
Sample Collection/Lysing Device
[0130] This device is used for the first step of blood collection
and/or lysing. Any of the following collection devices can be used
for blood collection and/or pre-lysis. FIG. 20 describes a
capillary device which is commonly used for blood collection. The
device can be coated on the inside with lysing reagents and
anticoagulants to prevent clotting and to lyse the blood as soon as
it is collected.
[0131] FIG. 21 shows a collection device also commonly used in the
blood collection industry and is a composite of a capillary and
microtube wherein the microtube contains dried lysing reagents and
anti-coagulants.
[0132] In addition to the above, a novel blood collection device
very similar to a plastic transfer pipette can also be used as
shown in FIG. 22. The following blood collection device can be made
of plastic and has a bulb which can be used as a lysing chamber
after blood collection. After pre-lysis, the bulb can be squeezed
to inject the blood lysate onto a separation module. The bulb is
coated with lysing material via freeze-drying or gel-encapsulation.
Alternatively, a bead of lysing material can be dropped into the
device. The bulb has the same volume as the required volume of
blood.
[0133] When lysis of blood is not performed, collection devices
need not be used for blood collection. The separation module can be
held up to the body (e.g., an infant's heel or an adult's finger)
to collect blood after puncture using a lancet.
Separation Module
[0134] The separation module consists of a separation device and a
sample SIM also referred to as the sample introduction module
(SIM). FIG. 23 shows a SIM where the separation membrane is bonded
to a plastic SIM that introduces the analyte of interest (cells or
nucleic acids) into a reaction vessel. The separation membrane can
be bonded to the plastic SIM via ultrasonic welding using energy
directors (FIG. 24) or by laser welding or by adhesive bonding.
[0135] The aforementioned SIMs mate with a separation device that
houses an absorbent material which could be a blotter pad. FIG. 25
shows a separation device that ensures intimate contact between the
separation membrane and the blotter material so as to enable fast
wicking. The plastic device can also have a visualization ring
printed or molded on top as an indicator of blood volume collected
when a blood collection device is not used. The top surface of the
device could be polished so that the operator can visualize how
much blood has been collected and ascertain if the ring has been
filled.
[0136] Alternatively blood can be collected via one of the
aforementioned collection devices with a fixed volume and added
onto the separation device in which case printed/molded
visualization rings are note needed. When lysis of blood is not
performed, collection devices need not be used for blood
collection. The separation module can be held up to the body (e.g.,
an infant's heel or an adult's finger) to collect blood after
puncture using a lancet.
[0137] In another embodiment of the separation module, the module
may have a spring fixture that improves contact between the blotter
material and the separation membrane. The module also has a tab
that can be pressed to release the SIM after sample processing. In
yet another embodiment, the separation module can be a part of the
reaction vessel. The composite device has a rail on which the SIM
can slide and enter the reaction vessel after sample
processing.
Wash Dropper/Container
[0138] This container stores the wash buffer and could be any of
the following:
[0139] a) Plastic/glass dropper bottle
[0140] b) Squeeze bottles
[0141] c) Spout liquid pouches
[0142] d) Liquid blister packs
[0143] e) Liquid dispensers
The above containers may be lined with aluminum foil to minimize
evaporation.
[0144] All publications, patents, patent applications and sequences
identified by accession numbers mentioned in the above
specification are herein incorporated by reference in their
entirety. Although the invention has been described in connection
with specific embodiments, it should be understood that the
invention as claimed should not be unduly limited to such specific
embodiments. Modifications and variations of the described
compositions and methods of the invention that do not significantly
change the functional features of the compositions and methods
described herein are intended to be within the scope of the
following claims.
* * * * *