U.S. patent application number 10/162131 was filed with the patent office on 2003-01-09 for modified microporous membrane for non-specific and sequence-specific nucleic acid capture and methods of use.
Invention is credited to Arnold, Todd Edward, Chesterson, Richard S., Meyering, Mark T..
Application Number | 20030006190 10/162131 |
Document ID | / |
Family ID | 25362111 |
Filed Date | 2003-01-09 |
United States Patent
Application |
20030006190 |
Kind Code |
A1 |
Arnold, Todd Edward ; et
al. |
January 9, 2003 |
Modified microporous membrane for non-specific and
sequence-specific nucleic acid capture and methods of use
Abstract
Disclosed herein is a novel multi-layered, composite microporous
membrane comprising in at least one layer a highly electropositive
hydrophilic material distributed throughout wherein the material is
capable of irreversibly binding nucleic acid and, optionally, at
least one layer where the material is associated with
sequence-specific peptide nucleic acids, permitting the
simultaneous or sequential capture, amplification and/or
identification of specific nucleic acid sequences of interest. Also
disclosed herein are methods of use of the composite membranes of
the invention in applications based on the sequence-specific
capture and/or amplification and identification of nucleic acid
from complex biological samples.
Inventors: |
Arnold, Todd Edward;
(Glastonbury, CT) ; Meyering, Mark T.;
(Middlefield, CT) ; Chesterson, Richard S.;
(Meriden, CT) |
Correspondence
Address: |
Cummings & Lockwood
700 State Street
P. O. Box 1960
New Haven
CT
06509-1960
US
|
Family ID: |
25362111 |
Appl. No.: |
10/162131 |
Filed: |
June 4, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10162131 |
Jun 4, 2002 |
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09873675 |
Jun 4, 2001 |
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Current U.S.
Class: |
210/638 ;
264/48 |
Current CPC
Class: |
C12Q 1/6834 20130101;
B01D 2325/022 20130101; B01D 2325/36 20130101; B01D 2323/36
20130101; C12Q 1/6834 20130101; B01D 69/12 20130101; B01D 71/56
20130101; B01D 2323/12 20130101; B01D 69/141 20130101; B01D 67/0011
20130101; B01D 67/0088 20130101; C12Q 2525/107 20130101; C12Q
2531/113 20130101 |
Class at
Publication: |
210/638 ;
264/48 |
International
Class: |
B01D 015/04; C02F
001/42; B29D 001/00 |
Claims
What is claimed is:
1. A multi-layer, composite microporous membrane, wherein at least
one layer of the membrane comprises a highly electropositive
material operatively positioned on or within the microporous
membrane, and wherein the material has associated therewith a
sequence-specific peptide nucleic acid (PNA).
2. The membrane of claim 1, wherein the polyamide is nylon.
3. The membrane of claim 2, wherein the nylon is nylon 6,6.
4. The membrane of claim 1, wherein the microporous membrane has a
high surface area.
5. The membrane of claim 1, wherein one of the layers comprises a
reinforcing material.
6. The membrane of claim 1, wherein the highly electropositive
hydrophilic material comprises an element selected from the group
consisting of: silicon (Si), boron (B), titanium (Ti) and aluminum
(Al), and wherein the material has been rendered hydrophilic by
treatment with functionalizing groups capable of imparting
sufficient hydrophilicity to the electropositive material.
7. The membrane of claim 6, wherein the electropositive material is
an oxide.
8. The membrane of claim 7, wherein the electropositive material is
aluminum oxide.
9. The membrane of claim 6, wherein the electropositive material is
functionalized with hydroxyl (--OH) groups.
10. The membrane of claim 1, wherein the PNA is capable of
associating with target nucleic acid to form a PNA-nucleic acid
complex on or within the microporous membrane.
11. The membrane of claim 10, wherein the PNA is labeled to
facilitate identification or recognition of the PNA-nucleic acid
complexes.
12. The membrane of claim 1, wherein the membrane comprises at
least one layer void of any highly electropositive material.
13. The membrane of claim 1, wherein the highly electropositive
hydrophilic material is free of association with a
sequence-specific peptide nucleic acid (PNA).
14. A method of making the membrane of claim 1, wherein the method
comprises the steps of combining a highly electropositive
hydrophilic material with a microporous membrane dope during
formation of the dope; modifying the hydrophilic material by
association with a sequence specific peptide nucleic acid (PNA);
and using the combined dope and highly electropositive hydrophilic
material to form the membrane.
15. A method for confirming the presence of target nucleic acid in
a sample known to comprise the target nucleic acid, wherein the
sample is derived from animal and vegetable tissues and cells
containing nucleic acid and other substances, the method
comprising: (a) providing the membrane of claim 1; (b) contacting
the sample with the membrane, wherein two or more layers comprise a
highly electropositive hydrophilic material, wherein at least one
layer is free of the highly electropositive hydrophilic material,
wherein at least one layer comprising the hydrophilic material
further comprises PNA associated with the electropositive material,
and wherein the PNA is capable of hybridizing with the target
nucleic acid; (c) subjecting the membrane to conditions sufficient
to hybridize the nucleic acid to the PNA; (d) removing all
non-hybridized substances from the membrane; (e) treating the
membrane so as to dissociate the hybridized nucleic acid from the
PNA; and (f) collecting the nucleic acid.
16. The method of claim 15, wherein the method comprises the
further step of storing the membrane containing the nucleic acid
hybridized to the PNA before execution of steps (e) and (f).
17. The method of claim 15, wherein the nucleic acid-containing
sample is derived from a nucleic acid/protein mixture, a
biotechnical preparation of bacteria or viruses, a bodily fluid or
matter, animal or vegetable tissue, a cell lysate or homogenate, or
degradation products thereof.
18. The method of claim 15, wherein the at least one layer free of
highly electropositive hydrophilic material further comprises a
bacteriocide.
19. The method of claim 15, wherein the at least one layer free of
highly electropositive hydrophilic material further comprises a
cell lysing agent.
20. The method of claim 15, wherein the method further comprises
the step of determining the quantity of target nucleic acid present
in the sample.
21. A method for the combined separation and amplification of
target nucleic acid comprising the steps of: (a) providing the
membrane of claim 1, wherein a layer comprising the hydrophilic
material has been treated to associate a PNA with the material,
wherein the PNA is capable of hybridizing with the target nucleic
acid; (b) contacting a sample known to comprise the target nucleic
acid with the membrane under conditions sufficient to hybridize the
nucleic acid to the PNA; (c) amplifying the hybridized nucleic
acid; and (d) collecting at least a portion of the amplified
nucleic acid.
22. The method of claim 21, wherein amplification of the target
nucleic acid is achieved by a polymerase chain reaction (PCR)
technique.
23. The method of claim 21, wherein amplification of the target
nucleic acid is achieved by isothermic methods.
24. A method for separating target nucleic acid from a sample
suspected of containing the nucleic acid comprising the steps of:
(a) providing the membrane of claim 1, wherein the PNA is capable
of hybridizing to the nucleic acid of interest; (b) contacting the
membrane with the sample under conditions sufficient to permit
hybridization of the nucleic acid of interest with the PNA to form
a PNA-nucleic acid complex; and (c) detecting the presence of
PNA-nucleic acid complexes.
25. The method of claim 24, wherein the nucleic acid-containing
sample is derived from a bodily fluid or matter, animal or
vegetable tissue, or a cell lysate or homogenate, or degradation
products thereof.
26. A multi-layer, composite microporous membrane, wherein one or
more of the layers of the membrane have been modified so as to
confer on the layer a capability to associate therewith target
nucleic acid.
27. The membrane of claim 26, wherein the one or more of the layers
has been physically modified so as to confer on the layer the
capability to associate therewith target nucleic acid.
28. The membrane of claim 26, wherein the one or more of the layers
has been chemically modified so as to confer on the layer the
capability to associate therewith target nucleic acid.
29. The membrane of claim 27, wherein the physical modification
comprises addition of one or more heterogeneous substances to the
layer.
30. The membrane of claim 29, wherein the one or more heterogeneous
substances comprise a hydrophilic, highly electropositive
material.
31. The membrane of claim 29, wherein the one or more heterogeneous
substances comprise sequence specific peptide nucleic acid.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under Title 35, U.S.C.
.sctn.119(e), of U.S. application Ser. No. 09/873,675, filed Jun.
4, 2001.
BACKGROUND OF THE DISCLOSURE
[0002] The present disclosure relates to articles of manufacture
comprising a multi-layer composite microporous membrane, wherein at
least one layer of the membrane has associated therewith highly
electropositive solid phase hydrophilic materials useful for highly
efficient and irreversible binding of nucleic acids, optionally
modified with sequence specific peptide nucleic acids (PNA's);
methods of fabricating such articles of manufacture; and methods of
using such articles of manufacture to identify, separate and/or
amplify target nucleic acid and to optionally store the membrane
and bound nucleic acid for archival purposes.
[0003] Detection of Nucleic Acid Through Use of Probe
Complementarity
[0004] The molecular structure of nucleic acids provides for
specific detection by means of complementary base pairing of
oligonucleotide probes or primers to sequences that are unique to
specific target organisms or tissues. Since all biological
organisms or specimens containing nucleic acid of specific and
defined sequences, a universal strategy for nucleic acid detection
has extremely broad applications in a number of diverse research
and development areas as well as commercial industries. The
potential for practical uses of nucleic acid detection has been
greatly enhanced by the description of methods to amplify or copy,
with fidelity, precise sequences of nucleic acid found at low
concentration to much higher copy numbers, so that they are more
readily observed by available detection methods.
[0005] Amplification of Nucleic Acid
[0006] The original amplification method is the polymerase chain
reaction (PCR) described by Mullis et al. (U.S. Pat. Nos.
4,683,195, 4,683,202, and 4,965,188, all of which are specifically
incorporated herein by reference). Subsequent to the introduction
of PCR, a wide array of strategies for amplification have been
described. See, for example, U.S. Pat. No. 5,130,238 to Malek,
nucleic acid sequence based amplification (NASBA); U.S. Pat. No.
5,354,668 to Auerbach, isothermal methodology; U.S. Pat. No.
5,427,930 to Buirkenmeyer, ligase chain reaction; and U.S. Pat. No.
5,455,166 to Walker, strand displacement amplification (SDA); all
of which are specifically incorporated herein by reference. Some of
these amplification strategies, such as SDA or NASBA, require a
single stranded nucleic acid target. The target is commonly
rendered single stranded via a melting procedure using high
temperature prior to amplification.
[0007] Extraction of Nucleic Acid From Sample
[0008] Prior to nucleic acid amplification and detection, the
target nucleic acid must be extracted and purified from the
biological specimen such that inhibitors of amplification reaction
enzymes are removed. Further, a nucleic acid target that is freely
and consistently available for primer annealing must be provided. A
wide variety of strategies for nucleic acid purification are known.
These include, for example, phenol-chloroform extraction and/or
ethanol precipitation (Sambrook et al. (1989) Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.; high salt precipitation (Dykes (1988)
Electrophoresis 9:359-368); proteinase K digestion (Grimberg et al.
(1989) Nucleic Acids Res. 22:8390); chelex and other boiling
methods (Walsh et al. (1991) Bio/techniques 10:506-513); and solid
phase binding and elution (Vogelstein and Gillespie (1979) Proc.
Nat. Acad. Sci. USA 76:615-619), all of which teachings are
specifically incorporated herein by reference and representative of
knowledge attributable to one of ordinary skill in the relevant
area of art.
[0009] Analysis of Nucleic Acid in Complex Samples
[0010] The analysis of nucleic acid targets, therefore, generally
consists of three steps: nucleic acid extraction/purification from
biological specimens, direct probe hybridization and/or
amplification of the specific target sequence, and specific
detection thereof. In currently employed conventional protocols
each of these three steps is performed separately, making nucleic
acid analysis labor intensive. Further, numerous manipulations,
instruments and reagents are necessary to perform each step of the
analysis.
[0011] For analysis purposes, nucleic acid must frequently be
extracted from extremely small specimens in which it is difficult,
if not impossible, to obtain a second confirmatory specimen.
Examples include analysis of crime scene evidence or fine needle
biopsies for clinical testing. In such examples, the extent of the
genetic testing and confirmation through replica testing is, thus,
limited by the nucleic acid specimen size. Using conventional
extraction protocols for these small specimens, the nucleic acid is
often lost or yields are such that only a single or few
amplification analyses are possible.
[0012] Specimens that contain high levels of endogenous or
background nucleic acid such as blood are extremely difficult to
analyze for the presence of low level specific targets. Solid
phases with high nucleic acid avidity can be utilized to
irreversibly capture oligonucleotide or probe sequences. By
changing buffer conditions these materials can then selectively
capture target sequences even in the presence of high levels of
background nucleic acid.
[0013] Nucleic Acid Binding to Solid Phase Supports
[0014] The requirements for binding of DNA and other nucleic acid
to solid phases and subsequently being able to elute them therefrom
have been described by Boom (U.S. Pat. No. 5,234,809, specifically
incorporated herein by reference) and Woodard (U.S. Pat. Nos.
5,405,951, 5,438,129, 5,438,127, all of which are specifically
incorporated herein by reference). Specifically, DNA binds to solid
phases that are electropositive and hydrophilic.
[0015] Since conventional purification methods require elution of
the bound nucleic acid, these solid phase materials are widely
considered to be of little use for DNA purification. In fact,
considerable effort has been expended to derive solid phase
materials sufficiently electropositive and hydrophilic to
adequately bind nucleic acid and yet allow for its elution
therefrom (See, for example, U.S. Pat. Nos. 5,523,392, 5,525,319
and 5,503,816 all to Woodard, and all of which are specifically
incorporated herein by reference).
[0016] Solid-phase reversible immobilization (SPRI) is a widely
used technique for purifying nucleic acid of interest. SPRI uses
carboxyl-coated magnetic particles (that form the base material for
most magnetic particle manufacture) to bind nucleic acid. Under
conditions of high polyethylene glycol and salt concentration, SPRI
magnetic particles have been found to bind both single- and
double-stranded DNA, including PCR products. The nucleic acid
typically may be eluted with water, 10 mM Tris or formamide.
[0017] Other types of functionalized particles may be used for
binding template nucleic acid molecules, such as hydroxylated beads
and reverse phase resins. These particles are available from a wide
variety of commercial sources (e.g., Ansys, Waters, and
Varian).
[0018] U.S. Pat. No. 4,921,805 discloses a capture reagent bound to
a solid support useful for the separation and isolation of nucleic
acids from complex unpurified biological solutions. The nucleic
acid capture reagent comprises a molecule capable of intercalation
into a DNA helix, and is attached to the solid support via a
molecular linker. The capture reagent-nucleic acid complexes are
isolated from the sample by centrifugation, filtration or by
magnetic separation. Nucleic acids are separated from the isolated
complexes by, for example, treating the capture reagent-nucleic
acid complexes with dilute alkali.
[0019] Solid-phase amplification systems are also known. The
so-called DIAPOPS (Detection of Immobilized Amplified Product in
One Phase System) combines solid phase PCR and detection by
hybridization. DIAPOPS is used to covalently bind a PCR primer to a
well. Nucleic acid is covalently bound to the solid phase by a
carbodiimide condensation reaction. Manipulation is simplified and
contamination diminished since the transfer of the amplicon from
the amplification system to the detection system is eliminated.
[0020] "Standard" solid phase-anchored amplification techniques use
specific oligonucleotides coupled to a solid phase as primers for
cDNA synthesis (prepared from a mRNA molecule). This amplification
results in the production of a cDNA that is covalently linked to a
solid phase such as agarose, acrylamide, magnetic, or latex beads.
A solid phase with cDNA attached, generated using oligo (dT) as a
primer, contains sequence information similar to a cDNA library;
thus, it represents a "solid phase library." The cDNA that is
attached to the solid phase can be used directly as a template for
PCR or can be modified enzymatically prior to the PCR or other
amplification procedure. Oligonucleotides that are attached to a
solid phase can also function in affinity purification of RNA. RNA
isolated this way can be directly reverse transcribed, using the
primer that is coupled to the solid phase. Subsequent amplification
can employ this primer with or without additional internal primers.
Since the cDNA is coupled to a solid phase, changing buffer
conditions or primer composition is conveniently achieved by
washing the solid phase and re-suspending it in a different PCR
mixture.
[0021] Multiple-sample nucleic acid hybridization analysis has been
conducted on a variety of filter and solid support formats (see G.
A. Beltz et al., in Methods in Enzymology, Vol. 100, Part B, R. Wu,
L. Grossman, K. Moldave, Eds., Academic Press, New York, Chapter
19, pp. 266-308 (1985)). One format, the so-called "dot blot"
hybridization, involves the non-covalent attachment of target
nucleic acid to a filter, which is subsequently hybridized with a
radioisotope labeled probe(s). "Dot blot" hybridization gained
widespread use, and many versions were developed (see M. L. M.
Anderson and B. D. Young, in Nucleic Acid Hybridization--A
Practical Approach, B. D. Hames and S. J. Higgins, Eds., IRL Press,
Washington D.C., Chapter 4, pp. 73-111, (1985)). The "dot blot"
hybridization has been further developed for multiple analysis of
genomic mutations (D. Nanibhushan and D. Rabin, in EPA 0228075,
Jul. 8, 1987) and for the detection of overlapping clones and the
construction of genomic maps (U.S. Pat. No. 5,219,726).
[0022] Carboxylated latex beads having a plurality of first and
second nucleic acids are used in the so-called "Bridge
Amplification" technique to similarly allow amplification,
separation and detection in the same system. Such system is
described in detail in U.S. Pat. No. 5,641,658, the disclosure of
which is hereby incorporated specifically by reference.
[0023] Polyamides
[0024] Presently, extensive use is made of polyamide matrices, in
particular nylon matrices, as solid support for immobilization and
hybridization of nucleic acid. Various types of polyamide matrices
are known to bind nucleic acid irreversibly and are far more
durable than nitrocellulose. As nucleic acid can be immobilized on
polyamide matrices in buffers of low ionic strength, transfer of
nucleic acid from gels to such matrices can be carried out
electrophoretically, which may be performed if transfer of DNA by
capillary action or vacuum is inefficient.
[0025] Two basic types of polyamide membranes are commercially
available: unmodified nylon and charge-modified nylon.
Charge-modified nylon is preferred for transfer and hybridization
as its increased positively charged surface has a greater capacity
for binding nucleic acids. See, e.g., U.S. Pat. No. 4,473,474, the
disclosure of which is herein incorporated specifically by
reference. Generally, nylon membranes must be treated, however, to
immobilize the DNA after it has been transferred, as by way of
thorough drying, or exposure to low amounts of ultraviolet
irradiation (at 254 nm), and such immobilization is not
irreversible.
[0026] Nylon Filter Membranes
[0027] Polyamide membranes, and in particular nylon membranes,
offer many advantages in the filtration of materials in general.
Nylon, as other polyamides, has a natural hydrophilicity, but a
narrow wicking rate. It is also particularly strong. In addition,
nylon can be cast as a liquid film and then converted to a solid
film that presents a microporous structure when dried (See, e.g.,
U.S. Pat. No. 2,783,894). Such microporous structures permit micron
and submicron size solid particles to be separated from fluids and
provide an exceedingly high effective surface area for filtration.
Microporous polyamide structures may be manufactured so as to be
multi-layered or multi-layered so as to provide for different
filter characteristics in each layer. See, e.g., U.S. Pat. No.
6,090,441 (the "'441 patent"), the disclosure of which is hereby
incorporated specifically by reference.
[0028] Labeled Probes
[0029] The detection of amplified nucleic acid for clinical use
relies largely on hybridization of the amplified product and
detection with a probe labeled with a variety of enzymes and
luminescent reagents. U.S. Pat. No. 5,374,524 to Miller,
specifically incorporated herein, describes a nucleic acid probe
assay that combines nucleic acid amplification and solution
hybridization using capture and reporter probes. These techniques
require multiple reagents, several washing steps, and specialized
equipment for detection of the target nucleic acid. Moreover, these
techniques are labor intensive and require technicians with
considerable expertise in molecular biology techniques.
[0030] Nucleic acids modified with biotin (U.S. Pat. No. 4,687,732
to Ward et al.; European Patent No. 063879; both specifically
incorporated herein), digoxin (European Patent No. 173251,
specifically incorporated herein) and other haptens have also been
used. For example, U.S. Pat. No. 5,344,757 to Graf, specifically
incorporated herein, uses a nucleic acid probe containing at least
one hapten as a label for hybridization with a complementary target
nucleic acid bound to a solid membrane. The sensitivity and
specificity of these assays is based on the incorporation of a
single label in the amplification reaction which can be detected
using an antibody specific to the label. The usual case involves an
antibody conjugated to an enzyme. Furthermore, the addition of
substrate generates a calorimetric or fluorescent change which can
be detected with an instrument.
[0031] Attachment of Oligonucleotides to Solid Supports
[0032] Mechanisms for attachment of oligonucleotides to
microparticles in hybridization assays and for the purification of
nucleic acids is well known in the art. European Patent No. 200133,
specifically incorporated herein, describes the attachment of
oligonucleotides to water-insoluble particles less than 50
micrometers in diameter used in hybridization assays for the
capture of target nucleotides; U.S. Pat. No. 5,387,512 to Wu,
specifically incorporated herein, describes the use of
oligonucleotide sequences covalently bound to microparticles as
probes for capturing PCR amplified nucleic acids. U.S. Pat. No.
5,328,825, to Findlay, specifically incorporated herein, also
describes an oligonucleotide linked by way of a protein or
carbohydrate to a water-insoluble particle. The oligonucleotide
probe is covalently coupled to the microparticle or other solid
support. The sensitivity and specificity of all of the
above-reference patents is based on hybridization of the
oligonucleotide probe to the target nucleic acid.
[0033] Detection of Nucleic Acid at Low Copy Number in Complex
Samples
[0034] Using the current nucleic acid hybridization formats and
stringency control methods, it remains difficult to detect low copy
number (i.e., 1-100,000) nucleic acid targets even with the most
sensitive reporter groups (enzymes, fluorophores, radioisotopes,
etc.) and associated detection systems (fluorometers, luminometers,
photon counters, scintillation counters, etc.)
[0035] This difficulty is caused by several underlying problems
associated with direct probe hybridization. One problem relates to
the stringency control of hybridization reactions. Hybridization
reactions are usually carried out under stringent conditions in
order to maximize hybridization specificity. Methods of stringency
control involve primarily the optimization of temperature, ionic
strength, and denaturants in hybridization and subsequent washing
procedures. Unfortunately, the application of these stringency
conditions results in a concomitant decrease in the number of
hybridized probe/target complexes remaining for detection.
[0036] Another problem relates to the high complexity of DNA in
most samples, particularly in human genomic DNA samples. When a
sample is composed of an enormous number of sequences that are
closely related to the specific target sequence, even the most
unique probe sequence has a large number of partial hybridizations
with non-target sequences.
[0037] A third problem relates to the unfavorable hybridization
dynamics between a probe and its specific target. Even under the
best conditions, most hybridization reactions are conducted with
relatively low concentrations of probes and target molecules. In
addition, a probe often has to compete with the complementary
strand for the target nucleic acid.
[0038] A fourth problem for most present hybridization formats is
the high level of non-specific background signal. This is caused by
the affinity of DNA probes to almost any material.
[0039] These problems, either individually or in combination, lead
to a loss of sensitivity and/or specificity for nucleic acid
hybridization in the above described formats. This is unfortunate
because the detection of low copy number nucleic acid targets is
necessary for most nucleic acid-based clinical diagnostic
assays.
[0040] Available Combined Purification/Amplification Systems
[0041] Qiagen, one market leader in nucleic acid sample
preparation, produces and markets a variety of DNA and RNA sample
preparation devices. Typically such devices are based upon glass
fiber sheets where the biological sample must be clarified prior to
its being applied to the binding matrix. The nucleic acid is
typically captured in the presence of high salt buffer (anion
exchange); the nucleic acid extensively washed; and the nucleic
acid recovered by exposing the bound nucleic acid to a low ionic
strength solution (e.g., Tris-EDTA (10 mM Tris-HCl, pH 7.5-8.0; 1
mM EDTA) or deionized water). The nucleic acid is then transferred
to another vessel for amplification or further analysis. Other
companies selling nucleic acid sample preparation devices include:
Millipore (a membrane-based size exclusion ultra-filtration
system), Promega, Bio-Rad, Invitrogen, and MWG (anion
exchange-based systems).
[0042] A simplified, combined purification and amplification system
is available from CpG-Biotech of Lincoln Park, N.J.
(http://www.cpg-biotech.- com/). This system utilizes a proprietary
cell lysis solution (Release-IT.TM.), which permits cell lysis and
amplification to occur in the same reaction tube. Release-IT
sequesters cell lysis products that might inhibit polymerases and
the supplier claims that this improves the specificity and
amplification yield. The CpG-Biotech Release-IT system eliminates
the need for a separate genomic DNA purification step prior to
amplification.
[0043] Combined Purification, Amplification and Detection
Systems
[0044] Combined purification, amplification, and detection systems
are also known in the art. Such systems permit the processes of
isolation and purification of nucleic acids from complex samples,
amplification of target nucleic acid, and detection of the
amplified products to occur in a self-contained environment.
[0045] U.S. Pat. No. 5,955,351 discloses a self-contained device
integrating nucleic acid extraction, amplification, and detection.
The system integrates the extraction and amplification of the
nucleic acid allowing both procedures to be performed in one
chamber, detection in another chamber and collection of waste in
yet another chamber. The reaction chambers are functionally
distinct, sequential and compact. Xtrana, Inc. (Denver, Colo.)
manufactures an embodiment of such a device, referred to as the
SCIP cartridge. U.S. Pat. No. 6,153,425 similarly discloses a
self-contained device integrating nucleic acid extraction,
amplification and detection. Such device comprises a first hollow
elongated cylinder with a single closed end and a plurality of
chambers therein, and a second hollow elongated cylinder positioned
contiguously inside the first cylinder capable of relative
rotation. Sample is introduced into the second cylinder for
extraction. The extracted nucleic acid is bound to a solid phase,
and therefore not eluted from the solid phase by the addition of
wash buffer. Amplification and labeling takes place in the second
cylinder. Finally, the labeled, amplified product is reacted with
microparticles conjugated with receptor specific ligands for
detection of the target sequence.
[0046] A commercial product known as Xtra Amp.TM. (Xtrana, Inc.,
Denver, Colo.) permits nucleic acid extraction, amplification and
detection to be performed in a single microcentrifuge tube. Xtra
Amp employs a proprietary material, known commercially as Xtra
Bind.TM., to extract and irreversibly bind nucleic acid in a
sample. As is disclosed in U.S. Pat. No. 6,291,166 (the "'166
patent"), the disclosure of which is hereby incorporated
specifically by reference, Xtra Bind is capable of binding both DNA
and RNA in single-stranded form. Captured nucleic acid can be
amplified directly on the solid phase material by a variety of
amplification strategies including those requiring single-strand
initiation. Specific selection of low copy nucleic acid targets
present in complex specimens can be performed by binding specific
hybridization probes to the solid phase beads.
[0047] Peptide Nucleic Acids
[0048] The structure of DNA identified by Watson and Crick in 1953
has had a great impact on life sciences such as molecular biology,
biochemistry, etc. DNA is a biopolymer with four different bases of
adenine (A), cytosine (C), guanine (G), thymine (T), sugar
(deoxyribose) and phosphate, to build a very stable double helix
structure: The phosphate-sugar forms the backbone, and nucleotide
bases attached to the sugars are paired with complementary bases,
such as A to T, and G to C, in the opposing paired strand, which
pairing is stabilized by hydrogen bond formation between the
complementary bases in the double helix. The specific/complementary
hydrogen bonds between bases plays a very important role in
nucleotide drug treatment strategies such as antisense and gene
therapy, in particular, for genetic disease, cancer and cardiac
diseases.
[0049] Peptide nucleic acid monomers have a N-(2-aminoethyl)
glycine backbone to which adenine, cytosine, guanine, or thymine
bases are linked by amide bonds. See FIG. 7. Peptide nucleic acids
are synthesized by creating an amide bond between an amino group of
the backbone and a carboxyl group of another peptide nucleic acid
monomer. Currently, peptide nucleic acid monomers protected by an
acid-labile t-butyloxycarbonyl protecting group or alkali-labile
fluoromethyloxycarbonyl protecting group are commercially
available, where exocyclic amino groups of adenine, cytosine and
guanine are protected by acid-stable dipenylmethyloxycarbonyl or
benzyloxycarbonyl protecting groups.
[0050] Peptide nucleic acid synthesis is generally carried out in a
similar manner as the oligonucleotide synthesis method
conventionally known in the art. See Acc. Chem. Res. 24:278 (1991);
see also U.S. Pat. No. 6,357,163, the disclosure of which is hereby
incorporated specifically by reference. Nielson et al. synthesized
oligopeptide nucleic acid by using a solid-phase matrix as follows:
First, the amino group on the solid support is reacted with the
carboxyl group of the specified base (A, C, G or T), whose amino
group in the backbone is protected by acid- or base-labile
functional groups, in order to link to each other. Next, the
resultant structure is treated with acid or base to eliminate the
amino protecting groups to reveal the amino group, which is
subsequently reacted with the carboxyl group of the peptide nucleic
acid of specified base, whose amino group in the backbone is
protected by acid- or base-labile functional groups, in order to
link to each other in the form of an amide bond. The steps are
repeated to obtain an oligonucleotide of desired base sequence and
number, and finally treated with strong acid to separate the
exocyclic amino protecting group from the solid support by chemical
reaction. This method is desirable in a sense that it assures
complete reaction of excessive peptide nucleic acids (5
equivalents) as much as possible and provides easy purification of
peptide nucleic acid on an organic-solvent-resistant solid support
by filtering the residual monomers and reactants and washing with
organic solvent.
[0051] The DNA mimetic, peptide nucleic acid (PNA), has the
potential to detect single-base substitution in sample DNA. Peptide
nucleic acid is a fully synthetic DNA-recognizing ligand with a
neutral peptide-like backbone that is structurally homomorphous to
the deoxyribose phosphate backbone of DNA, and purine- and
pyrimidine-based nucleobases (i.e., adenine, cytosine, thymine and
guanine). Sequence specific hybridization of PNA to complementary
DNA occurs through Watson-Crick H-bonding between the
nucleobases.
[0052] The neutrality of the PNA backbone results in stronger
binding of PNA to DNA as compared to DNA-DNA binding. Using the
mutations associated with cystic fibrosis (CF) as a model system,
it has been demonstrated, for example, that PNA can distinguish
normal and mutant sequences in the CF gene.
SUMMARY OF THE INVENTION
[0053] In one aspect, the present invention provides a multi-layer,
composite microporous membrane, wherein at least one layer of the
membrane comprises a highly electropositive material operatively
positioned on or within the microporous membrane, and wherein the
material has associated therewith a sequence-specific peptide
nucleic acid (PNA), wherein the PNA is capable of associating with
target nucleic acid to form a PNA-nucleic acid complex. Preferably,
each layer of the microporous membrane comprises a polymeric
material. Preferably, the polymeric material is a polyamide. More
preferably, the polyamide is nylon. More preferably still, the
nylon is nylon 6,6. A preferred method for preparation of the nylon
membrane of the invention is a phase inversion process. Preferably,
the polymeric material of the membranes will have a high surface
area. More preferably, the surface area of the microporous membrane
is at least 60 m.sup.2/g. In an alternative embodiment, the
multi-layer membrane of the invention comprises a reinforcing
material. Preferably, the reinforcing material is a polyolefin.
[0054] In another aspect of the invention, the highly
electropositive hydrophilic material of which one layer of the
multi-layer membrane is comprised, comprises an element selected
from the group consisting of: silicon (Si), boron (B), titanium
(Ti) and aluminum (Al). Preferably, the highly electropositive
material has been rendered hydrophilic by treatment with
functionalizing groups capable of imparting sufficient
hydrophilicity to the electropositive material. Preferably, the
electropositive material is functionalized with hydroxyl (--OH)
groups. In one embodiment, the electropositive material is in a
crystalline form. In an alternative embodiment, the electropositive
material is in an amorphous form. Preferably, the electropositive
material is an oxide. More preferably, the electropositive material
is aluminum oxide. In yet another alternative embodiment, the
electropositive material is in an elemental state.
[0055] According to the practice of the present invention, the
PNA-nucleic acid complex formed with the target nucleic acid
comprises deoxyribonucleic acid (DNA). Preferably, the nucleic acid
is complementary deoxyribonucleic acid (cDNA). Alternatively, the
nucleic acid is ribonucleic acid (RNA). In this alternative
embodiment, the ribonucleic acid is, preferably, messenger
ribonucleic acid (mRNA). In yet another aspect of the invention,
the PNA can be radiolabeled to facilitate identification or
recognition of the PNA-nucleic acid complexes. In an alternative
version of this embodiment, the target nucleic acid can be
radiolabeled to facilitate identification or recognition of the
PNA-nucleic acid complexes.
[0056] In yet another aspect of the present invention, the
multi-layer composite membrane comprises at least one layer
comprising a highly electropositive material capable of associating
with one or more nucleic acids. In such an embodiment, wherein
highly electropositive hydrophilic material of the least one layer
is free of association with a sequence-specific peptide nucleic
acid (PNA). In addition, the present invention contemplates that
the multi-layer composite membrane comprise at least one layer void
of any highly electropositive material. Consistent with the
invention, each of the layers is individually characterized in
terms of porosity, average pore size, pore size distribution,
three-dimensionality of pore distribution, and loading with
heterogeneous materials.
[0057] In another aspect, the present invention provides a method
of making the a multi-layer composite membrane, wherein the method
comprises the steps of combining a highly electropositive
hydrophilic material with a microporous membrane dope during
formation of the dope; modifying the hydrophilic material by
association with a sequence specific peptide nucleic acid (PNA);
and using the combined dope and highly electropositive hydrophilic
material to form the membrane. In this embodiment, the invention
contemplates that the electropositive material is modified with PNA
prior to combining the electropositive material with the membrane
dope. In this fashion, the PNA may be synthesized using the
electropositive material as a solid-phase medium. Alternatively,
the electropositive material is modified with PNA prior to
formation of the membrane and after mixing of the electropositive
material with the membrane dope. In yet another alternative, the
electropositive is modified with PNA after formation of the
membrane.
[0058] In another embodiment, the present invention provides a
method for confirming the presence of target nucleic acid in a
sample known to comprise the target nucleic acid, wherein the
sample is derived from animal and vegetable tissues and cells
containing nucleic acid and other substances, the method comprising
the steps of (a) providing the multi-layer microporous membrane of
the invention; (b) contacting the sample with the membrane, wherein
two or more layers of the membrane comprise a highly
electropositive hydrophilic material, wherein at least one layer is
free of the highly electropositive hydrophilic material, wherein at
least one layer comprising the hydrophilic material further
comprises PNA associated with the electropositive material, and
wherein the PNA is capable of hybridizing with the target nucleic
acid; (c) subjecting the membrane to conditions sufficient to
hybridize the nucleic acid to the PNA; (d) removing all
non-hybridized substances from the membrane; (e) treating the
membrane so as to dissociate the hybridized nucleic acid from the
PNA; and (f) collecting the nucleic acid. Alternatively, the method
comprises the further step of storing the membrane containing the
nucleic acid hybridized to the PNA before execution of steps (e)
and (f). This embodiment of the invention contemplates that the
target nucleic acid is hybridized to the PNA under stringent
hybridization conditions. According to this embodiment, the sample
contacts the at least one layer free of the highly electropositive
material subsequent to contacting the at least one layer comprising
a highly electropositive material associated with the PNA.
Furthermore, this embodiment of the invention contemplates that the
sample will contact the at least one layer comprising highly
electropositive material having PNA capable of hybridizing the
specific nucleic acid associated therewith prior to contacting a
layer free of the highly electropositive hydrophilic material, and
prior to contacting a layer comprising highly electropositive
material free of association with PNA. According to the method of
the invention, the target nucleic acid comprises DNA. Preferably,
the target nucleic acid comprises complementary DNA (cDNA). In an
alternative aspect, the target nucleic acid comprises RNA.
Preferably, the target nucleic acid comprises messenger RNA (mRNA).
According to the practice of the method of the invention, the
nucleic acid-containing sample is derived from a nucleic
acid/protein mixture, a biotechnical preparation of bacteria or
viruses, a bodily fluid or matter, animal or vegetable tissue, a
cell lysate or homogenate, or degradation products thereof.
[0059] In an alternative aspect, the present invention contemplates
that the nucleic acid not hybridized to the PNA is collected
separately. Additionally, the at least one layer free of highly
electropositive hydrophilic material may further comprise a
bacteriocide. In addition, the at least one layer free of highly
electropositive hydrophilic material may further comprise a cell
lysing agent. Also contemplated in the practice of the method of
the present invention is the further step of determining the
quantity of target nucleic acid present in the sample. According to
this aspect of the invention, the quantity of target nucleic acid
present is determined by a method selected from the group
consisting of fluorescence, chemiluminescence, and radioisotopic
assay. Still further, the method of the present invention
contemplates that determination of the quantity of target nucleic
acid present in the sample is performed prior to the steps of
treating the hybridized target nucleic acid to dissociate it from
the PNA, and collecting the target nucleic acid.
[0060] In another alternative embodiment, the present invention
provides a method for the combined separation and amplification of
target nucleic acid comprising the steps of (a) providing the
multi-layer composite microporous membrane of the invention,
wherein a layer comprising the hydrophilic material has been
treated to associate a PNA with the material, wherein the PNA so
associated is capable of hybridizing with target nucleic acid; (b)
contacting a sample known to comprise the target nucleic acid with
the membrane under conditions sufficient to hybridize the nucleic
acid to the PNA; (c) amplifying the hybridized nucleic acid; and
(d) collecting at least a portion of the amplified nucleic acid.
According to the practice of the method of the invention,
amplification of the target nucleic acid is achieved by a
polymerase chain reaction (PCR) technique. Alternatively,
amplification of the target nucleic acid is achieved by isothermic
methods. Preferably, the isothermic method is selected from the
group consisting of NASBA, RCAT and SDA. This embodiment of the
invention also contemplates that the method further comprises the
step of dissociating the hybridized nucleic acid prior to
collection of the amplified nucleic acid. In addition, the method
contemplates that at least one of the layers of the microporous
membrane is free of association with PNA.
[0061] In still another alternative embodiment, the present
invention contemplates a method for separating target nucleic acid
from a sample suspected of containing the nucleic acid comprising
the steps of (a) providing the multi-layer composite microporous
membrane of the invention, wherein PNA associated with highly
electropositive material in at least one layer of the membrane is
capable of hybridizing to the nucleic acid of interest; (b)
contacting the membrane with the sample under conditions sufficient
to permit hybridization of the nucleic acid of interest with the
PNA; and (c) removing the nucleic acid hybridized to the PNA. In
general, the nucleic acid-containing sample is derived from a
bodily fluid or matter, animal or vegetable tissue, or a cell
lysate or homogenate, or degradation products thereof.
Alternatively, the nucleic acid-containing sample comprises a
nucleic acid/protein mixture, or a biotechnical preparation of
bacteria or viruses.
[0062] According to this aspect of the present invention, the
nucleic acid of interest interacts with the PNA under stringent
hybridization conditions. Also contemplated by the present
invention is a self-contained device for extraction, amplification
and detection of nucleic acid of interest comprising the membrane
of the invention.
[0063] In still another alternative embodiment, the present
invention provides a multi-layer, composite microporous membrane,
wherein one or more of the layers of the membrane have been
modified so as to confer on the layer a capability to associate
therewith target nucleic acid. Preferably, the one or more of the
layers has been physically modified so as to confer on the layer
the capability to associate therewith target nucleic acid. More
preferably, the physical modification comprises addition of one or
more heterogeneous substances to the layer. According to this
aspect of the invention, the one or more heterogeneous substances
are added to the layer prior to fabrication of the multi-layer
membrane. Alternatively, the one or more heterogeneous substances
are added to the layer after fabrication of the multi-layer
membrane.
[0064] In accord with an additional aspect of the invention, the
one or more heterogeneous substances comprise a hydrophilic, highly
electropositive material. Alternatively, the one or more
heterogeneous substances comprise sequence specific peptide nucleic
acid. In an alternative aspect of this embodiment of the invention,
the one or more of the layers has been chemically modified so as to
confer on the layer the capability to associate therewith target
nucleic acid. Preferably, chemical modification of the one or more
layers comprises charge modification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] The above description of the present disclosure will be more
fully understood with reference to the following detailed
description when taken in conjunction with the accompanying
drawings, wherein:
[0066] FIG. 1 is an illustration of a method for isolating and
amplifying nucleic acids from a crude biological sample using a
nylon membrane imbued with a highly electropositive solid phase
hydrophilic material.
[0067] FIGS. 2A, 3A and 4A are scanning electron photomicrographs
of a microporous membrane of the present disclosure illustrating
the membrane imbued with a highly electropositive solid phase
hydrophilic material at 500.times., 2,500.times., and
5,000.times.;
[0068] FIGS. 2B, 3B and 4B are scanning electron photomicrographs
of a control microporous membrane free of the highly
electropositive solid phase hydrophilic material of FIGS. 2A, 3A
and 4A at 500.times., 2,500.times., and 5,000.times.;
[0069] FIG. 5 is a photograph of an agarose gel stained with
ethidium bromide;
[0070] FIG. 6 is a photograph of the lower portion of the gel of
FIG. 5;
[0071] FIG. 7 is a comparison of the structures of peptide nucleic
acids (PNA's) and deoxyribonucleic acid (DNA);
[0072] FIG. 8 is a schematic representation of an embodiment of the
present invention illustrating a three-layer composite membrane
material where one layer comprises PNA, a second layer is free of
both PNA and a highly electropositive hydrophilic material, and a
third layer comprises a heterogeneous, highly electropositive,
hydrophilic material.
[0073] FIG. 9, in six panels over six pages, illustrates
schematically the practice of the present invention with the
membrane of FIG. 8 for the capture and selective release of
sequence specific nucleic acid.
[0074] FIG. 10 illustrates schematically the practice of the
present invention with the membrane of FIG. 8, having varying pore
sizes between layers, used for the separation of genetic material
of interest from a complex biological sample.
DETAILED DESCRIPTION OF THE INVENTION
[0075] Unless indicated otherwise, the terms defined below have the
following meanings:
[0076] Xtra Bind.TM. solid phase matrix available from Xtrana, Inc,
Denver, Colo. Xtra Bind is a hydrophilic and electropositive solid
phase matrix.
[0077] PCR (Polymerase Chain Reaction). A method for amplifying a
DNA base sequence using a heat-stable polymerase and two 20
nucleotide primers, one complementary to the (+)-strand at one end
of the sequence to be amplified and the other complementary to the
(-)-strand at the other end. Because the newly synthesized DNA
strands can subsequently serve as additional templates for the same
primer sequences, successive rounds of primer annealing, strand
elongation, and dissociation produce rapid and highly specific
amplification of the desired sequence. PCR also can be used to
detect the existence of the defined sequence in a DNA sample.
[0078] Affinity chromatography: A technique of analytical chemistry
used to separate and purify a biological molecule from a mixture,
based on the attraction of the molecule of interest to a particular
ligand which has been previously attached to a solid, inert
substance. The mixture is passed through a column containing the
ligand attached to the stationary substance, so that the molecule
of interest stays within the column while the rest of the mixture
continues through to the end. Then, a different chemical is flushed
through the column to detach the molecule from the ligand and bring
it out separately from the rest of the mixture.
[0079] Hybridization: a single strand of a nucleic acid molecule
(DNA or RNA) is joined with a complementary strand of nucleic acid,
again DNA or RNA, to form a double-stranded molecule (or one which
is partly double-stranded, if one of the original single strands is
shorter than the other).
[0080] Probe: A single-stranded nucleic acid molecule with a known
nucleotide sequence which is labeled in some way (for example,
radioactively, fluorescently, or immunologically) and used to find
and mark certain DNA or RNA sequences of interest to a researcher
by hybridizing to it.
[0081] Rolling Circle Amplification (RCA): an amplification process
driven by DNA polymerase which can replicate circular
oligonucleotide probes with either linear or geometric kinetics
under isothermal (single temperature) conditions. In the presence
of two suitably designed primers, a geometric amplification occurs
via DNA strand displacement and hyperbranching to generate
10.sup.12 or more copies of each circle in 1 hour. In addition to
grossly amplifying a signal, this method--called
Exponential-RCA--is adequately sensitive to detect point mutations
in genomic DNA. Additional information is available on the
Molecular Staging Website at www.molecularstaging.com.
[0082] cDNA: complementary DNA--synthesized from an RNA template
using reverse transcriptase.
[0083] Reverse transcriptase: an enzyme found in retroviruses that
enables the virus to make DNA from viral RNA.
[0084] mRNA: messenger RNA--RNA that serves as a template for
protein synthesis.
[0085] Nucleotide: A subunit of DNA or RNA consisting of a
nitrogenous base (adenine, guanine, thymine, or cytosine in DNA;
adenine, guanine, uracil, or cytosine in RNA), a phosphate
molecule, and a sugar molecule (deoxyribose in DNA and ribose in
RNA). Thousands of nucleotides are linked to form a DNA or RNA
molecule.
[0086] Oligonucleotide: a compound comprising a nucleotide linked
to phosphoric acid. When polymerized, it gives rise to a nucleic
acid.
[0087] Primer: a short pre-existing polynucleotide chain to which
new deoxyribonucleotides can be added by DNA polymerase.
[0088] Template: a molecular mold or pattern for the synthesis of
another molecule. Specifically, the DNA molecule from which a PCR
or amplification product is generated.
[0089] Intercalating dye: a planar dye molecule that binds to
nucleic acid in a non-covalent fashion by inserting itself between
the stacked bases of the nucleic acid helix. Fluorescent dyes, like
ethidium bromide, can be used to visualize DNA and RNA molecules in
gel matrices.
[0090] The present disclosure enables the practice of the present
invention to overcome many of the problems associated with the
isolation of nucleic acids from large, complex sample volumes, and
the amplification of such isolated nucleic acids.
[0091] The invention of the present disclosure combines the
attributes of highly electropositive hydrophilic materials that
irreversibly bind with one or more nucleic acids with those of
microporous membranes having a very high effective surface area.
The hybrid structure comprising the microporous membrane imbued or
coated with the highly electropositive hydrophilic materials allows
for increased presentation of the electropositive materials to
permit enhanced nucleic acid binding. As indicated herein, such
captured nucleic acid molecules may be used as templates for
enzymatic amplification. The further incorporation or modification
of the membrane of the present invention to include
sequence-specific PNA's provides further capabilities in the
practice of sequence-specific nucleic acid capture, amplification
and/or identification that heretofore possible in the prior
art.
[0092] The membrane of the present invention may be placed into
microtiter plates (e.g., 96-, 384-, 1536-wells) thereby allowing
for capture of individual nucleic acid samples from biological
sources and may be placed into a thermal cycler for PCR, or into a
constant temperature incubator for isothermal amplification
procedures.
[0093] Advantageously, the highly electropositive material capable
of irreversibly binding one or more nucleic acids is selected from
the group consisting of silicon (Si), boron (B), titanium (Ti), and
aluminum (Al). Such material can be rendered sufficiently
hydrophilic by methods well known to those of ordinary skill in the
art, as for example by the addition of hydroxyl groups. A
particularly useful compound of the present disclosure is a
composition known as Xtra Bind (Xtrana, Inc. Denver, Colo.), a
composition having significant DNA binding affinity and avidity.
Suitable electropositive matrices have been disclosed, containing
silicon (Si), boron (B), titanium (Ti), or aluminum (Al), which
have been rendered sufficiently hydrophilic by hydroxyl (--OH) or
other groups, to result in a surface that irreversibly binds DNA
(See for example WO98/46797, the disclosure of which is herein
incorporated by reference). Examples of such matrices have been
demonstrated using aluminum oxide, silica, or titania. Aluminum
oxide particles are particularly useful as this matrix including,
but not limited to, alpha (.alpha.) aluminum oxide in hexagonal
crystal form, which can be milled and classified in a variety of
particle sizes. Such materials are available from various
commercial sources, such as Washington Mills Electro Minerals
Corporation, Niagara Falls, N.Y., as Duralum Special White; also
from Atlantic Equipment Engineers, Bergenfield, N.J., as fused
alpha aluminum oxide high purity powders.
[0094] Combining the highly electropositive material capable of
irreversibly binding one or more nucleic acids (Xtra Bind) with
single-layer or multi-layer membranes, results in an enabling
platform for isolation and capture of nucleic acids from complex
biological samples. The nucleic acid, once captured, can then be
analyzed using amplification procedures known to those skilled in
the art (PCR, NASBA, RCA) thereby enabling the detection of minute
quantities of analyte, such as, for example, nucleic acid, from
large sample volumes. When a layer in the multi-layer membrane,
with or without highly electropositive material, is further
modified to comprise sequence-specific PNA's, additional
capabilities involving the capture, identification and/or
quantification of specific polynucleotide sequences in a biological
sample comprising myriad sources of genetic material are made
possible that have heretofore not been possible in the prior
art.
[0095] The present disclosure encompasses, at least in part, a
microporous multi-layer membrane that comprises a highly
electropositive material capable of irreversibly binding single- or
multiple-strand nucleic acid (non-sequence specific capture). Such
a membrane provides a solid phase platform for the essentially
simultaneous capture and amplification of nucleic acid that is
capable of handling large sample volumes so as to isolate nucleic
acid found in low quantity in the sample volume. When utilizing an
embodiment comprising sequence-specific PNA's, then the membrane of
the present invention makes it possible to not only detect the
presence of nucleic acid in a complex sample comprising many other,
possibly interfering, components but also to detect the presence,
both qualitatively and quantitatively, of specific polynucleotide
sequences in a sample comprising multiple sources of genetic
material, even when the specific nucleic acid of interest is
present in the sample at a low copy number in comparison to the
total nucleic acid content of the sample.
[0096] In one representative embodiment, there are provided one or
more microporous membranes, such as a microporous polyamide
membrane, comprising (on both interior and exterior surfaces) a
highly electropositive material having hydrophilic properties which
is capable of irreversibly binding nucleic acid (DNA, RNA, etc.).
Preferably, the microporous membrane is a microporous phase
inversion membrane, such membranes being well known in the art.
Microporous phase inversion membranes are porous solids, which
contain microporous interconnecting passages that extend from one
surface to the other. The passages provide tortuous tunnels or
paths through which the liquid that is being filtered must pass.
Due to the high effective surface area of such membranes, such
constructs provide a much enhanced capture of nucleic acids from a
given volume of sample. Such membranes also permit enhanced
amplification of bound nucleic acid when used as a solid
amplification medium. In addition, such membranes may provide
additional advantages in being able to effectively filter out, by
size exclusion, solid phase components of complex samples.
[0097] Such membranes may function in sample preparation wherein
one captures nucleic acid from any number of sources (bacteria,
fungi, blood samples, etc.) on the membrane, and the captured
nucleic acids are amplified and identified using specific probe
molecules, which probe molecules may be PNA's specifically
synthesized to be complementary to polynucleotides of particular
interest.
[0098] By "phase inversion support" it is meant a polymeric support
that is formed by the gelation or precipitation of a polymer
membrane structure from a "phase inversion dope." A "phase
inversion dope" consists of a continuous phase of dissolved polymer
in a solvent, coexisting with a discrete phase of one or more
non-solvent(s) dispersed within the continuous phase. The formation
of the polymer membrane structure generally includes the steps of
casting and quenching a thin layer of the dope under controlled
conditions to effect precipitation of the polymer and transition of
discrete (non-solvent phase) into a continuous interconnected pore
structure. This transition from discrete phase of non-solvent
(sometimes referred to as a "pore former") into a continuum of
interconnected pores is generally known as "phase inversion." Such
membranes are well known in the art. Particular attention is drawn
to the '441 patent the disclosure of which teaches the preparation
of a supported, multi-layer microporous membrane such as used in
the present invention. Typically, a phase inversion support is
formed by dissolving the polymer(s) of choice in a mixture of
miscible solvent(s) and non-solvent(s), casting a support pre-form,
and then placing the surface of the support preform in contact with
a non-solvent (liquid or atmosphere) diluent miscible with the
solvent(s) (thereby precipitating or gelling the porous
structure.
[0099] Advantageously, the electropositive material capable of
irreversibly binding one or more nucleic acids is highly
electropositive and is selected from the group consisting of
silicon (Si), boron (B) and aluminum (Al). Such material can be
rendered sufficiently hydrophilic by methods well known to those of
ordinary skill in the art, as for example by the addition of
hydroxyl groups or by formation of an oxide.
[0100] A presently preferred phase inversion support comprises
polyamides--organic polymers formed by the creation of amide bonds
between monomers of one or more types. Particularly useful
polyamides in the present disclosure are nylons. Nylons comprise
aliphatic carbon chains, usually alkylene groups, between amide
groups. The amide groups in nylons are very polar and can hydrogen
bond with each other, and are essentially planar due to the partial
double-bond character of the C--N bond. Nylons are polymers of
intermediate crystallinity; crystallinity being due to the ability
of the NH group to form strong hydrogen bonds with the C.dbd.O
group. Nylon typically consists of crystallites of different size
and perfection. By way of example, nylon 6,6 is typically
synthesized by reacting adipic acid with hexamethylene diamine, and
is a particularly presently preferred nylon useful with the
practice of the present invention as disclosed herein.
[0101] The present inventors have discovered that hydrophilic
electropositive materials may be dispersed into polyamide materials
so as to be operatively positioned therein to produce superior
nucleic acid binding matrices. Particularly useful matrices are
microporous in nature, more particularly microporous membranes
having asymmetric pores. Such microporous membranes facilitate
capture of nucleic acids contained in relatively very low
concentration in relatively large volume of sample fluid and allow
the relatively large volumes of sample fluid to be filtered due to
the high effective surface areas thereof. Further modification of
the membranes to include sequence-specific PNA's renders the
membranes of the present invention useful for the capture and
identification of specific nucleotides in samples with complex
mixtures of nucleic acid.
[0102] The nucleic acid of interest, once irreversibly bound to the
membrane, can function as a template for enzymatic amplification
procedures, including, but not limited to, PCR, NASBA, RCA and
other isothermic amplification methods, as presently known in the
art, or as may become known. Such use of the microporous membrane
comprising the highly electropositive material capable of
irreversibly binding one or more nucleic acids, as disclosed
herein, enables the detection of minute quantities of analyte, such
as, for example, nucleic acid, from large sample volumes, for
example allowing detection of a single organism from a large input
volume. As is known to those skilled in the art, such detection is
not easily performed using currently available technologies, such
as those described above. Furthermore, the incorporation of
sequence-specific PNA into the membrane makes possible the capture
and detection of specific polynucleotides at low copy number even
from samples comprising multiple sources of nucleic acid.
[0103] The membranes of the present disclosure may also be placed
into a container or other structure to optimize sample flow and
handling, as well as for amplification and detection. Such
structures may include, but are not limited to, a microcentrifuge
or centrifuge tube, a multiwell plate, a filter housing, or a
manifold, or other devices as would be known to those skilled in
the art.
[0104] In another representative embodiment, there is disclosed a
multi-layer membrane having one or more layers that do not include
any significant amounts of highly electropositive hydrophilic
material(s) capable of irreversibly binding nucleic acids in
conjunction, with one or more additional layers that include the
electropositive materials, optionally modified by the presence of
PNA's. The membrane layers that do not include any electropositive
material can be used to remove debris from the sample prior to
exposing the nucleic acid fraction with the membrane layer
comprising the electropositive material and/or PNA. Discrete layers
in the membrane may be produced that include the electropositive
material. The problem of isolating small quantities of a nucleic
acid molecules from a large sample volume can be greatly reduced by
incorporating the electropositive material in a membrane layer
downstream of a membrane layer without the propensity for binding
nucleic acids, by removing debris that might interfere with nucleic
acid binding. A multi-layer microporous membrane that might be used
for such purposes may be produced, for example, by the methods
described in the '441 patent to Vining, et al. A presently
preferred multi-layer membrane comprises one or more microporous
polyamide layers, more preferably one or more microporous nylon
layers.
[0105] In yet another representative embodiment, there is disclosed
a multi-layer membrane having one or more layers individually
functionalized to facilitate the capture of specific nucleic acid
molecules. Such individually functionalized layers optionally may
comprise highly electropositive hydrophilic material(s) capable of
irreversibly binding nucleic acids and/or sequence-specific PNA's.
Furthermore, it is possible to modify the individual layers of the
multi-layer membrane to facilitate binding, either specific or
non-specific, of nucleic acid. Such modification can include both
physical modification, such as the incorporation of highly
electropositive hydrophilic material, as well as chemical
modification, such as derivitization of exterior or interior
surfaces, or charge-transfer treatment, such as with nylon membrane
materials.
[0106] In a presently preferred embodiment, the individually
functionalized layers are used to remove nucleic acids that are not
desired to be detected in a subsequent membrane layer or layers.
For example, a multi-layer membrane of such representative
embodiment may comprise a first or outer layer individually
functionalized so as to be capable of removing bacterial nucleic
acid from a sample containing human nucleic acid, the human nucleic
acid being desired to be enriched in an inner membrane layer of the
multi-layer membrane. That is, the layers can be arranged with
respect to each other such that undesired nucleic acid can be
removed upstream of a membrane layer in which a particular analyte
of interest (such as nucleic acid) is desired to be collected. A
multi-layer microporous membrane of such embodiment may be
produced, for example, by the methods described in the '441 patent
referenced above.
[0107] In yet another representative embodiment, there is provided
a nucleic acid archival substrate comprising a microporous membrane
imbued or coated with a highly electropositive hydrophilic material
capable of irreversibly binding one or more nucleic acid types, and
optionally modified to comprise sequence-specific PNA's. Nucleic
acid bound to such substrate can be stored for long periods of
time. Nucleic acid storage can be particularly useful, for example,
when samples may need to be compared to known samples obtained in
the future, such as when biological material is isolated at a crime
scene without a suspect being immediately identifiable.
[0108] In still another representative embodiment, there is
provided one or more microporous membranes, such as, a microporous
polyamide membrane, comprising a highly electropositive material
with hydrophilic properties that is capable of irreversibly binding
nucleic acid and further comprising another nucleic binding
material, e.g., anion exchange resin, intercalating dye, PNA,
etc.
[0109] In a presently preferred embodiment, the highly
electropositive hydrophilic material capable of irreversibly
binding one or more nucleic acid types has a particle size in the
range of about one nanometer (1 nm) to about one thousand microns
(1000.mu.). Such particle sizes have been found to provide enhanced
efficacy with respect to nucleic acid binding per unit area of the
membrane, which membrane, being microporous, comprises significant
interior as well as exterior surface area.
[0110] Methods for preparing such microporous membranes are also
disclosed. In a presently preferred representative method, a dope
is prepared with the highly electropositive hydrophilic material
capable of irreversibly binding one or more nucleic acid types
operatively dispersed therein, and the dope is used in the
production of microporous membrane by methods well known in the
art. In another method, the highly electropositive hydrophilic
material is placed in a polymer that is coated onto, or saturated
into, a preformed microporous membrane.
[0111] By dispersing the highly electropositive hydrophilic
material capable of irreversibly binding one or more nucleic acid
types into the material to be used in the formation of a
microporous membrane, a composite membrane is formed which permits
high surface area for the capture and/or removal of nucleic acids.
Alternatively, but less desirably (due to the difficulty in
providing a uniform coating throughout the microporous structure),
the microporous membrane may be coated with material, such as a
resin, comprising the highly electropositive hydrophilic
material.
[0112] As would be recognized by one of skill in the appropriate
art, it is possible to synthesize PNA's with complementarity to
specific nucleotide sequences of interest on solid supports. These
art-recognized methods of PNA synthesis can be adapted to prepare
sequence-specific PNA's directly on the highly electropositive
hydrophilic materials prior to the incorporation of such material
into a layer or layers of the membranes of the present invention.
Preferably, the synthesis of such PNA's on the solid phase material
renders the highly electropositive hydrophilic material capable of
binding to only the specific nucleic acid of interest and not to
all sources of nucleic acid present in the sample, at least within
a specific layer of the multi-layer membrane.
[0113] As referenced above, the composite membrane may be used not
only to capture the nucleic acid, but may be used as a platform for
amplifying the bound nucleic acid, and detecting the same. The
captured nucleic acid associated with the microporous membrane may
function as a solid phase template for amplification, enabling
detection of minute quantities of nucleic acid in a large sample
volume. The microporous membrane having the captured nucleic acid
may also be saved for archival purposes, with amplification and
detection being performed at a later date, either on the membrane
of after removal of the nucleic acid from the archival
membrane.
[0114] Turning to FIG. 1, there is shown an illustrative,
representative, method for amplifying nucleic acids using the
composite microporous membranes of the present disclosure. In step
A, the microporous membrane comprising the highly electropositive
hydrophilic material capable of irreversibly binding the nucleic
acid of interest is exposed to a complex biological sample
containing cellular debris and nucleic acid. Nucleic acid is
irreversibly captured on the membrane that is washed several times
to remove non-bound proteins and cellular debris. In step B, the
bound nucleic acid is amplified by known techniques with the
addition of, for example, primers, deoxynucleotide triphosphate
molecules (dNTPs), buffer, etc., producing amplified product (step
C). The membrane having the bound nucleic acid can be used as a
template for further amplification cycles, or be stored for
archival purposes.
[0115] In a presently preferred representative embodiment, a
multi-layer microporous membrane is employed, having at least one
layer incorporating the highly electropositive hydrophilic material
capable of binding irreversibly to one or more nucleic acid types
and one or more layers of the membrane being void of any of the
highly electropositive hydrophilic material capable of binding
irreversibly to nucleic acid. Those layers that do not incorporate
the electropositive materials can be used to remove debris from the
sample prior to exposure of the nucleic acid fraction to a layer
including the electropositive material. A further variation of the
membrane structure, and one that is preferred for certain unique
application of the use of the membranes of the present invention,
incorporates a layer, separate from the layer comprising highly
electropositive material alone, wherein the layer is modified to
comprise PNA's. These embodiments of the membrane of the present
invention make possible the solution of problems associated with
the isolation of very small quantities of a nucleic acid molecule
from a large sample volume in a manner not possible in the prior
art. These ends can be more readily reached by incorporating the
electropositive material in a layer downstream of a layer without
the capacity to bind nucleic acid, whereby the upstream layer
functions to remove debris that might interfere with nucleic acid
binding, and/or amplification, and/or detection in the layer
comprising the electropositive material.
[0116] In another presently preferred representative embodiment,
there is disclosed a multi-layer membrane having at least one layer
functionalized for the capture of specific nucleic acid molecules
and at least one layer comprising highly electropositive
hydrophilic material(s) capable of irreversibly binding nucleic
acids. The functionalized layers optionally may comprise highly
electropositive hydrophilic material(s) capable of irreversibly
binding nucleic acids. In a presently preferred type of such
representative embodiment, the functionalized layers are used to
remove nucleic acids that are not desired to be detected in a
subsequent layer of the multi-layer membrane. In a more preferred
embodiment, one of the layers is modified to comprise
sequence-specific PNA's capable of selectively binding to a
specific polynucleotide of interest from a sample comprising
multiple sources of nucleic acid.
[0117] It is presently preferred that the membrane in which the
highly electropositive hydrophilic material is incorporated has
pore sizes in the range of about 0.04 microns to about 20 microns.
Preferably, the membrane is a phase inversion microporous membrane.
Such membrane preferably comprises nylon (such as nylon 6,6), but
may comprise other materials used in the fabrication of
single-layer and multiple-layer phase inversion microporous
membranes as would be known to those of ordinary skill in the
art.
EXAMPLE 1
Preparation of Nylon Membrane Comprising Highly Electropositive
Hydrophilic Material
[0118] A dope formulation comprising approximately 16.1% by weight
Nylon 6,6 (Monsanto.RTM. Vydyne.TM. 66Z), 77.1% by weight formic
acid, and about 6.8% by weight methanol, was produced using the
methods disclosed in U.S. Pat. Nos. 3,876,738 and 4,645,602, the
disclosure of each of which is herein incorporated specifically by
reference. This is the standard formulation and method used to
produce the control (white) membrane.
[0119] To produce the multi-layer composite microporous membrane
comprising a highly electropositive hydrophilic material (Xtra
Bind) of the present invention, the method is similar to the above,
but with the additional step of adding the Xtra Bind prior to the
addition of Nylon, and changing the mixing apparatus to facilitate
uniform dispersion and uniform suspension of the heterogeneous Xtra
Bind material in the dope. Briefly, the altered method consists of
the following steps: a dope formulation comprising approximately
75.1% by weight formic acid and 6.3% by weight methanol (to final
weight of dope) was mixed in a Silverson.RTM. Model
#L4SRT.backslash.SU (Sealed Unit) one-half liter sealed vessel with
high dispersion mixing head for about 15 minutes at about 400 rpm.
To this mixture, 3.1% Xtra Bind material (500 mesh Xtra Bind
Matrix) at an intended ratio of about a 1:5 parts by weight of Xtra
Bind:Nylon was added. The resultant composition was mixed for about
10 minutes using the same mixing apparatus at about 2000 rpm. The
resultant was then dispensed into a 16 oz. glass jar. To this
resultant about 15.5% by weight Nylon-66 (Monsanto.RTM. Vydyne.TM.
66Z) (to final weight of dope) was added. The resulting composition
was mixed with a 1.25 inch diameter three-blade propeller mounted
on a T-line.RTM. Model #134-1 laboratory mixer. A cap with a
sealing arrangement for the propeller shaft was fabricated to
minimize volatile losses. Mixing occurred at ambient temperatures.
The mix cycle began with an initial mix at about 350 rpm for about
one-half hour; then the mixer was slowed to about 70 rpm for about
another two hours to homogenize the dope. After the resultant was
mixed, the glass jar was removed from the mixer, and sealed with a
cap. The sealed vessel and it's contents were rolled on a rolling
mill jar mixer, submerged in a waterbath at about 34.degree. C. for
several hours to ensure a uniform thermal history (maximum mix
temperature) of the dope, and maintain the suspension of Xtra Bind
material in the mix. The rolling mill was then removed from the
water bath. The jar and its contents were allowed to cool to room
temperature while rolling (again, to maintain the suspension).
Gentle rolling continued until the dope was used to form a
microporous membrane.
[0120] To gain an appreciation for the pore size of a microporous
nylon membrane with Xtra Bind cast directly from this dope, a small
portion (.about.20 cc) of the dope was cast and quenched in a
laboratory apparatus which simulates the casting process described
in U.S. Pat. No. 3,876,738, to Marinaccio and Knight, to produce a
single layer, nominally 5 mil thick, wet, non-reinforced layer of
microporous nylon membrane. This membrane was washed in deionized
water, then folded over onto itself (about 10 mils, wet) and dried
under conditions of restraint to prevent shrinkage in either the
machine direction (x-direction) or cross direction (y-direction).
This produced a small sample of dried double layer non-reinforced
microporous nylon membrane having a combined thickness of about
eight (8) mils after shrinkage in thickness (z-direction) of the
collapsing wet pore structure was complete (actual thickness shown
in Table 1, below). An Initial Bubble-Point and Foam-All-Over-Point
test was performed, as described in U.S. Pat. No. 4,645,602, using
deionized water as a wetting fluid.
[0121] A second casting was also produced via cast, quench, and
wash. It was not folded over onto itself, but dried under
conditions of restraint as a single layer, to produce a small
sample of dried single layer non-reinforced microporous nylon
membrane. This sample was produced for Scanning Electron Microscopy
(SEM) analysis.
[0122] The control (white) membrane was similarly cast, quenched,
washed, and dried in both single and double layer samples, and
tested. The results of such testing are provided in Table 1,
below.
1TABLE 1 For SEM analysis Double layer Single layer Xtra Bind:
Nylon Dry Dry Content IBP psig FAOP psig Thickness (mils) Thickness
(mils) 0:100 30.5 34.0 8.5 4.5 Control (White) 1:5 39 44 8.2
4.3
[0123] The dry single-layer versions of the control (white)
membrane and the Xtra Bind-containing membrane were submitted for
SEM analysis in cross section. The results are shown in FIGS. 2A,
3A and 4A. From a review of the SEMs, it is evident that the Xtra
Bind matrix is embedded within the pore structure of the nylon
membrane, in such a way that the surfaces of the heterogeneous Xtra
Bind particles are accessible to fluids within the pores;
therefore, the binding functionality of the Xtra Bind is
maintained.
[0124] As can be clearly seen from the SEMs of FIGS. 2B, 3B and 4B,
the Control sample contains no irregularly shaped objects/particles
in the passages or tortuous tunnels or paths formed in the final
membrane, while the Test sample clearly shows non-membrane
material, in this case the highly electropositive hydrophilic
material capable of irreversibly binding one or more nucleic acids,
positioned in with the passages, tortuous tunnels or paths formed
in the final membrane.
[0125] A sample similar to the second casting was also produced via
cast, quench, and wash. This casting was not folded over onto
itself, but dried under conditions of restraint as a single layer,
to produce a small sample of dried single layer non-reinforced
microporous nylon membrane. This sample was utilized for
determining if the non-reinforced microporous nylon membrane having
the highly electropositive hydrophilic material is capable of
irreversibly binding one or more nucleic acid types. The results of
these tests are reported in Example 2 below.
EXAMPLE 2
Binding of Nucleic Acid to Membranes of Example 1
[0126] To assess whether microporous nylon membrane containing the
highly electropositive hydrophilic material (Xtra Bind.TM.)
irreversibly binds nucleic acid and if the captured nucleic acid is
capable of functioning as a template for PCR, the following
experiment was performed.
[0127] Known amounts of K562 cells were lysed and diluted in water.
Either 10.0-ng or 1.0-ng samples of genomic DNA was contacted with
an Xtra Bind-containing microporous nylon membrane or unmodified
nylon microporous membrane (without Xtra Bind) and incubated in the
lysis/binding buffer for an appropriate time in microcentrifuge
tubes. The first two lanes of a data set are duplicates of 10.0-ng
samples; the second two are duplicates of 1.0-ng samples. The
membranes were then washed with buffer and the membranes were
combined with appropriate components to support DNA amplification
using the polymerase chain reaction (PCR). Forward and reverse
primers directed against the human leukocyte antigen DR.beta.
(HLA-DR.beta.) were used to amplify the product of interest.
[0128] FIG. 5 is a photograph of an agarose gel stained with
ethidium bromide. The first four lanes (PCR Controls) are controls
indicating that the PCR is functional for the production of the
product of interest. The next four lanes contain the samples of
reaction product when the genomic DNA is incubated in the presence
of the unmodified nylon, washed and then amplified by the PCR. It
can be readily seen that no product is detected. The last four
lanes are negative controls--PCR's that lack DNA template,
indicating that any product seen is not the result of contaminating
DNA in any of the buffer components used in the reaction.
[0129] FIG. 6 is a photograph of the lower portion of the gel in
FIG. 5. This sample is one where the genomic DNA was incubated in
the presence of membrane containing the Xtra Bind material, washed
and then amplified by the PCR. PCR product is readily seen in these
lanes indicating that K562 genomic DNA was retained by the membrane
and that this genomic DNA is functional as a template for enzymatic
amplification.
[0130] Clearly a difference is seen between the Xtra
Bind-containing microporous nylon membrane and the unmodified
microporous nylon membrane demonstrating the superior performance
of the Xtra Bind-containing microporous nylon membrane in retaining
nucleic acid as a functional template for the PCR.
[0131] Thus, it should be apparent from the above example that the
microporous membranes disclosed herein and the methods of making
and using same provides improved membranes and methods for
separating nucleic acids from liquid biological samples and
amplifying the same.
[0132] It should be pointed out that the capture of nucleic acids
using highly electropositive hydrophilic material capable of
irreversibly binding one or more nucleic acids, such as, for
example, Xtra Bind, is the irreversible binding of the nucleic
acids to the highly electropositive hydrophilic material. This
enables the membrane containing the highly electropositive
hydrophilic material of the present disclosure to be used, among
other uses, as an archiving system. Additionally, it should be
clear that a large volume of sample can be processed using the,
presently preferred, nylon microporous membrane containing the,
presently preferred, Xtra Bind material.
EXAMPLE 3
Capture and Identification of Single Nucleotide Polymorphisms
(SNPs) in a Target Sequence
[0133] As the results from Example 2, above, indicate, microporous
nylon membranes modified to comprise a highly electropositive,
hydrophilic material such as Xtra Bind are capable of the capture
and subsequent amplification of target nucleic acid. The results of
example 2, coupled with the teachings of the '441 patent,
illustrate that multi-layer composite membranes of the present
invention can provide a unique combination of characteristics that
makes possible a powerful set of applications heretofore
unavailable in the prior art. Example 3, and the examples that
follow, illustrate a representative selection of these
applications.
[0134] A rapid and accurate detection of genetic variants,
including single-base mismatches, is essential for the detection of
genetic diseases. Even a single-base substitution in a human gene
can result in deleterious effects in humans. Thus, there is a need
for a sensitive nucleic acid-based diagnostic technique for the
detection of genetic diseases which will include the ability to
ascertain whether an individual is heterozygous or homozygous
allelic for a genetic disease.
[0135] Single nucleotide polymorphisms (SNPs) are the most common
type of genetic variation found in the human genome, occurring
approximately once per 1000 bases. Since they are single base
changes in the primary DNA sequence they are easily detected by
sequencing analysis. They can be useful as genetic markers for
navigating the genome. As unrelated entities, the value of SNP's in
disease manifestation is questionable. However, it is believed that
if a SNP linkage map can be built, SNP's that travel together may
be indicators of disease states or predispositions for developing a
particular disease. In addition, it is hypothesized that SNP's may
be useful in designing patient-specific pharmaceutical therapies to
optimize the efficacy of the drug and to avoid adverse drug
reactions (ADR's).
[0136] Nucleic acid sample (DNA or RNA) is filtered through the
membrane as it appears in FIG. 9A. The sample can be lysed prior to
filtration or on the membrane itself. Buffer is functional for
lysis and does not affect nucleic acid binding to the PNA. Nucleic
acid is bound in a sequence-specific fashion in Layer 1 (FIG. 9B)
using membrane-bound PNA's directed against the sequence(s) of
interest. Buffer for binding in Layer 1 is designed so as to select
against binding in Layer 3. Should strand invasion be required, PNA
clamps can be employed in Layer 1, as well.
[0137] PNA-nucleic acid hybrids have a much higher thermal
stability than DNA-DNA DNA-RNA, or RNA-RNA species. In addition,
PNA duplexes are not affected by ionic strength. This feature
allows for formation of duplexes under conditions that do not favor
formation of standard nucleic acid duplex molecules (e.g., cell
extracts and serum). PNA's also exhibit greater specificity in
binding to a complementary DNA or RNA molecule, since a PNA-DNA
mismatch is significantly more destabilizing than a mismatch in a
DNA duplex species. Mismatches, which correlate to SNP's in the
complementary region between the PNA and target nucleic acid, can
be dissociated from sequences lacking the nucleotide variants at
temperatures .about.10.degree. C. below the temperature that
denatures a perfect hybrid molecule. This feature will allow for
the specific release of the bound SNP-containing variant DNA while
leaving the wild-type sequence attached to the PNA in Layer 1 of
the membrane (FIG. 9C).
[0138] A buffer at the appropriate temperature is passed through
the membrane that releases the DNA/mRNA molecule containing a
nucleotide variant from the PNA tethers in Layer 1. This buffer
also functions to optimize binding of the released nucleic acid to
the Xtra Bind material in Layer 3.
[0139] Once freed from Layer 1, the SNP-containing sequence
variants pass through Layer 2 and are bound irreversibly in Layer 3
of the membrane on the Xtra Bind material. The bound DNA can then
be amplified or analyzed as desired, utilizing techniques well
known in the art.
EXAMPLE 4
Separation of Victim's Vaginal/Cervical Epithelia From
Perpetrator's Sperm Cells
[0140] A problem frequently encountered in forensic analyses of
genetic material, such as in identifying the attacker in a rape
case, is that the epithelial cells collected during a vaginal swab
often overwhelm the PCR and prevent the detection of the
perpetrator's DNA. A means for enriching for Y-chromosome-specific
sequences by achieving the separation of epithelia from sperm would
significantly reduce the amount of interfering DNA in the
sample.
[0141] In this Example, an asymmetric, three-layered membrane is
employed for separating epithelial cells from sperm in Layer 1,
capture of sperm cells in Layer 2, and sequence-specific capture of
Y-chromosome sequences using PNA incorporated in Layer 3. The PNA's
used for capture of Y-chromosome-specific sequences are derived
from nucleotide sequence databases in the public domain. The PNA
capture probes are designed to recognize short tandem repeats
(STR's) unique to the male Y-chromosome.
[0142] A swab from victim is placed into PBS to release the cells.
The sample is then filtered through the membrane where the
epithelial cells are captured as described above. Alternatively, as
would be recognized by one of skill in the relevant art, the
membrane can be contained in a "collection tube" or other similar
apparatus, and effect the same result. It is also possible to
utilize an integral construction and reduce concerns of cross
contamination. The sperm cells are washed through the first
membrane layer by either subsequent `forward` washes or repeated
cycles of reverse and forward PBS washes. Sperm cells are lysed in
Layer 2, and the released DNA is captured on the PNA contained in
Layer 3. Contaminating DNA from Layer 1-captured cells would not
bind and can be washed away using stringency washes. The captured
sperm DNA can then be released from the membrane or analyzed in
situ by PCR or other method(s).
EXAMPLE 5
Capture and Identification of the Presence of Genetically Modified
Organisms (GMOs)
[0143] The presence of GMOs is not only a concern for uncontrolled
proliferation of artificially modified genetic material, with an
accompanying potential for loss of native species by GM species
with greater robustness, but also a concern for possible allergic
reactions to gene products not usually present in a particular food
(e.g., peanut proteins in potatoes). In this application, an
asymmetric membrane containing PNA's specific for the transgene
contained in the GMO of interest are contained in Layer 3. DNA is
prepared from organisms to be evaluated for the presence of
sequences indicating incorporation of genetically-modified loci,
for example, the presence of genes encoding virus resistance
proteins and genes improving resistance to herbicides in food
plants. A concern, particularly in the European Union (EU), is gene
transfer among species by cross pollination.
[0144] Use of a membrane similar to that described in Example 3
above would improve the ability of researchers and agricultural
agencies to monitor more rapidly and efficiently the extent of gene
transfer and identify plants/animals harboring the exogenous
gene(s).
[0145] Samples are prepared, and then contacted with membranes
containing PNA's to capture the sequence of interest by placing the
PNA-containing membrane in a multi-well plate (96-, 384-,
1536-well), creating, in essence, a two-dimensional, semi-micro
array. In this fashion, a large number of samples could be screened
rapidly for one or several exogenous sequences.
[0146] The process and membrane is as depicted in FIG. 10. DNA
samples are introduced to the membrane and captured in Layer 1,
containing PNA's directed toward the transgenic locus. Non-specific
DNA is easily washed away using appropriately stringent conditions.
DNA captured on the PNA's located in Layer 1 are released and
captured irreversibly in the Xtra Bind-modified membrane of Layer 3
downstream. Again, Layer 2 functions, in effect, as a wash layer as
described in Example 3 and in FIG. 8.
[0147] The DNAs bound in the Xtra Bind matrix optionally can be
amplified for analysis. As would be recognized by one of skill in
the art, the combined capture and amplification of the target DNA
would be desirable in those situations where the transgene would be
present in total nucleic acid at very low levels. In addition, the
PNA-bound DNA can be examined for sequence variation and provide
insight into how these modifications affect the expression of the
transgene.
[0148] While the disclosure has been described with respect to
presently preferred embodiments, those skilled in the art will
readily appreciate that various changes and/or modifications can be
made to the disclosure without departing from the spirit or scope
of the disclosure as defined by the appended claims. All references
cited in this specification are herein incorporated by reference to
the same extent as if each individual reference was specifically
and individually indicated to be incorporated by reference.
* * * * *
References