U.S. patent application number 09/726627 was filed with the patent office on 2001-04-05 for solid medium and process for the storage and rapid purification of nucleic acid.
Invention is credited to Davis, James C., Iyer, Mridula, Qu, Daqing, Smith, Martin A..
Application Number | 20010000149 09/726627 |
Document ID | / |
Family ID | 26822100 |
Filed Date | 2001-04-05 |
United States Patent
Application |
20010000149 |
Kind Code |
A1 |
Smith, Martin A. ; et
al. |
April 5, 2001 |
Solid medium and process for the storage and rapid purification of
nucleic acid
Abstract
A medium for storage and subsequent analysis of a genetic
material includes a support for immobilizing the genetic material
thereon and allowing subsequent elution of the genetic material
therefrom and a coating functionally associated with the support
for enabling cellular lysis and releasing the genetic material from
the lysed cells while stabilizing the immobilized released genetic
material. A method of storing the genetic material and subsequently
analyzing the genetic material includes the steps of immobilizing
the genetic material on a support while enabling cellular lysis and
release of genetic material from the lysed cells and stabilizing
the immobilized released genetic material on the support. The
genetic material is then eluted to generate a soluble genetic
material fraction. The eluted genetic material can be analyzed.
Inventors: |
Smith, Martin A.;
(Brookline, MA) ; Iyer, Mridula; (Acton, MA)
; Qu, Daqing; (Newton, MA) ; Davis, James C.;
(Rockland, MA) |
Correspondence
Address: |
KOHN & ASSOCIATES
Suite 410
30500 Northwestern Highway
Farmington Hills
MI
48334
US
|
Family ID: |
26822100 |
Appl. No.: |
09/726627 |
Filed: |
November 30, 2000 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09726627 |
Nov 30, 2000 |
|
|
|
09507548 |
Feb 18, 2000 |
|
|
|
09507548 |
Feb 18, 2000 |
|
|
|
09398625 |
Sep 18, 1999 |
|
|
|
60130716 |
Apr 22, 1999 |
|
|
|
60123990 |
Mar 11, 1999 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
435/6.14 |
Current CPC
Class: |
B01L 2300/0822 20130101;
B01L 2300/163 20130101; C12N 15/1006 20130101; B01L 3/5023
20130101; C12Q 1/6806 20130101; Y10T 436/143333 20150115; C12N 1/06
20130101; C12Q 1/6806 20130101; C12Q 2565/625 20130101; G01N 1/405
20130101; B01L 3/5029 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method of releasing genetic material from a support by eluting
the genetic material from the support into solution in heated water
at different temperatures to generate soluble populations of
genetic material fractions at each temperature.
2. A method as in claim 1, wherein said eluting step further
includes: heating the water containing the support having the
genetic sample immobilized therein to varying temperatures in the
range of 65.degree. C. to 100.degree. C.; and releasing the heated
genetic material from the heated support and into the water
solution at each temperature.
3. The method as in claim 1 further including the step of washing
the support having the genetic material immobilized therein prior
to the eluting step.
4. The method as in claim 1 further including the steps of:
collecting a genetic material sample in a container containing the
support; contacting the support with the genetic material; and
conducting said immobilizing and eluting steps within the
container.
5. The method as in claim 1 wherein the eluting step includes
disposing the support having the genetic material therein into a
container.
6. The method as in claim 1 wherein the eluting step includes
disposing the support having the genetic material therein into a
multi-well plate.
7. The method as in claim 1 wherein the eluting step includes
disposing the support having the genetic material therein into a
water bath.
8. The method as in claim 1 wherein said eluting step includes
eluting genetic material from a support having a fiber matrix and a
surfactant deposited onto the fiber matrix for protecting the
genetic material, applied to the fiber matrix, from
degradation.
9. The method as in claim 5 wherein eluting step further includes
eluting genetic material from the fiber matrix having synthetic
glass fibers and filaments.
10. The method as in claim 5 wherein the eluting step further
includes eluting genetic material from the support having the
surfactant that further includes an anionic detergent such as
sodium dodecyl sulphate.
11. The method as in claim 1 wherein said method includes the step
of analyzing the eluted populations of genetic material.
12. The method as in claim 8 wherein said analyzing step is further
defined as amplifying the various populations of soluble genetic
material and visualizing the amplified populations of genetic
material.
13. The method as in claim 8 wherein said analyzing step is further
defined as amplifying the various populations of soluble genetic
material and labeling the amplified populations of genetic material
with fluorescent probes.
14. The method as in claim 8 wherein said analyzing step is further
defined as amplifying and fluorescently labeling various
populations of genomic DNA in the solution thereof.
15. The method as in claim 8 wherein said analyzing step further
includes the step of genetic genotyping.
16. The method as in claim 1 wherein the genetic material source is
selected from the group consisting essentially of blood, saliva,
sputum, and buccal epithelial cells.
17. The method as in claim 1 further including the steps of
applying a genetic material sample to the support wherein the
support is disposed within a spin basket of a spin microfuge
device, spinning the basket, discarding a produced filter to
perfect said immobilization steps and subsequently performing said
eluting step.
18. The method as in claim 14 wherein said eluting step is further
defined as adding heated water to the basket containing the support
subsequent to said discarding step, spinning the basket and
collecting the water containing the eluted nucleic acid therein.
Description
CROSSREFERENCE TO RELATED APPLICATIONS
1. This present application is a continuation application of U.S.
Ser. No. 09/507,548, filed Feb. 18, 2000, which is a divisional
application of U.S. Ser. No. 09/398,625, filed Sep. 18, 1999, which
is a conversion of U.S. Provisional patent application Ser. No.
60/130,716, filed Apr. 22, 1999, and that claims the benefit of
U.S. Provisional application Ser. No. 60/123,990, filed on Mar. 11,
1999, which applications are incorporated herein by reference.
FIELD OF THE INVENTION
2. The present invention relates to medium and methods for storage
and subsequent purification of nucleic acids or genetic material
from whole cells. In particular, the invention relates to the
storage and purification of nucleic acids from a biological mixture
of molecules in a fluid phase on a support. The purified nucleic
acid may then be utilized for a variety of analyses such as
amplification by the polymerase chain reaction (PCR) (PCR
Technology: Principles and Applications for DNA Amplification, H.
Erlich (ed) Stockton Press 1989), genotyping, sequencing (Sanger et
al (1977) DNA Sequencing with Chain Terminating inhibitors P. N. A.
S. 74: 5463), optical density quantitation, southern and northern
blotting, fluorescent detection, making molecular probes, and
cloning (Molecular Cloning, Sambrook, et al. (1989)).
DESCRIPTION OF BACKGROUND ART
3. Genotyping is the discipline of identifying an individual's
genome in relation to disease specific alleles and/or mutations
that occur as an effect of parental linkage. The rapid purification
of human genomic DNA is an essential part of a genotyping process;
the genomic DNA of an individual being the structural unit for the
entire DNA sequence of every allele expressed.
4. Human genomic DNA cannot be directly sequenced. In order to
carry out sequence analysis on regions of the chromosomes that may
contain portions of mutation or disease specific sequences,
selected portions are amplified via PCR and the amplified products
sequenced. The selected portions of the chromosomes that are
amplified are dictated by the specific sequence of the primers used
in the PCR amplification. The primer sets that are used in
genotyping studies are commercially available and are
representative for the chromosome under examination. Therefore, if
linkage studies identify that a disease bearing sequence is on a
particular chromosome, then many primer sets will be utilized
across that chromosome in order to obtain genetic material for
sequencing. The resultant PCR products may well represent the
entire chromosome under examination. Due to the large length of
chromosomes, many PCR reactions are carried ocut on the genomic DNA
template from a single patient.
5. Human genomic DNA is purified by a variety of methods (Molecular
Cloning, Sambrook et al. (1989)). Consequently, many commercial kit
manufacturers provide products for such techniques, for example:
AmpReady.TM. (Promega, Madison, Wis.), DNeasy.TM. (Qiagen,
Valencia, Calif.), and Split Seconds.TM. (Roche Molecular
Biochemicals, Indianapolis, Ind.). These products rely on the use
of specialized matrices or buffer systems for the rapid isolation
of the genomic DNA molecule.
6. More recently, microporous filter-based techniques have surfaced
as tools for the purification of genomic DNA as well as a whole
multitude of nucleic acids. The advantage of filter-based matrices
are that they can be fashioned into many formats that include
tubes, spin tubes, sheets, and microwell plates. Microporous filter
membranes as purification support matrices have other advantages
within the art. They provide a compact, easy to manipulate system
allowing for the capture of the desired molecule and the removal of
unwanted components in a fluid phase at higher throughput and
faster processing times than possible with column chromatography.
This is due to the fast diffusion rates possible on filter
membranes.
7. Nucleic acid molecules have been captured on filter membranes,
generally either through simple adsorption or through a chemical
reaction between complementary reactive groups present on the
filter membrane or on a filter-bound ligand resulting in the
formation of a covalent bond between the ligand and the desired
nucleic acid.
8. Porous filter membrane materials used for non-covalent nucleic
acid immobilisation have included materials such as nylon,
nitrocellulose, hydrophobic polyvinylidinefluoride (PVDF), and
glass microfiber. A number of methods and reagents have also been
developed to also allow the direct coupling of nucleic acids onto
solid supports, such as oligonucleotides and primers (eg. J. M.
Coull et al., Tetrahedron Lett. Vol. 27, page 3991; B. A. Conolly,
Nucleic Acids Res., vol. 15, page 3131, 1987; B. A. Conolly and P.
Rider, Nucleic Acids Res., vol. 12, page 4485, 1985; Yang et al
P.N.A.S. Vol. 95: 5462-5467). UV cross-linking of DNA (Church et
al., PNAS, vol. 81, page 1991, 1984), The Generation Capture Column
Kit (Gentra Systems, Minneapolis, Minn.) and RNA (Khandjian, et
al., Anal. Biochem, Vol. 159, pages 227, 1986) to nylon membranes
have also been reported.
9. Many chemical methods have been utilized for the immobilization
of molecules such as nucleic acids on filter membranes. For
example, activated paper (TransBind.TM, Schleicher & Schuell
Ltd., Keene, N.H.) carbodimidazole-activated hydrogel-coated PVDF
membrane (Immobilin-IAV.TM, Millipore Corp., Bedford, Mass.), MAP
paper (Amersham, Littlechalfont Bucks, Wis.), activated nylon
(BioDyne.TM, Pall Corp., (Glen Cove, N.Y.), DVS- and cyanogen
bromide-activated nitrocellulose. Membranes bound with specific
ligands are also known such as the SAM2TM Biotin Capture Membrane
(Promega) which binds biotinylated molecules based on their
affinity to streptavidin or MAC affinity membrane system (protein
A/G) (Amicon, Bedford, Mass.) Some of the disadvantages of covalent
attachment of biomolecules onto activated membranes are:
10. a) Molecule immobilization is often slow requiring 20-180
minutes for reaction completion.
11. b) High ligand and biomolecule concentration is needed for fast
immobilization.
12. c) Constant agitation is needed during the immobilization
process that may result in biomolecule denaturation and
deactivation.
13. d) Once the immobilization process is complete, often a
blocking (capping) step is required to remove residual covalent
binding capacity.
14. e) Covalently bound molecules can not be retrieved from the
filter membrane.
15. There is a need for a nucleic acid immobilization procedure
that exhibits the high specificity of covalent immobilization onto
the filter membrane without the use of harsh chemical reactions and
long incubation times. In particular there is a need for the
capture and separation of nucleic acids from a mixture in a fluid
phase onto a filter membrane matrix. Of special interest is the
ability to store or archive the bound nucleic acids on the filter
membrane matrix.
16. More recently, glass microfiber, which has been shown to
specifically bind nucleic acids from a variety of nucleic acid
containing sources very effectively (for example see: itch at al
(1997) Simple and rapid preparation of plasmid template by
filtration method using microtiter filter plates. NAR, vol. 25, No.
6: 1315-1316; Andersson, B. et al (1996) Method for 96-well M13 DNA
template preparations for large-scale sequencing. BioTechniques
vol. 20: 1022-1027). Under the correct salt and buffering
conditions, nucleic acids will bind to glass or silica with high
specificity.
17. Based on U.S. Pat. Nos. 5,496,562, 5,756,126, and 5,307,527, it
has been demonstrated that nucleic acids or genetic material can be
immobilized to a cellulosic-based dry solid support or filter (FTA
filter). The solid support described is conditioned with a chemical
composition that is capable of carrying out several functions: (i)
lyse intact cellular material upon contact, releasing genetic
material, (ii) enable and allow for the conditions that facilitate
genetic material immobilization to the solid support (probably by a
combination of mechanical and chaotrophic), (iii) maintain the
immobilized genetic material in a stable state without damage due
to degradation, endonuclease activity, UV interference, and
microbial attack, and (iv) maintain the genetic material as a
support-bound molecule that is not removed from the solid support
during any down stream processing (as demonstrated by Del Rio et al
(1995) BioTechniques. Vol. 20: 970-974).
18. The usefulness of the so called FTA cellulosic filter material
described in U.S. Pat. Nos. 5,496,562, 5,756,126, and 5,807,527 has
been illustrated or several nucleic acid techniques such as
bacterial ribotyping (Rogers, C & Burgoyne, L (1997) Anal.
Biochem. Vol. 247: 223-227), detection of single base differences
in viral and human DNA (Ibrahim et al (1998) Anal. Chem. Vol. 70:
2013-2017), DNA databasing (Ledray et al (1997) J. Emergency
Nursing. Vol. 23, No. 2: 156-158), automated processing for STR
electrophoresis (Belgrader, B & Marino, M (1996) L.R.A. vol. 9:
3-7, Belgrader et al (1995) BioTechniques. Vol. 19, No. 3:
427-432), and oligonucleotide ligation assay for diagnostics (Baran
et al (1996) nature Biotech. Vol 14: 1279-1282).
19. It has been shown that nucleic acid or genetic material applied
to, and immobilized to, FTA filters cannot be simply removed, or
eluted from the solid support once bound (Del Rio et al (1995)
BioTechniques. Vol. 20: 970-974). This is a manor disadvantage for
applications where several downstream processes are required from
the same sample, such a STR profiling and genotyping.
20. Currently, cellular material is applied to FTA filter media,
and generally the cellular material, once applied forms a spot on
the FTA filter. From this spot, small punches can be taken; each
small punch will have immobilized to it enough nucleic acid or
genetic material to facilitate a single downstream process such as
a PCR reaction. As the two primers administered to a PCR reaction
are presented in solution, it is of no consequence that the
cellular nucleic acid template is immobilized to the filter. All
amplicon will be formed in solution. Amplicon can then be readily
removed from the reaction by aspirating the liquid phase away from
the FTA solid filter punch. Therefore, for multiple processing from
a single sample, many punches have to be taken. Multiple punching
is very time consuming, and as yet, has not lent itself to
simplified automation.
21. It is much more desirable to provide nucleic acid as a soluble
fraction from which aliquots can be readily dispensed to as many
reactions as required. Automated liquid handling of this type is a
fundamental technique within the pharmaceutical and other
industries (for example see: Armstrong et al (1998) J. Biomolecular
Screening. Vol. 3, No. 4: 271-275).
SUMMARY OF THE INVENTION
22. In accordance with the present invention, there is provided a
medium for storage and subsequent analysis of a genetic material,
the medium including a support for immobilizing a genetic material
thereon and for allowing subsequent elution of genetic material
therefrom. A coating is functionally associated with the support
for enabling cellular lysis and releasing the genetic material from
the lysed cells while stabilizing the immobilized released genetic
material. A method for storing the genetic material and
subsequently analyzing the genetic material includes the steps of
immobilizing the genetic material on the support while enabling
cellular lysis and release of genetic material from the lysed
calls. The immobilized released genetic material is stabilized. The
genetic material is then eluted to generate a soluble genetic
material fraction. The eluted genetic material is subsequently
analyzed.
BRIEF DESCRIPTION OF THE DRAWINGS
23. FIG. 1 is a digital representation of a gel showing the effect
of different heat elution regimes on blood genomic DNA bound to the
filter membrane of the invention with respect to Amelogenin PCR
amplification; PCR products being noted at 218 bp, lane 1: blood
spotted 1 mm filter disk with 82.degree. C., 10 minute incubation,
lane 2: 82.degree. C. eluted fraction, lane 3: blood spotted 1 mm
filter disk with 95.degree. C., 10 minute incubation, lane 4:
95.degree. C. eluted fraction, lane 5: no DNA control;
24. FIG. 2 is a digital representation of a gel showing the results
of a full elution protocol for blood genomic DNA bound to the
filter membrane of the invention with respect to Amelogenin PCR
amplification, PCR products being noted at 218 bp, lane 1: blood
spotted 1 mm filter disk processed with no elution step, lane 2:
blood spotted 1 mm filter disk processed with an elution step, lane
3: eluted fraction, lane 4: wash step 1, lane 5: wash step 2;
25. FIG. 3a shows OliGreen Fluorescent probe ss genomic DNA
standard curve;
26. FIG. 3b shows Relative Fluorescent Units (RFU) and calculated
yields of eluted ss genomic DNA from blood spotted to the filter
material of the invention and cellulosic FTA filter card;
27. FIG. 4a shows average calculated total yields of eluted genomic
DNA from different quantities of saliva applied to the filter
membrane of the invention;
28. FIG. 4b shows a digital representation of an Amelogenin PCR
amplification of six individual genomic DNA purifications from
female saliva using the filter membrane of the invention, a PCR
product of 218 bp being expected, lane MW: pGEM molecular weight
markers, lanes 1-6: individual female saliva genomic DNA
samples;
29. FIG. 5a is a digital representation of a gel showing tissue
typing using HLA-A primers on individual male blood samples. A PCR
product of 900 bp is expected to be amplified, lane MW: pGEM
molecular weight markers, lanes 1-6: individual male blood genomic
DNA samples;
30. FIG. 5b shows a digital representation of a gel of tissue
typing using HLA-B primers on individual male blood samples, a PCR
product of 1090 bp is expected to be amplified, lane MW: pGEM
molecular weight markers, lane 1-6: individual male blood genomic
DNA samples;
31. FIG. 6a is a digital representation of a gel showing results
from Amelogenin PCR amplification of DNA purified from 6 individual
saliva samples using the filter membrane of the invention in the
format of a 7 mm free-floating disc in a microtube, lane MW: pGEM
molecular weight markers, lanes 1-6: individual saliva genomic
DNA;
32. FIG. 6b shows Amelogenin PCR amplification of DNA purified from
7 individual blood samples using the filter membrane of the
invention in the format of a microcentrifuge spin basket, lane MW:
pGEM molecular weight markers, lanes 1-7: individual blood genomic
DNA;
33. FIG. 6c shows Amelogenin PCR amplification of DNA purified from
a buccal scrape sample using the filter membrane of the invention
in the format of a swab, lane 1: swab after elution step, lane 2:
eluted fraction, lane 3: no DNA PCR control;
34. FIG. 7 is a table of protocol steps and total time required for
genomic DNA prepared from blood using commercially available kits
compared to the filter membrane of the invention;
35. FIG. 7b is a table of the yields of genomic DNA prepared from
blood using commercially available kits compared to the filter
membrane of the invention;
36. FIG. 7c is a digital representation of a gel showing Amelogenin
PCR amplification of purified genomic DNA from blood using
commercially available kits compared to the filter membrane of the
invention, lane 1 filter membrane of he invention, lane 2: Roche
kit, lane 3: Promega kit;
37. FIG. 8a is a digital representation of a gel showing Amelogenin
PCR amplification of genomic DNA purified from day 1 spotted blood
using the filter membrane of the invention, lane 1: blood spotted 1
mm filter disk processed with no elution step, lane 2: blood
spotted 1 mm filter disk following an elution step, lane 3: eluted
fraction;
38. FIG. 8b shows Amelogenin PCR amplification of genomic DNA
purified from 19 week old spotted blood using the filter membrane
of the invention, lane MW: pGEM molecular weight markers, lane 1:
eluted fraction, lane 2: blood spotted 1 mm filter disk following
an elution step;
39. FIG. 9 is a cross-sectional view of a filter membrane made in
accordance with the present invention; and
40. FIG. 10 is a cross-section of a device made in accordance with
the present invention.
DETAILED DESCRIPTION OF THE INVENTION
41. The present invention most generally provides a medium for
storage and subsequent analysis of the genetic material, the medium
generally including a support for immobilizing a genetic material
thereon and allowing subsequent elution of the genetic material
therefrom. A coating is functionally associated with the support
for enabling cellular lysis and releasing the genetic material from
the lysed cells while stabilizing the immobilized released genetic
material. A method is also provided of storing a genetic material
most generally including the steps of immobilizing a genetic
material on the support which allows subsequent elution of the
genetic material and lysing cells and releasing the genetic
material from the lysed cells while stabilizing the immobilized
released genetic material. The genetic material can then be
analyzed in solution as opposed to being immobilized on the
support.
42. The chemical composition of the support facilitates the lysis
of whole cells and the subsequent capture of the released nucleic
acids. The chemical composition further aids in their long term
storage. The composition of the support is such that the rapid
purification of the captured nucleic acid can be carried out. That
is, the support itself allows for the release of nucleic acid by an
elution step thereby providing a soluble nucleic acid fraction. As
disclosed in more detail below and exemplified in the following
examples, the present invention is most efficient with regard to
elution of total DNA from the sample.
43. Preferably, the support of the present invention is a porous
material in the form of a filter membrane as described and defined
below.
44. Unexpectedly, it has now been discovered that a support, when
processed in accordance with the invention, to provide a nucleic
acid eluting filter material provides a number of advantages and
applications as described hereinafter over the prior art discussed
above. Thus, use of the media of the present invention now provides
advantages of faster processing of nucleic acid-containing
biological fluids as well as multiple processing of fluids.
45. The present invention, generally shown at 10 in FIG. 9,
includes the following components:
46. (i) a suitable support, preferably a filter membrane 12;
and
47. (ii) a chemical coating 14.
48. Reaction of the filter membrane with the chemical coating
solution produces the filter membrane of the invention. If the
membrane is fibrous, this coating is a coating of the filter
fibers, not the filter surface.
49. The term "filter membrane" as used herein means a porous
material or filter media formed, but not limited to, either fully
or partly from glass, silica or quartz including their fibers or
derivatives thereof. Other materials from which the filter membrane
can be composed also include cellulose-based (nitrocellulose or
carboxymethylcellulose papers), hydrophilic polymers including
synthetic hydrophilic polymers (eg. polyester, polyamide,
carbohydrate polymers), polytetrafluoroethylene, and porous
ceramics.
50. The media used for the filter membrane of the invention
includes any material that does not inhibit the sorption of the
chemical coating solution and which does not inhibit the storage,
elution and subsequent analysis of nucleic acid-containing material
added to it. This includes flat dry matrices or a matrix combined
with a binder. It is preferred that the filter membrane of the
invention be of a porous nature to facilitate immobilization of
nucleic acid. Unlike prior art supports, the support of the present
invention allows for elution of the genetic material therefrom in a
state that allows for subsequent analysis. Unexpectedly, such
elution is a time efficient step thereby providing for almost
immediate analysis.
51. The term "chemical coating solution" as used herein means a
chemical composition that is able to sorb to the aforementioned
filter membrane. The composition of the chemical coating solution
is as described and relates to that outlined in U.S. Pat. Nos.
5,756,126, 5,807,527, and 5,496,562. Absorption of the chemical
coating solution to the selected filter membrane results in the
formation of the filter membrane of the invention.
52. More specifically, the chemical coating solution includes a
protein denaturing agent and a free radical trap. The denaturing
reagent can be a surfactant that will denature proteins and the
majority of any pathogenic organisms in the sample. Anionic
detergents are examples of such denaturing reagents. The chemical
solution can include a weak base, a chelating agent, and the
anionic surfactant or detergent, and optionally uric acid and urate
salt as discussed in detail in the above-cited U.S. Pat. No.
5,807,527. More preferably, the weak base can be a Tris,
trishydroxymethyl methane, either as a free base or as the
carbonate, and the chelating agent can be EDTA, and the anionic
detergent can be sodium dodecyl sulfate. Other coatings having
similar function can also be utilized in accordance with the
present invention.
53. The term "functionally associated with" means that the coating
is disposed, sorbed, or otherwise associated with the support of
the present invention such that the support and coating function
together to immobilize nucleic acid thereon through an action of
cellular lysis of cells presented to the support. That is, the
coating can be adsorbed, absorbed, coated over, or otherwise
disposed in functional relationship with the media. For example,
the support, in the form of a filter membrane, can be disposed in a
solution containing the chemical solution. As stated above, the
support of the present invention is preferably a porous filter
media and can be in the form of a flat, dry media. The media can be
combined with a binder, examples of binders well-known in the art
being polyvinylacrylamide, polyvinylacrylate, polyvinylalcohol,
gelatin, for example.
54. It is critical that the support of the present invention be
capable of releasing the genetic material immobilized thereto by a
heat elution. Preferably, such a heat elution is accomplished by
the exposure of the support having three genetic material stored
thereon to heated water, the water being nuclease free. This
capacity to allow for elution characterizes the various support
materials of the present invention.
55. The term "filter membrane of the inventions" as used herein
means functional solid supports or matrices that enables the
specific immobilization of nucleic acid, through an action of
cellular lysis. Nucleic acid may be presented to it in the form of
nucleic acid-containing material such as blood, cultured mammalian
cells, saliva, urine, cultured bacterial cells, yeast, solid
tissue, faeces, lymphatic fluid, amniotic fluid, plant issue, and
the like. The filter membrane of the invention is such that nucleic
acid immobilized to it can remain so in a stable form, not exhibit
degradation, shearing, endonuclease digestion, nor UV damage.
56. The filter membrane of the invention is such that at any point
during a storage regime, it allows for the rapid purification of
immobilized nucleic acid. The invention is such that immobilized
nucleic acid is collected in the form of a soluble fraction
following a simplified elution process, during which immobilized
nucleic acid is released from the filter membrane of the invention.
The filter membrane of the invention yields nucleic acid of
sufficient quality that it does not impair downstream analyses such
as polymerase chain reaction (PCR), ligase chain reaction (LCR),
transcription mediated amplification (TMA), reverse transcriptase
initiated PCR, DNA or RNA hybridization techniques, sequencing, and
the like.
57. Nucleic acid immobilized to a solid filter support, although a
suitable template for singular PCR reactions, cannot be measured or
detected by traditional techniques such as optical density or
fluorescence. Nucleic acid has to be in solution for these
techniques. Other post purification techniques where nucleic acid
is desired in the soluble form includes: cloning, hybridization
protection assay, bacterial transformation, mammalian transfection,
transcription-mediated amplification, and the like. The present
invention provides nucleic acid in such a soluble form.
58. The filter membrane of the invention can possess the same
chemical component as FTA that enables the action of cellular lysis
and nucleic acid release upon sample application. The chemical
component ensures nucleic acid stability via protein denaturants, a
free radical trap, and viral/microbial inhibitors. The difference
between prior art FTA solid supports and the filter membrane of the
invention is that the base solid support, or filter, has been
changed compared to that described for FTA products. This change in
solid support material, or filter, has enabled, upon a simplified
heat elution step, bound nucleic acid to be removed from the filter
membrane of the invention whereas it cannot be removed from FTA
solid support (see Del Rio et al (1995) BioTechniques. Vol. 20:
970-974). The nucleic acid released from the filter membrane of the
invention is thus presented as a soluble fraction that can be
readily aliquoted to multiple downstream processes such as PCR
amplification. The eluted soluble nucleic acid can also be entered
into techniques where soluble nucleic acid is a necessity such as
optical density analysis, fluorescence detection, cloning,
transformation, and the like. This added technique of elution
enables high throughput multiple processing regimes, such as
genotyping.
59. As discussed below in the experimental section, it can be
advantageous to provide a device for storage and subsequent
analysis of genetic material wherein a sample can be collected,
such as a fluid sample in the form of blood or saliva. As shown in
FIG. 10, the device can include a container, such as a tube 16,
containing the media 10 constructed in accordance with the present
invention. The container must be non-reactive with the genetic
material. Examples of such containers can be a tube 16 made from a
polymer selected from the group consisting of common polypropylene,
but also polysulphone. As shown in FIG. 10, a sample 18 has been
disposed within the tube 16 thereby exposing the media disk, in a
free floating form within the tube 16, to the sample. As discussed
below in greater detail in the experimental section, the method of
the present invention can be utilized to immobilize genetic
material from the sample onto the media 10.
60. The present invention further provides, most generally, a
method for storing the genetic material and subsequently analyzing
genetic material by the steps of immobilizing the genetic material
on the support while enabling cellular lysis and release of the
genetic material from the lysed cells. The chemical coating on the
support, in the form of the filter media, enables the lysing of the
genetic material and stabilization of the immobilized released
genetic material. The support allows for eluting of the genetic
material to generate the soluble genetic material fraction, thereby
allowing for subsequent analysis of the genetic material, as
discussed above.
61. The eluting step can be accomplished by heating the support
having the genetic sample immobilized thereon, the support
releasing the heated genetic material therefrom and into solution,
preferably into a nuclease free water. Most preferably, this is
accomplished by disposing the support having the genetic
immobilized thereon into heated water, the water being heated
preferably between 65.degree. C. and 100.degree. C.
62. As discussed in greater detail in the examples below, various
washes can be performed in various types of buffers. Preferably,
the washing buffers can be selected from the group including
Tris/EDTA; 70% ethanol; STET; SSC; SSPE FTA purification reagent,
and the like.
63. The present invention can find utility in many areas of
genomics. For example, the present invention provides the
capability to elute bound genetic material for the rapid
purification of the genetic material to be utilized in any number
of forensic applications, such as identification,
paternity/maternity identification, and at the scene of a
crime.
64. Prisoners from many countries are required to give a genetic
sample (blood or buccal sample) for DNA fingerprinting purposes.
The use of the present invention provides a means for the long term
storage of prisoner genetic material. If necessary, the genetic
material can be tested as soon as it is taken or many years after
storage. The genetic material can be obtained as either a soluble
or solid phase fraction once isolated on the filter media of the
present invention.
65. The present invention can be utilized for paternity/maternity
identification having a particular use for a mother or hospital
wherein a newborn has been mislaid in the hospital. The rapid
ability of the present invention to provide for a purified genetic
sample provides even greater utility in such instances where a
speedy identification of a mislaid child would be most
propitious.
66. The present invention is a significant contribution to current
methodology for the preparation of soluble genetic material which
are otherwise time consuming and often result in inadequate
template that is damaged or contaminated. The present invention
provides high yield of purified genetic material of superior
quality in less than twenty minutes of laboratory time. The rapidly
purified generic material can be utilized for any number of
food/agricultural applications such as tracing, breeding,
identification, and cloning.
67. The efficiency with which food manufacturers detect pathogenic
outbreak in both their livestock and finished product is the
measure of a successful company. The use of the present invention
as a swab that can be simply pressed against food or the use of a
card onto which carcass blood can be spotted enables uses of the
present invention to rapidly isolate and detect for the presence of
pathogenic genetic material. Time consuming prior art assay
techniques and involved nucleic acid preparations do not need to be
performed if the present invention is utilized. Collected
pathogenic nucleic acid can be used as a soluble fraction or solid
phase fraction with the choice of an elution step.
68. Tracing carcass material, whether for legal or health issues,
enables manufacturers to keep control of their products. At the
point of kill in a slaughter house, a card utilizing the present
invention can be attached to the carcass onto which its blood has
been spotted. At the same time, a second card can be spotted with
the same blood and kept as an archive at the slaughter house. If an
identification issue arises for a certain carcass, genetic records
on both the carcass and the slaughterhouse can be utilized. If the
carcass card is inadvertently removed, identification can still be
carried out by simply pressing a carcass flesh onto such a
card.
69. Identifying the desired genes and characteristics that are
required for a subsequent generation of a plant or animal requires
the time effective and reliable generation nucleic acid from
potential parents. The present invention can be used for the
isolation of either soluble or solid phase genetic material to
provide effective and reliable results in such a need. Likewise,
the present invention, in the form of microplates, a tube or a
chip, can be used for the generation and detection of genetic
material. The present invention provides methodology for superior
template preparation time (whether soluble or solid) and cost
effective archiving.
70. Pressing a media, in the form of a swab or otherwise, enables
the user to pick up any contaminated microbes on food products of
any type. Genetic material isolated from the media can then be
utilized for any manner of diagnostic procedure depending on
whether soluble or solid phase genetic material is required. This
analysis can be done almost effectively immediately, as opposed to
prior art techniques.
71. By the use of genetic manipulation techniques, food stuff has
been produced with increased size, flavor, ripening, and sugar
content. Many countries prohibit the sale of genetically modified
food products and therefore require testing to be carried out.
Since one is looking for specific genes that generate these
characteristics, genetic material is required. The present
invention can be used to provide rapid purification of both soluble
and solid phase genetic material.
72. In view of the above, the present invention finds utility in
various areas of genomics.
73. The present invention can further be utilized in the areas of
purification from a patient's whole blood. Currently, genomic DNA
is typically purified from a patient's whole blood the genetic
material present in the leukocyte population. Methods of genomic
DNA extraction often involve many steps and involve several buffers
and purification matrices. Recently, several new methodologies for
genomic DNA extraction have been available. One is the FTA 31 ET
isolation exploited by Fitzco-Whatman. Another is the method
described by Cambridge Molecular Technologies Ltd., UK (CMT), using
Whatman F58301 (GF/L) material. The Fitzco-Whatman method utilizes
an FTA coat on 31 ET cellulosic material that spontaneously lyses
leukocytes releasing the genomic DNA. This promotes integration and
binding with the media. The DNA is fixed permanently to the media
as no methodology for elution of DNA from the prior art FTA coated
31 ET was determined. For many applications, the fact that the
genomic DNA bound to the 31 ET media cannot be eluted poses no
problem whatsoever. PCR and RFLP are readily performed on the bound
template. However, for genotyping experimental where many PCR
reactions are carried on the same DNA population, the process of
having to punch out different 1 millimeter disks for every primer
set used is too time consuming to be efficient.
74. The present invention provides an ideal solution by allowing
for elution of the DNA thereby providing a soluble DNA for each of
the reactions performed.
75. Specifically, the CMT method utilizes Whatman GF/L glass fiber
that has been shown to specifically capture leukocytes from whole
blood application. Upon cellular capture, a lysis buffer is
introduced and the released genomic DNA binds to the GF/L. The
genomic DNA-GF/L binding is a strong enough interaction to
withstand several washing steps. After washing, the GF/L bound
genomic DNA is eluted with the application of water or TE buffer to
the filter at preferably 82.degree. C. As discussed above, a range
of temperatures and buffers can be used. The GF/L media ensures
leukocyte capture from whole blood. The coating of the present
invention promotes lysis of the cells without the addition of
inconvenient lysis buffers and steps. The genomic DNA stays bound
to the GF/L media during washing steps. Full elution of the bound
genomic DNA is achieved with the addition of water or buffer at the
appropriate temperature, preferably 80.degree. C.
76. With the genomic DNA in a soluble format, many PCR reactions
can be carried out from the same DNA population with simple
alaquating of the template rather than cumbersome punching.
Likewise, an FTA coated GF/L matrix can be incorporated into a
single tube, as discussed above, of a microplate device depending
on the degree of throughput required.
77. The above examples show the various utilities of the present
invention and are not meant to be limiting.
EXAMPLES
Example 1
Heat Elution
78. Several drops of freshly finger-stick drawn blood was spotted
to the filter membrane of the invention and allowed to air-dry for
two minutes. Once dried two 1 mm diameter punches were taken from
the dried blood spot and applied to individual 2000 ul
polypropylene PCR tubes. To each tube containing a single 1 mm
blood punch, 200 ul of FTA Purification Reagent (Fitzco, Inc) was
added. Per 500 ml: 0.29 g NaCl; 5 ml 1 M Tris pH 7.5; 1 ml 0.5 M
EDTA; 2.5 ml Triton x-100. Tubes were incubated for five minutes at
room temperature with no shaking. Following incubation the FTA
purification Reagent was aspirated from the tube. A second aliquot
of 200 ul of FTA Purification Reagent was added to each tube. The
tubes were incubated for five minutes at room temperature without
shaking. Following incubation the FTA Purification Reagent was
aspirated from both tubes. 200 ul of TE (10 mM Tris-HCl, 1 mM EDTA,
pH 8.0) buffer was then added to each tube. The tubes were
incubated for five minutes at room temperature without shaking. The
TE buffer was then fully aspirated from both tubes, leaving the now
washed 1 mm disc at the bottom of each tube. 20 ul of nuclease free
water was then applied to both tubes. One tube was incubated at
82.degree. C. for 10 minutes; the other was incubated at 95.degree.
C. for 10 minutes in a Biometra thermacycler. Following heat
incubations the 20 ul of nuclease free water was aspirated from
each tube and retained. An Amelogenin PCR amplification master mix
was made up according to manufacturer's instructions (Promega),
with a 25 ul aliquot applied to both tubes containing the 1 mm
punches, and a 5 ul aliquot applied to both 20 ul nuclease free
water samples. PCR was carried out following parameters described
by the manufacturing of the Amelogenin primer set (Promega).
Following PCR 10 ul of each PCR reaction was visualized on a 1.5%
agarose gel stained with ethidium bromide.
79. It can be seen from the Amelogenin amplification results (FIG.
1), that nucleic acid immobilized to the filter membrane of the
invention is not readily removed from the solid support following
82.degree. C. heat incubation. Amplification product is noted from
the 1 mm solid punch, but is not present in the 20 ul nuclease free
water fraction. At 95.degree. C. heat incubation we see that
nucleic acid is eluted from the filter membrane of the invention.
Amplification product is not detected from the 1 mm solid punch,
but is present in the nuclease free water fraction.
Example 2
Full Elution Protocol
80. Several drops of freshly finger-stick drawn blood were spotted
to the filter membrane of the invention and allowed to air-dry for
two minutes. Once dried two 1 mm diameter punches were taken from
the dried blood spot and applied to individual 200 ul polypropylene
PCR tubes. To each tube containing a single 1 mm blood punch, 200
ul of FTA Purification Reagent (Fitzco, Inc) was added. Tubes were
incubated for five minutes at room temperature with no shaking.
Following incubation the FTA purification Reagent was aspirated
from the tube, 20 ul of the aspirate was retained. A second aliquot
of 200 ul of FTA Purification Reagent was added to each tube. The
tubes were incubated for five minutes at room temperature without
shaking. Following incubation the FTA Purification Reagent was
aspirated from both tubes, 20 ul of the aspirate was retained. 200
ul of TE buffer was then added to each tube. The tubes were
incubated for five minutes at room temperature without shaking. The
TE buffer was then fully aspirated from both tubes, leaving the now
washed 1 mm disc at the bottom of each tube.
81. To one of the tubes, 20 ul of nuclease free water was then
applied, and then incubated at 95.degree. C. for 10 minutes in a
Biometra thermacycler. Nothing was added to the other tube
containing a 1 mm punch. Following heat the incubation of one of
the tubes; the 20 ul of nuclease free water was aspirated and
retained. An Amelogenin PCR amplification master mix was made up
according to manufacture's instructions (Promega), with a 25 ul
aliquot applied to the tube containing the 1 mm punch that had not
been subjected to heat incubation, and also the 1 mm punch that had
been subjected to heat incubation. A 5 ul aliquot of master mix was
applied to the 20 ul nuclease free water samples of the heat
incubation punch, as well as the 20 ul aliquots taken from both FTA
Purification Reagent incubations. PCR was carried out following
parameters described by the manufacturer of the Amelogenin primer
set (Promega). Following PCR 10 ul of each PCR reaction was
visualized on a 1.5% agarose gel stained with ethidium bromide.
82. It can be seen from the Amelogenin amplification results (FIG.
2) that nucleic acid from a whole cell source is immobilized to the
filter membrane of the invention and does not elute from the solid
support during washing steps. This is illustrated with
amplification product detected from the 1 mm punch processed with
no heat elution step, and the lack of amplification product
detected in both FTA Purification Reagent washing steps. Complete
elution, or release, of the immobilized nucleic acid following heat
incubation is illustrated by amplification product detected in the
20 ul nuclease free water aspirate, and none detected from the 1 mm
punch subjected to heat incubation. Example 2 indicates that all of
the nucleic acid that has been initially immobilized to the filter
membrane of the invention remains bound during washing steps, and
is fully recovered into a soluble fraction following 95.degree. C.
heat incubation.
Example 3
Comparison of Elution
83. Single stranded DNA can be readily detected with the use of
OliGreen.RTM. (Molecular Probes, Inc), a fluorescent probe specific
for the single stranded molecule. By using OliGreen the total
single stranded DNA eluted from the filter membrane of the
invention can be determined, as well as other single stranded DNA
purification methods. A standard curve for single stranded genomic
DNA was constructed according to manufacture's instructions
(Molecular Probes, Inc) (see FIG. 3a).
84. 5 ul of freshly finger-stick drawn blood was spotted to a 7 mm
disk of the filter membrane, composed of a chemically coated porous
glass microfiber filter membrane, of the invention and also to a 7
mm disk of the commercially available FTA solid support. Both spots
were allowed to air-dry for two minutes. Once dried, the punches
were applied to individual 1.5 ml polypropylene Eppendorf tubes. To
each tube containing a single 7 mm blood punch, 1 ml of FTA
Purification Reagent (Fitzco, Inc) was added.
85. Tubes were incubated for five minutes at room temperature with
no shaking. Following incubation the FTA purification Reagent was
aspirated from both tubes. A second 1 ml aliquot of FTA
Purification Reagent was added to each tube. The tubes were
incubated for five minutes at room temperature without shaking.
Following incubation, the FTA Purification Reagent was aspirated
from both tubes. 1 ml of TE buffer was then added to each tube. The
tubes were incubated for five minutes at room temperature without
shaking. The TE buffer was then fully aspirated from both tubes,
leaving the now washed 7 mm punches at the bottom of each tube. To
both tubes, 200 ul of nuclease free water was applied, and then
incubated at 100.degree. C. for ten minutes in a water bath.
86. Following heat incubation, the nuclease free water fraction
were aspirated from both tubes and immediately chilled on ice. 50
ul of the nuclease free water fraction of both samples was then
subjected to OliGreen fluorometric quantitation according to the
manufacturer's instructions (Molecular Probes, Inc). Relative
fluorescent units (RFU) were taken for each sample, and with use of
the standard curve (FIG. 3a) the total yields of the eluted DNA
calculated (dilution factor for quantitation is 4-fold, total
volume of eluate is 200 ul).
87. Typically from 5 ul of whole blood one can expect between
35,000 and 50,000 white blood cells; each cell containing
approximately 7 pg of genomic DNA (A. Eisenberg, personal
communication). Taking the upper limit, 350 ng of total genomic DNA
is expected from 5 ul of whole blood. From the OliGreen
quantitation data (FIG. 3b) it can be seen that 300 ng of total
genomic DNA is recovered from the filter membrane of the invention,
representing almost 100% of the expected yield. 60.2 ng of total
genomic DNA is recovered from 5 ul of whole blood spotted to FTA
solid support. Example 3 illustrates the filter membrane of the
invention exhibits a nucleic acid elution characteristic that is
not apparent for the FTA solid support. Also from 5 ul of whole
blood, approaching 100% of the available genomic DNA present within
the sample cells can be isolated as a soluble fraction.
Example 4
Genomic DNA Preparation from Saliva
88. Genomic DNA can be readily purified from many different cell
sources. One of the most common sources, particularly in forensics
and for its non-invasive collection, is saliva containing buccal
epithelial cells. Although easy to collect, saliva does exhibit
some difficult for genomic DNA purification in that it is extremely
viscous and not easily applied to column chromatography. Also PCR
inhibitors are present within the mucus of saliva.
89. 5, 10, 50, and 100 ul of female saliva were applied to
individual 7 mm punches of the filter membrane of the invention.
The saliva-spotted 7 mm punches were air dried for two minutes.
Once dried, the punches were applied to individual 1.5 ml
polypropylene Eppendorf tubes. To each tube containing a single 7
mm saliva punch, 1 ml of FTA Purification Reagent (Fitzco, Inc) was
added. Tubes were incubated for five minutes at room temperature
with no shaking. Following incubation, the FTA purification Reagent
was aspirated from both tubes. A second 1 ml aliquot of FTA
Purification Reagent was added to each tube.
90. The tubes were incubated for five minutes at room temperature
without shaking. Following incubation the FTA Purification Reagent
was aspirated from both tubes. 1 ml of TE buffer was then added to
each tube. The tubes were incubated for five minutes at room
temperature without shaking. The TE buffer was then fully aspirated
from both tubes, leaving the now washed 7 mm punches at the bottom
of each tube. To both tubes, 200 ul of nuclease free water was
applied, and then incubated at 100.degree. C. for ten minutes in a
water bath. Following heat incubation, the nuclease free water
fraction were aspirated from all tubes and immediately chilled on
ice. 50 ul of the nuclease free water fraction of all samples was
then subjected to OliGreen fluorometric quantitation according to
the manufacturer's instructions (Molecular Probes, Inc). Relative
fluorescent units (RFU) were taken for each sample, and with use of
the standard curve (3a) the total yields of the eluted DNA
calculated (dilution factor for quantitation is 4-fold, total
volume of eluate is 200 ul). 10 ul of the nuclease free water
fraction of six female saliva samples (5 ul) spotted to the filter
membrane of the invention and subjected to same processing steps as
above, was utilized as the template for 25 ul reaction volume
Amelogenin PCR amplification according to manufacturer's
instructions (Promega).
91. It can be seen (FIG. 4a) that genomic DNA can be isolated as a
soluble fraction from saliva spotted to the filter membrane of the
invention. The relationship between saliva starting volume and
total DNA yield is given. The increase in yield of soluble genomic
DNA does not show a linear relationship with respect to starting
saliva volume, this was probably due to the filter membrane of the
invention becoming saturated with saliva volume, with the capacity
reaching a maximum at around 50 ul.
92. Amelogenin PCR amplification can be demonstrated for all of
nuclease free water elution fractions from 5 ul of starting saliva
spotted to the filter membrane of the invention (FIG. 4b).
Example 5
Downstream Use of Eluted DNA
93. Tissue typing is a generic genotyping technique that is common
practice within the clinical community. Often blood is taken from a
potential donor and their tissue type determined by a combination
of allele-specific PCR followed by hybridization.
94. 5 ul of freshly acquired male whole blood was applied to six 7
mm punches of the filter membrane of the invention. The punches
were then applied to a 1.5 ml polypropylene Eppendorf tubes. To the
tubes containing a single 7 mm blood punch, 1 ml of FTA
Purification Reagent (Fitzco, Inc) was added. The tubes were
incubated for five minutes at room temperature with no shaking.
Following incubation, the FTA purification Reagent was aspirated
from the tubes. A second 1 ml aliquot of FTA Purification Reagent
was added to the tubes. The tubes were incubated for five minutes
at room temperature without shaking. Following incubation the FTA
Purification Reagent was aspirated from the tubes. 1 ml of TE
buffer was then added. The tubes were then incubated for five
minutes at room temperature without shaking. The TE buffer was then
fully aspirated from the tubes, leaving the now washed 7 mm punches
at the bottom. 200 ul of nuclease free water was applied, and the
tubes then incubated at 100.degree. C. for 10 minutes in a water
bath. Following heat incubation the nuclease free water fraction
was aspirated from the tubes and 10 ul of each used for either
HLA-A PCR amplification or HLA-B PCR amplification. Both PCR
amplifications were carried out according to manufacture's
directions (Lifecodes, Inc). 10 ul of each amplification reaction
was visualized on a 1.5% agarose gel with ethidium bromide
staining.
95. All of the nuclease free water fractions acquired from the
blood samples spotted to the filter membrane of the invention give
PCR amplification product for HLA-A (FIG. 5a), and HLA-B (FIG. 5b).
Example 5 illustrates the validity of soluble genomic DNA
purification from blood using the filter membrane of the invention,
providing a soluble DNA fraction that can be utilized for typical
genotyping amplification reactions. The generation of a 1 kb
amplification product illustrates the high quality of the isolated
soluble genomic DNA fraction.
FIG. 6
Filter Membrane of the Invention Formats
96. A main advantage of the filter membrane of the invention is
that it is manufactured in the form of a filter paper reel. Filter
paper manufactured in this way is capable of being formatted to a
variety of devices. Other genomic DNA purification media such as
polymeric resin for example cannot be formatted in the same way.
For example it is suitable for a filter material to be designed in
a swab configuration--it would be extremely difficult to propose
the same format for chromatographic resin. The desired format for
the filter membrane of the invention is dependent upon the
application.
97. Saliva represents a very difficult sample in that it is a
viscous fluid. Traditional column chromatography, or spin tubes are
not devices for handling it. To that end, the filter membrane of
the invention was formatted in the configuration of a 7.5 mm
free-floating disk held within a 2 ml Eppendorf tube. With such a
device, saliva can be directly administered from the donor's mouth.
Because the filter membrane of the invention is free floating
within the tube, there will be no change of filter clogging which
results in poor recovery.
98. Six saliva samples of approximately 100 ul each were directly
administered to individual tubes containing a free-floating disk of
the filter membrane of the invention. To each tube containing a
single 7.5 mm saliva disks 1 ml of FTA Purification Reagent
(Fitzco, Inc) was added. Tubes were incubated for five minutes at
room temperature with no shaking. Following incubation the FTA
Purification Reagent was aspirated from the tubes. A second 1 ml
aliquot of FTA Purification Reagent was added to each tube. The
tubes were incubated for five minutes at room temperature without
shaking. Following incubation, the FTA Purification Reagent was
aspirated from the tubes. 1 ml of TE buffer was then added to each
tube. The tubes were incubated for five minutes at room temperature
without shaking. The TE buffer was then fully aspirated from the
tubes, leaving the now washed 7.5 mm disks at the bottom of each
tube.
99. To all tubes, 200 ul of nuclease free water was applied, and
then incubated at 100.degree. C. for 10 minutes in a water bath.
Following heat incubation the nuclease free water fractions were
aspirated from all the tubes and 10 ul of each eluate applied to 25
ul Amelogenin PCR amplification reactions according to
manufacturer's instructions (Fromega). 10 ul of each reaction was
visualized on a 1.5% agarose gel and ethidium bromide staining
(FIG. 6a). The free-loading disk for the filter membrane of the
invention can be utilized for recalcitrant samples such as
saliva.
100. The filter membrane of the invention can be formatted to a
spin microfuge device. Such a device has been shown to be an
extremely quick tool for the isolation of nucleic acids (see Qiagen
catalog). Three layers of 7.4 mm disks of the filter membrane of
the invention were configured into the spin basket of seven spin
microfuge devices. 5 ul of seven individual male blood samples were
applied to the filter disk of each spin basket. 0.5 ml of FTA
Purification Reagent (Fitzco Inc) was added to the basket. The
microfuge tubes containing the basket was then centrifuged at
6000.times.g for one minute. The resultant filtrates were discarded
from the microfuge tubes. 0.5 ml of FTA Purification Reagent was
again added to the filter baskets and again the tubes centrifuged
at 6000.times.g for one minute. Following the removal of the
filtrates from the microfuge tubes, 0.5 ml of TE buffer was added
to the baskets. This was followed by the same centrifugation regime
described above. After the TE buffer centrifugation step, 200 ul of
nuclease free water was added to the baskets of the microfuge
tubes. The microfuge tubes was then incubated at 100.degree. C. for
fifteen minutes in a water bath. Following heat incubation the
microfuge tubes were centrifuged at 12,000.times.g for two minutes
to recover the nuclease free water fractions. 10 ul of each
nuclease free water fraction was applied to 25 ul Amelogenin PCR
amplification reactions according to manufacturer's instructions
(Promega). 10 ul of each reaction was visualized on a 1.5% agarose
gel and ethidium bromide staining (FIG. 6b).
101. The spin microfuge device that contains the filter membrane of
the invention can be utilized to isolate soluble genomic DNA from
whole blood in less than 20 minutes. The quality of the genomic DNA
isolated is demonstrated with 100% PCR amplification of the samples
evaluated.
102. Buccal scrapes are often used as a means for the collection of
nucleic acid containing samples such as epithelial cells,
particularly in population field studies such as offender
identification. The filter membrane of the invention can be
formatted in the configuration of a swab that can be directly
administered into the mouth of the donor. By scraping the swab
along the inside of the donor's cheek, epithelial cells can be
collected on the filter membrane of the invention. Other genomic
DNA purification tools such as chromatographic resin cannot be
readily configured to swabs that are applied to donors' mouths. A
small piece of the filter membrane of the invention was configured
into the stem of a commercially available oral swab (Fitzco Inc).
The swab was placed into the mouth of a male donor and scraped
along the inside of the cheek for ten seconds. Following scraping,
the filter membrane of the invention that constitutes the swab head
was placed into a 1.5 ml Eppendorf tube. To the tube containing the
swab head, 1 ml of FTA Purification Reagent (Fitzco, Inc) was
added. The tube was incubated for five minutes at room temperature
with no shaking. Following incubation, the FTA purification Reagent
was aspirated from the tube. A second 1 ml aliquot of FTA
Purification Reagent was added to each tube. The tube was incubated
for five minutes at room temperature without shaking. Following
incubation, the FTA Purification Reagent was aspirated from the
tube. 1 ml of TE buffer was then added to the tube. The tube was
incubated for five minutes at room temperature without shaking. The
TE buffer was then fully aspirated from the tube, leaving the now
washed swab head at the bottom of each tube. To all tubes, 200 ul
of nuclease free water was applied, and then incubated at
100.degree. C. for ten minutes in a water bath. Following heat
incubation the nuclease free water fraction was aspirated from the
tube and 10 ul of the eluate applied to 25 ul Amelogenin PCR
amplification reaction according to manufacturer's instructions
(Promega). A 1 mm punch was taken from the swab head following heat
incubation and applied to a 25 ul Amelogenin PCR amplification
according to manufacturer's instructions (Promega). 10 ul of each
reaction was visualized on a 1.5% agarose gel and ethidium bromide
staining (FIG. 6c). The filter membrane of the invention can be
configured in the form of a swab that can be utilized to purify
genomic DNA from buccal scrapes. The isolated soluble genomic DNA
is of suitable quality for PCR amplification.
Example 7
Product Comparisons
103. There are many genomic DNA purification systems that are
commercially available. To illustrate the validity of the filter
membrane of the invention, the device of the present invention was
compared directly to the genomic DNA purification kits available
from Roche Molecular Biochemicals (Split Second.TM.) and Promega
(AmpReady.TM.).
104. 5 ul of freshly drawn finger-stick blood was applied to both
commercial kits and to the filter membrane of the invention.
Procedure was followed according to manufacturer's directions for
both of the commercial kits. 5 ul of freshly finger-stick drawn
blood was spotted to a 7 mm disk of the filter membrane of the
invention. The punch was applied to a 1.5 ml polypropylene
Eppendorf tube. To the tube containing a single 7 mm blood punch, 1
ml of FTA Purification Reagent (Fitzco, Inc) was added. The tube
was incubated for five minutes at room temperature with no shaking.
Following incubation the FTA purification Reagent was aspirated
from the tube. A second 1 ml aliquot of FTA Purification Reagent
was added to the tube. The tube was incubated for five minutes at
room temperature without shaking.
105. Following incubation, the FTA Purification Reagent was
aspirated from the tube. 1 ml of TE buffer was then added to the
tube. The tubes were incubated for five minutes at room temperature
without shaking. The TE buffer was then fully aspirated from the
tube, leaving the now washed 7 mm punches at the bottom of the
tube.
106. To the tube, 200 ul of nuclease free water was applied, and
then incubated at 100.degree. C. for ten minutes in a water bath.
Following heat incubation the nuclease free water fraction was
aspirated from both tubes and immediately chilled on ice. 50 ul of
the nuclease free water fraction was then subjected to OliGreen
fluorometric quantitation according to the manufacturer's
instructions (Molecular Probes, Inc).
107. The same OliGreen quantitation was carried out for both the
Roche Molecular Biochemical and Promega purified genomic DNA
samples. Relative fluorescent units (RFU) were taken for all
samples, and with use of the standard curve (3a) the total yields
of the eluted DNA calculated (dilution factor for quantitation is
4-fold, total volume of eluate is 200 ul) (see FIG. 7b). A 10 ul
aliquot of the genomic DNA produced from each protocol was applied
to 25 ul Amelogenin PCR amplification reactions according to
manufacturer's instructions (Promega). 10 ul of each reaction was
visualized on a 1.5% agarose gel and ethidium bromide staining.
108. A table outlining the various steps for each protocol has been
constructed (FIG. 7a) and illustrates that the filter membrane of
the invention requires fewer hands on operations and can be
completed in a similar, or faster, amount of time as the commercial
kits.
109. Amelogenin PCR amplification is successful for all
methodologies evaluated (FIG. 7c). The filter membrane of the
invention provides a method for genomic DNA isolation that is
comparable to commercially available kits in terms of speed, yield,
and PCR template quality.
Example 8
Archiving Blood Samples
110. The ability to archive nucleic acid containing samples such as
blood or bacterial plasmid clones is extremely important for
procedures where downstream processes have failed, "look-back"
regimes in transfusion medicine are required, or genotyping of a
patient that is no longer alive is needed. It has been demonstrated
in U.S. Pat. Nos. 5,496,562; 5,756,126; and 5,807,527 that blood
samples applied to FTA solid support can be kept stable, without
nucleic acid damage, at room temperature for extended length of
time. The characteristic is due in part to the chemical composition
of the solid matrix. The filter membrane of the invention composes
of the exact chemical composition as the FTA solid support, but
differs to FTA with respect to the base filter material. The
following experiment demonstrates that the present invention
maintains the archiving capability of the prior art filters while
providing the unexpected improvements of the present invention.
111. Several drops of freshly finger-stick drawn blood was spotted
to the filter membrane of the invention and allowed to air-dry for
two minutes. Once dried two 1 mm diameter punches were immediately
taken from the dried blood spot and applied to individual 200 ul
polypropylene PCR tubes. The remainder of the blood spot was placed
into an airtight polypropylene bag and stored at room temperature
on a laboratory bench-top for 19 weeks.
112. After 19 weeks storage, one 1 mm punch was taken from the
blood spot and applied to a 200 ul polypropylene PCR tube. To each
tube containing a single 1 mm blood punch, 200 ul of FTA
Purification Reagent (Fitzco, Inc) was added. Tubes were incubated
for five minutes at room temperature with no shaking.
113. Following incubation, the FTA purification Reagent was
aspirated from the tube. A second aliquot of 200 ul of FTA
Purification Reagent was added to each tube. The tubes were
incubated for five minutes at room temperature without shaking.
Following incubation, the FTA Purification Reagent was aspirated
from the tubes. 200 ul of TE buffer was then added to each tube.
The tubes were incubated for five minutes at room temperature
without shaking. The TE buffer was then fully aspirated from both
tubes, leaving the now washed 1 mm disc at the bottom of each tube.
20 ul of nuclease free water was then applied to both tubes. Tubes
were then incubated at 95.degree. C. for 10 minutes. Following heat
incubations the 20 ul of nuclease free water was aspirated from
each tube and retained.
114. An Amelogenin PCR amplification master mix was made up
according to manufacturers instructions (Promega), with a 25 ul
aliquot applied to both tubes containing the 1 mm punches, and a 5
ul aliquot applied to 20 ul nuclease free water samples. PCR was
carried out following parameters described by the manufacturer of
the Amelogenin primer set (Promega). Following PCR 10 ul of each
PCR reaction was visualized on a 1.5% agarose gel stained with
ethidium bromide, and photographed using a Polaroid camera.
115. Example 8 illustrates that soluble genomic DNA isolation and
PCR amplification can be carried out from fresh blood spotted to
the filter membrane of the invention at day 1 (FIG. 8a). The same
blood sample spotted to the filter membrane of the invention can
also be processed to give soluble isolated genomic DNA that is
suitable for PCR amplification (FIG. 8b). The filter membrane of
the invention exhibits the same sample archive characteristics as
the FTA solid support. Along with the demonstrated archive
characteristic, the filter membrane of the invention differs from
FTA solid support in that immobilized nucleic acid can be readily
released from the solid filter matrix.
116. Throughout this application, various publications, including
United States patents, are referenced by author and year and
patents by number. Full citations for the publications are listed
below. The disclosures of these publications and patents in their
entireties are hereby incorporated by reference to this application
in order to more fully describe the state of the art to which this
invention pertains.
117. The invention has been described in an illustrative manner,
and it is to be understood that the terminology which has been used
is intended to be in the nature of words of description rather than
of limitation.
118. Obviously, many modifications and variations of the present
invention are possible in light of the above teachings. It is,
therefore, to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described.
* * * * *