U.S. patent application number 12/503500 was filed with the patent office on 2009-11-12 for rapid and efficient capture of dna from sample without using cell lysing reagent.
Invention is credited to Robert T. Belly, Jianbo Sun.
Application Number | 20090280498 12/503500 |
Document ID | / |
Family ID | 22454077 |
Filed Date | 2009-11-12 |
United States Patent
Application |
20090280498 |
Kind Code |
A1 |
Belly; Robert T. ; et
al. |
November 12, 2009 |
RAPID AND EFFICIENT CAPTURE OF DNA FROM SAMPLE WITHOUT USING CELL
LYSING REAGENT
Abstract
Nucleic acids can be made available for amplification or other
treatment after admixture of a sample with specific weakly basic
polymers to form a precipitate with the nucleic acids at acidic pH.
After removing non-precipitated materials, the pH is then made
basic, thereby releasing the nucleic acids from the polymer. This
method for preparing specimen samples is simple and quite rapid,
and the released nucleic acids can be further treated in
hybridization assays or amplification procedures. No surfactant or
other cell lysing reagents are employed. The weakly basic polymers
are water-soluble and cationic at acidic pH, but neutral in charge
at basic pH.
Inventors: |
Belly; Robert T.; (Webster,
NY) ; Sun; Jianbo; (Beijing, CN) |
Correspondence
Address: |
PHILIP S. JOHNSON;JOHNSON & JOHNSON
ONE JOHNSON & JOHNSON PLAZA
NEW BRUNSWICK
NJ
08933-7003
US
|
Family ID: |
22454077 |
Appl. No.: |
12/503500 |
Filed: |
July 15, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10019514 |
Feb 21, 2003 |
7262006 |
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12503500 |
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11613475 |
Dec 20, 2006 |
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10019514 |
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Current U.S.
Class: |
435/6.12 ;
435/6.15; 536/23.5; 536/24.33 |
Current CPC
Class: |
C12Q 1/6806 20130101;
C12Q 1/6806 20130101; C12N 15/1003 20130101; C12Q 1/6886 20130101;
C12Q 2600/16 20130101; C12Q 2527/125 20130101; C12Q 2600/156
20130101; C12Q 2527/119 20130101 |
Class at
Publication: |
435/6 ; 536/23.5;
536/24.33 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04 |
Claims
1. A kit for detection of K-ras mutation in a biological sample,
said kit comprising a diagnostic K-ras primer selected from the
group consisting of: TABLE-US-00011 <SEQ ID: NO 1> (a)
TGAATATAAA CTTGTGGTAC CTGGAGC T (5K15S), <SEQ ID: NO 2> (b)
ATATAAACTT GTGGTAGTTC CAGCTGGT (5K37), <SEQ ID: NO 3> (c)
GAATTAGCTG TATCGTCAAG GCACTC (3K42), <SEQ ID: NO 4> (d)
TCAGCAAAGA CAAGACAGGT A (5BK5), <SEQ ID: NO 5> (e) TATAGATGGT
GAAACCTGTT TGTTGG (5N12A), <SEQ ID: NO 6> (f) CTTGCTATTA
TTGATGGCAA CCACACAGA (3N13A)
(g) any combination of the foregoing.
2. The oligonucleotide TGAATATAAA CTTGTGGTAC CTGGAGC T <SEQ ID:
NO 1>.
3. The oligonucleotide ATATAAACTT GTGGTAGTTC CAGCTGGT <SEQ ID:
NO 2>.
4. The oligonucleotide GAATTAGCTG TATCGTCAAG GCACTC <SEQ ID: NO
3>.
5. The oligonucleotide TCAGCAAAGA CAAGACAGGT A <SEQ ID: NO
4>.
6. The oligonucleotide TATAGATGGT GAAACCTGTT TGTTGG <SEQ ID: NO
5>.
7. The oligonucleotide CTTGCTATTA TTGATGGCAA CCACACAGA <SEQ ID:
NO 6>.
8. A K-ras diagnostic primer comprising the oligonucleotide
TGAATATAAA CTTGTGGTAC CTGGAGC T<SEQ ID: NO 1>.
9. A K-ras diagnostic primer comprising the oligonucleotide
ATATAAACTT GTGGTAGTTC CAGCTGGT <SEQ ID: NO 2>.
10. A K-ras diagnostic primer comprising the oligonucleotide
GAATTAGCTG TATCGTCAAG GCACTC <SEQ ID: NO 3>.
11. A K-ras diagnostic primer comprising the oligonucleotide
TCAGCAAAGA CAAGACAGGT A <SEQ ID: NO 4>.
12. A K-ras diagnostic primer comprising the oligonucleotide
TATAGATGGT GAAACCTGTT TGTTGG <SEQ ID: NO 5>.
13. A K-ras diagnostic primer comprising the oligonucleotide
CTTGCTATTA TTGATGGCAA CCACACAGA <SEQ ID: NO 6>.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Application 60/132,443, filed May 4, 1999, and Non-Provisional
application U.S. Ser. No. 10/019,514 filed Feb. 21, 2003 under U.S.
Pat. No. 7,262,006, and Divisional application U.S. Ser. No.
11/613,475 filed Dec. 20, 2006, which is incorporated by reference
in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to a method for preparing a sample by
capture and selective release of nucleic acids for detection. In
particular, it relates to a method for capture and release of
nucleic acids for subsequent treatment such as amplification. It
also relates to a test kit for use in the method.
BACKGROUND OF THE INVENTION
[0003] Technology to detect minute quantities of nucleic acids has
advanced rapidly over the last two decades including the
development of highly sophisticated amplification techniques such
as polymerase chain reaction (PCR). Researchers have readily
recognized the value of such technology to detect nucleic acids
which are indicative of diseases and genetic features in human or
animal test specimens. The use of probes and primers in such
technology is based upon the concept of complementarity, that is,
the bonding of two strands of a nucleic acid by hydrogen bonds
between complementary nucleotides (also known as nucleotide pairs).
PCR is a significant advance in the art to allow detection of very
small concentrations of a targeted nucleic acid. The details of PCR
are described, for example, in U.S. Pat. No. 4,683,195 (Mullis et
al), U.S. Pat. No. 4,683,202 (Mullis) and U.S. Pat. No. 4,965,188
(Mullis et al), although there is a rapidly expanding volume of
literature in this field.
[0004] In order to effectively amplify and detect a target nucleic
acid, it is usually necessary to isolate that nucleic acid from
cellular and other specimen debris. Various lysing procedures are
known, including freezing, treatment with digesting enzyme such as
proteases (for example, Proteinase K), boiling, and use of various
detergents (see for example U.S. Ser. No. 178,202, filed Apr. 6,
1988 by Higuchi, and EP-A-0 428 197, published May 22, 1991),
solvent precipitations and heating protocols.
[0005] Circulating DNA has been detected in blood serum and plasma.
Nanogram quantities are detected in normal subjects (Steinman, C.
R., J. Clin. Invest. 56:512-515, 1975 and Raptis, L., et al., J.
Clin. Invest. 66:1391-1399, 1980), and increased levels are
detected in chronic autoimmune diseases (Leon, S. A., et al.,
Cancer Res., 37:646-650, 1977) and in cancer patients (Stroun, M.,
et al., Eur. J. Cancer Clin. Oncol. 28:707-712, 1987; Maebo, A.,
Jpn. J. Thorac. Dis. 28:1085-1091, 1990; Fournie, G. J., et al.,
Cancer Lett., 91:221-227, 1995; Lin, A., et al., BioTechniques
24:(6) 937-940, 1998; and Sorenson, G. D., et al., Cancer
Epidemiology, Biomarkers and Prevention 3:67-71, 1994). Recently,
it has become evident that free extracellular DNA present in blood
serum and plasma can be used for genotype analysis (Lin, A., et
al., BioTechniques 24:(6) 937-940, 1998), for detection of cancer
(Mulcahy, H. E., et al., Clin. Cancer Res. 4:271-275, 1998), and
DNA in maternal serum may be used in prenatal diagnostics (Lo
Dennis, et al., Am. J. Human Genet. 62:768-775, 1998). Mutations
present in a primary tumor, often can be detected using DNA from
blood plasma or serum DNA (Sorenson, G. D., et al., Cancer
Epidemiology, Biomarkers and Prevention 3:67-71, 1994; Vasyukhin,
V., et al., In Challenges of Modern Medicine, Vol. 5, Biotechnology
Today, R. Verna, and A. Shamoo, eds, 141-150. Aera-Serono Symposia
Publications, Rome; Mulcahy, H. E., et al., supra.; Kopreski, M.
S., et al., Brit. J. Cancer 76:1293-1299, 1997; Chen, X., et al.
Nature Medicine 2: 1033-1035, 1996; Vasioukin, V., et al., Brit. J.
Haematology 86:774-779, 1994; and Tada, M., et al., Cancer Res.
53:2472-2474, 1993). Thus, DNA present in serum and plasma
represents a minimally invasive source for information related to
cancer diagnosis, prognosis, and therapy.
[0006] To effectively amplify and detect a target nucleic acid, it
is usually necessary to separate the nucleic acid from interfering
substances present in a specimen of interest. Several different
approaches have been used to concentrate and purify DNA from blood
serum or plasma. Many of these methods involve multiple steps
including phenol, ether, and chloroform treatment, dialysis,
passage through Concanavalin A-Sepharose to remove polysaccharides
and then centrifugation in a cesium chloride gradient (Vasyukhin,
V., et al., supra.). More recently, Qiagen has commercialized a
system for DNA concentration and purification based on a spin
column protocol. The Quiagen protocol is complex, involving a total
of eight steps, treatment with a protease, incubations at
70.degree. C., and requires the use of at least 3 different
buffers, in addition to a silica spin column centrifugation
step.
[0007] Recently, Goecke et al.(WO 97/34015)reported the detection
of extracellular tumor-associated nucleic acid in blood plasma and
serum using nucleic acid amplification assays. In their preferred
method, DNA is co-precipitated from plasma and serum using a
multistep protocol involving an initial co-precipitation by
gelatin, followed by solvent treatment and centrifugation. Other
time-consuming and complex protocols involving the use of glass
beads, silica particles or diatomaceous earth for extraction of DNA
from serum and plasma are also described.
[0008] The use of weakly basic polymers for the capture and
selective release of nucleic acids has been described U.S. Pat. No.
5,622,822 (Ekeze et al.), U.S. Pat. No. 5,582,988 (Backus et al.),
and U.S. Pat. No. 5,434,270 (Ponticello, et al.). The protocols
described in the aforementioned patents depend upon the use of a
cell lysing agent or a cell lysing step. Surfactants are often used
as cell lysing agents.
[0009] The use of surfactants and other lysing agents results in
the release of nucleic acids from cells and cellular components in
blood; causing a large concentration of background DNA.
SUMMARY OF THE INVENTION
[0010] The problems associated with the use of lysing agents or
lysing steps in prior art methods have been overcome with the
method of the present invention.
[0011] The method of this invention involves the use of a weakly
basic polymer, as described in the above-indicated US patents, for
the capture and selective release of the captured nucleic acids
from the polymer, but without the use of a lysing step or lysing
agent, as performed using prior art methods.
[0012] According to one aspect of the invention, a simplified,
easy-to-use method for recovering DNA from blood serum and plasma
is provided. The method includes the use of a weakly basic polymer
for binding DNA from a sample such as blood serum or plasma. Upon
binding DNA, the polymer becomes insoluble. The polymer-bound DNA
is then separated from the liquid mixture which comprises
non-desirable soluble substances. DNA is then released from the
polymer by means of alkali addition. Thus the method of the present
invention requires only three steps: (a) contact of sample with
buffer, (b) contact and incubation of mixture formed in step (a)
with a weakly basic polymer, and (c) release of the DNA bound to
polymer in step (b) by contact with alkali. The method eliminates
the need for extraction with alcohol or other solvent and toxic
materials such as phenol or chloroform, and lysing agents are not
used. The method not only simplifies DNA recovery, but also results
in an improvement in yield of amplifiable target DNA. Although the
method is preferably used with serum and blood as the sample, it is
applicable to other body fluids including but not limited to urine,
bile, spinal fluid, bronchial lavage (BAL), colonic washes, and
stool. In addition, samples of any type can be used, including
those collected from animals, humans, environmental and microbial
specimens.
[0013] In another aspect the present invention relates to
amplification and detection of target DNA using the method of DNA
recovery described hereinabove.
[0014] The inventive methods of the invention comprise the steps
of:
A) at a pH of less than 7, contacting a sample suspected of
containing a nucleic acid with a water-soluble, weakly basic
polymer in an amount sufficient to form a water-insoluble
precipitate of the weakly basic polymer with all nucleic acids
present in the sample, B) separating the water-insoluble
precipitate from the sample, and C) contacting the precipitate with
a base to raise the solution pH to greater than 7, and thereby
releasing the nucleic acids from the weakly basic polymer, the
weakly basic polymer comprising recurring units derived by addition
polymerization of one or more ethylenically unsaturated
polymerizable monomers having an amine group which can be
protonated at acidic pH.
[0015] This invention also provides a method for the amplification
and detection of a target nucleic acid comprising:
I) providing a target nucleic acid using the steps of: [0016] A) at
a pH of less than 7, contacting a sample suspected of containing a
target nucleic acid with a water-soluble, weakly basic polymer in
an amount sufficient to form a water-insoluble precipitate of the
weakly basic polymer with all nucleic acids present in the sample,
including the target nucleic acid, [0017] B) separating the
water-insoluble precipitate from the sample, and [0018] C)
contacting the precipitate with a base to raise the solution pH to
greater than 7, and thereby releasing the nucleic acids, including
the target nucleic acid, from the weakly basic polymer, [0019] the
weakly basic polymer comprising recurring units derived by addition
polymerization of one or more ethylenically unsaturated
polymerizable monomers having an amine group which can be
protonated at acidic pH, II) amplifying the target nucleic acid
present among the released nucleic acids, and III) detecting the
amplified target nucleic acid. A test kit for amplification of a
target nucleic acid comprises, separately packaged: a) an
amplification reaction mixture comprising one or more amplification
reagents, and b) a weakly basic polymer comprising recurring units
derived by addition polymerization of one or more ethylenically
unsaturated polymerizable monomers having an amine group which can
be protonated at acidic pH.
[0020] The present invention provides a rapid, simple and effective
method for selectively isolating and providing nucleic acids for
further treatment, such as hybridization assays or amplification
procedures. This invention overcomes the problems noted above
relating to conventional isolation means, including the use of
polyethyleneimine. In addition, the problems presented by the use
of polyethyleneimine combined with a fluorinated phosphate
surfactant are also avoided because the surfactant is not needed.
The sample preparation method of this invention is not tedious and
requires a minimum of steps, thereby making it more readily
automated. It usually can be carried out within about 15 minutes
(preferably within 10 minutes).
[0021] These advantages are provided by using in place of the
polyethyleneimine a "weakly basic" polymer which is cationic and
water-soluble at acidic pH, but deprotonates at a basic pH which is
significantly above the pKa of the polymer. By "weakly basic" is
meant that the polymer pKa is less than 7, and more likely less
than 6.5. Thus, the polymer can be used at low pH to precipitate
nucleic acids because of the ionic interaction of the cationic
polymer and the anionic phosphate backbone of nucleic acids.
[0022] After removing noncomplexed materials, and upon a pH
adjustment to basic conditions, the nucleic acids are released (or
decomplexed) from the weakly basic polymer of the precipitate and
available for further treatment, such as amplification. The
amplification procedures can be carried out under basic
conditions.
BRIEF DESCRIPTION OF THE DRAWING
[0023] FIG. 1 illustrates a standard curve for DNA as evaluated by
TaqMan amplification with the .beta.-actin gene after 40 PCR
cycles.
[0024] FIG. 2 ILLUSTRATES THE RESULTS OF ANALYSIS FOR A k-12 ras
mutation as determined by gel electrophoresis after REMS-PCR in
accordance with Example 7.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention is especially suited for the
extraction and detection of one or more target nucleic acids
present in a sample of any type collected from animals, humans,
environmental or microbial specimens. The nucleic acids so obtained
can be further treated by subjecting them to conventional
hybridization assays, the procedures of which are well known in the
art (for example, U.S. Pat. No. 4,994,373, incorporated herein by
reference with respect to the hybridization technology).
[0026] However, for the sake of brevity, the remaining discussion
will be directed to preferred embodiments whereby the nucleic acids
are subjected to amplification procedures, particularly PCR.
However, the scope of this invention is not intended to be so
limited because other amplification techniques (such as LCR) can be
used also.
[0027] The general principles and conditions for amplification and
detection of nucleic acids using polymerase chain reaction are
quite well known, the details of which are provided in numerous
references including U.S. Pat. No. 4,683,195 (Mullis et al), U.S.
Pat. No. 4,683,202 (Mullis), U.S. Pat. No. 4,965,188 (Mullis et al)
and WO-A-91/12342. The noted U.S. patents are incorporated herein
by reference. In view of the teaching in the art and the specific
teaching provided herein, a worker skilled in the art should have
no difficulty in practicing the present invention by combining the
preparatory method of this invention with polymerase chain reaction
procedures, or with any other amplification procedure known in the
art.
[0028] Other amplification procedures which can be used in the
practice of this invention include, but are not limited to, ligase
chain reaction as described, for example, in EP-A-0 320 308
(published December, 1987) and EP-A-0 439 182 (published January,
1990).
[0029] Test specimens ("samples") can include body fluids or other
materials containing genetic DNA or RNA. The target nucleic acid
can be extracted from any suitable human, animal, microbial, viral
or plant source.
[0030] The advancement disclosed herein contemplates that prior to
contact with the weakly basic polymer defined herein, no extraction
of nucleic acids from the specimen is required. While the prior art
teaches various lysing procedures known in the art (including those
described by Laure et al in The Lancet, pp. 538-540 (Sep. 3, 1988),
Maniatis et al, Molecular Cloning: A Laboratory Manual, pp. 280-281
(1982), Gross-Belland et al in Eur. J. Biochem., 36, 32 (1973) and
U.S. Pat. No. 4,965,188 (noted above)). Extraction of DNA from
whole blood or components thereof is described, for example, in
EP-A-0 393 744 (published Oct. 24, 1990), U.S. Pat. No. 5,231,015
(Cummins et al) and U.S. Pat. No. 5,334,499 (Burdick et al); the
lysing procedure being dependent upon the type of specimen being
used as the source of nucleic acids; a preferred lysing procedure
includes heating the specimen in the presence of a suitable
nonionic surfactant, a number of which are well known in the art.
Another useful lysing procedure is described in U.S. Ser. No.
08/063,169 (filed May 18, 1993 by Ekeze and Kerschner) whereby a
whole blood specimen is mixed with a buffered solution of ammonium
chloride, followed by additional steps which includes a second
mixing with ammonium chloride, the methods of the instant invention
do not employ a lysing step.
[0031] The sample, first diluted and admixed with a buffer at below
pH of about 7.0, is admixed with a weakly basic polymer (defined
below) in an amount sufficient to complex with all nucleic acids
present in the sample, forming a water-insoluble precipitate. This
polymer is water-soluble at acidic pH. Generally, the amount of
polymer present is at least about 0.01 weight percent, with from
about 0.05 to about 0.5 weight percent preferred. Of course, a
skilled artisan would know how to adjust the amount of polymer to
accommodate any quantity of nucleic acids. Mixing can be carried
out in any suitable manner for up to 30 minutes (generally less
than 5 minutes) and at any suitable temperature (generally from
15.degree. to 35.degree. C.
[0032] Suitable buffers for admixture with sample include those
buffers having a pKa less than 7, more preferably less than pKa
6.5, including MES (2-[N-Morpholino]ethanesulfonic acid) at pK 6.1,
BIS-TRIS (bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;
2-bis[2-hydroxyethyl]amino-2-[hydroxymethyl]-1,3-propanediol) at pK
6.5, ADA (N-[2-Acetamido]-2-iminodiacetic acid;
N-[Carbamoylmethyl]iminodiacetic acid) at pK 6.6, ACES
(N-[Carbamoylmethyl]-2-aminoethanesulfonic acid;
N-[2-Acetamido]-2-aminoethanesulfonic acid) at pK 6.8, PIPES
(piperazine-N,n'-bis[2-ethanesulfcid];
1,4-piperazinediethanesulfonic acid) at pK 6.8, MOPSO
(3-[N-Morpholino]-2-hydroxypropanesulfonic acid) at pK 6.9,
BIS-TRIS Propane (1,3-bis[tris(Hydroxymethyl)methylamino]propane)
at pK 6.8, PBS (phosphate buffered saline), and TRIS
(tris(hydroxymethyl)aminomethane), the weakly basic polymer can be
used in its water-soluble free form, or attached to a
water-insoluble substrate, such as in an affinity column, or
attached to polymeric, glass or other inorganic particles. Thus,
the polymers can be attached using conventional means (for example,
absorption, covalent bonds or specific binding reactions) to a
suitable substrate, including glass, polymeric or magnetic
particles, filters or films. Where the weakly basic polymer is
water-insoluble even at basic pH, it can be removed through
filtration, centrifugation or other conventional means after the
nucleic acids are released.
[0033] While bound to the weakly basic polymer, however, the
nucleic acids are not useful. It is then necessary to separate the
water-insoluble precipitate from the remainder of the sample which
may contain considerable cellular debris and excess polymer. This
separation can be achieved using any of various conventional
procedures, including centrifugation or filtration after which the
liquid is discarded. Centrifugation is preferred in the practice of
this invention and can be carried out at greater than about
1,000.times.g, for one minute to 5 minutes.
[0034] After the separation step, the nucleic acids can be
decomplexed or released from the weakly basic polymer, by
contacting the precipitate with a base, with or without heating.
Strong bases may be used without heating, and they include, but are
not limited to, sodium hydroxide, potassium hydroxide, ammonium
hydroxide, lithium hydroxide, sodium carbonate, sodium bicarbonate,
a tertiary amine (such as triethylamine, diisopropylethylamine and
lutidine), tricine, bicine or any other organic or inorganic base
which would be readily apparent to one skilled in the art. Useful
weaker bases may include basic buffers such as
tris(hydroxymethyl)aminomethane (or acid addition salts thereof),
N,N-bis(2-hydroxyethyl)glycine,
N-tris(hydroxymethyl)methyl-glycine, and others well known in the
art. Heating may be necessary when weaker bases are used.
[0035] Such heating can be carried out for up to 15 minutes
(generally less than 5 minutes) at a temperature that is at least
about 50.degree. C., and preferably is from about 95.degree. to
about 125.degree. C., under suitable pressure. As used in this
paragraph, "about" refers to +/-0.5.degree. C.
[0036] In preferred embodiments, weaker bases can be used with
heating, to release the nucleic acids from the precipitate. This
provides a solution containing nucleic acids which are ready for
amplification without further treatment. Such weaker bases may be
buffers, such as tris(hydroxymethyl)aminomethane hydrochloride.
[0037] In some embodiments, the polymers used in such embodiments
are those (defined below) which are water-insoluble even at basic
pH. Such polymers can be removed from the system after release of
nucleic acids and prior to amplification if desired.
[0038] The resulting solution containing released nucleic acids has
a basic pH. In some instances, the nucleic acids can be further
treated without any further adjustment in pH. In other embodiments
where a strong base is used, the pH of the solution may be adjusted
(generally downward) to from about 6 to about 9 (preferably from
about 7.5 to about 9), using any suitable acid or buffer, such as
tris(hydroxymethyl)aminomethane hydrochloride,
N,N-bis(2-hydroxyethyl)glycine, N-tris(hydroxymethyl)methylglycine
and others which would be readily apparent to one skilled in the
art. The amounts of such materials needed to achieve the desired pH
would be readily apparent to one skilled in the art.
[0039] At basic pH, the polymer used for capture of nucleic acids
can be either water-soluble or water-insoluble, and monomers needed
for providing such properties are described below.
[0040] The described method of capturing and releasing nucleic
acids of this invention is typically carried out within about 20
minutes, and preferably within about 10 minutes.
[0041] As used herein, unless otherwise noted, the modifier "about"
refers to a variance of 110% of the noted values. When used with pH
values, "about" refers to +/-0.5 pH unit.
[0042] In a preferred embodiment of this invention, a method for
the amplification and detection of a target nucleic acid
comprises:
I) providing a sample suspected of containing a target nucleic
acid, II) subjecting the target nucleic acid to the steps of:
[0043] A) at a pH of less than 7, contacting the target nucleic
acid with a water-soluble, weakly basic polymer in an amount
sufficient to form a water-insoluble precipitate of the weakly
basic polymer with all nucleic acids present in the sample,
including the target nucleic acid, [0044] B) separating the
water-insoluble precipitate from the sample, and [0045] C)
contacting the precipitate with a base to raise the solution pH to
greater than 7, and thereby releasing the nucleic acids, including
the target nucleic acid, from the weakly basic polymer, [0046] the
weakly basic polymer comprising recurring units derived by addition
polymerization of one or more ethylenically unsaturated
polymerizable monomers having an amine group which can be
protonated at acidic pH, III) without further adjustment of pH,
amplifying the released target nucleic acid, and IV) detecting the
amplified target nucleic acid.
[0047] In the foregoing method, it is still more preferred that the
weakly basic polymer is water-insoluble at basic pH, and the method
further comprises the step of removing the water-insoluble polymer
after release of the target nucleic acid but prior to amplification
thereof.
[0048] The weakly basic polymer used in the practice of this
invention is prepared from one or more ethylenically unsaturated
polymerizable monomers, at least one of which has an amine group
which can be protonated at acidic pH. Thus, at acidic pH, the
polymer is protonated to form the acid addition salt of the amine.
At basic pH, the polymer exists as the free base.
[0049] Particular "weakly basic groups" which can be a part of
polymerizable monomers useful in this invention include, but are
not limited to, cyclic amine groups, or primary, secondary or
tertiary aminoalkyl groups which can be protonated at acidic pH.
Useful cyclic amine groups include, but are not limited to,
imidazolyl, isoxazolyl, pyridyl, piperidyl, piperazinyl, pyrazolyl,
triazolyl, tetrazolyl, oxadiazolyl, pyridazinyl, pyrimidyl,
pyrazinyl, quinolinyl and quinazolinyl groups. The preferred groups
are cyclic groups which are aromatic, and the imidazolyl group is
most preferred. Useful aminoalkyl or cyclic amine groups are linked
to vinyl groups of the monomers using convenient linking groups
including alkylene, amido or ester groups, and multiple alkylene
groups can be linked together with imino, oxy, amide, carbonyl or
ester groups.
[0050] Generally useful polymers for capturing nucleic acids are
comprised of recurring units derived by addition polymerization
of:
a) from about 15 to 100 weight percent of a water-soluble, weakly
basic ethylenically unsaturated polymerizable monomer having at
least one group which can be protonated at acidic pH and which is
selected from the group consisting of aminoalkyl, imidazolyl,
isoxazolyl, pyridyl, piperidyl, piperazinyl, pyrazolyl, triazolyl,
tetrazolyl, oxadiazolyl, pyridazinyl, pyrimidyl, pyrazinyl,
quinolinyl and quinazolinyl, b) from 0 to about 35 weight percent
of a nonionic, hydrophilic ethylenically unsaturated polymerizable
monomer, and c) from 0 to about 85 weight percent of a nonionic,
hydrophobic ethylenically unsaturated polymerizable monomer.
[0051] Preferably, the weakly basic polymer is comprised of
recurring units of from about 20 to about 100 weight percent of a),
from 0 to about 25 weight percent of b), and from 0 to about 80
weight percent of c).
[0052] A more specific class of monomers useful in a) above are
those represented by the structure (I):
##STR00001##
wherein R.sup.3 is hydrogen or methyl, and X is oxy or imino. In
addition, R.sup.4 is a divalent hydrocarbon linking group having
from 1 to 8 carbon and hetero atoms in the chain and comprising one
or more alkylene groups (such as methylene, ethylene, n-propylene,
isopropylene and n-pentylene), providing that when there is more
than one alkylene group, they are linked together in R.sup.4 with
one or more carbonyl, oxy, imino, ester or amido groups in any
operable combination. By "operable combination" is meant that those
groups can be combined with the alkylene groups in any chemically
possible configuration, and can be used in combination (connected
to each other) in chemically possible ways (such as oxycarbonyl,
carbonamido and others readily apparent to one skilled in the art).
It is also to be understood that R.sup.4 can be terminated (or
connected to R.sup.5) with a carbonyl, oxy, imino, ester or amido
group.
[0053] R.sup.5 is a cyclic amine or primary, secondary or tertiary
aminoalkyl group, as defined above, which can be protonated at
acidic pH.
[0054] Examples of useful type a) monomers include, but are not
limited to, 1-vinylimidazole, 2-methyl-1-vinylimidazole,
2-vinylpyridine, 1-hydroxy-6-vinyl-1H-benzotriazole, 2-aminoethyl
methacrylate hydrochloride, 2-aminoethyl acrylate hydrochloride,
N-(3-aminopropyl)methacrylamide, 2-vinylquinoline,
N-(3imidazolylpropyl)methacrylamide,
N-(2-imidazolylethyl)methacrylamide,
N-(3-imidazolylpropyl)acrylamide,
N-(1,1-dimethyl-3-N-imidazolylpropyl)acrylamide,
N-(imidazolylmethyl)acrylamide, 1-vinylpyrrolidinone,
3-(N,N-dimethylamino)propyl metharcylate and acid addition salts of
the noted free bases.
[0055] A class of novel monomers of type a) of this invention can
be used to prepare either homopolymers or copolymers. These
monomers are defined by the structure (II):
##STR00002##
wherein R is hydrogen or methyl. Preferably, R is methyl. In
addition, R.sup.1 is branched or linear alkylene of 1 to 3 carbon
atoms (such as methylene, ethylene, trimethylene or propylene).
Preferably, R.sup.1 is alkylene of 2 or 3 carbon atoms. More
preferably, R.sup.1 is trimethylene.
[0056] Particularly useful monomers having structure (II) include,
but are not limited to, N-(3-imidazolylpropyl)methacrylamide,
N-(2-imidazolylethyl)methacrylamide,
N-(3-imidazolylpropyl)acrylamide,
N-(1,1-dimethyl-3-N-imidazolylpropyl)acrylamide,
N-(imidazolylmethyl)acrylamide, and their acid addition salts. Of
the novel monomers described herein, the first compound is most
preferred.
[0057] Preferred type a) monomers include 1-vinylimidazole and
N-2-methyl-1-vinylimidazole.
[0058] If the monomers of type a) have low or no water solubility,
they can also be polymerized in the form of their acid addition
salts (such as the hydrochloride or hydrobromide).
[0059] Monomers identified as type b) monomers are those which are
defined herein as "hydrophilic", meaning those which, when
homopolymerized, provide homopolymers which are water-soluble at pH
7 or above. Generally, such monomers have hydrophilic groups such
as hydroxy, amine (primary, secondary, tertiary and cyclic), amide,
sulfonamide and polyethyleneoxy groups, but it is not necessary
that they comprise such groups if the noted homopolymer
water-solubility parameter is met.
[0060] Representative monomers of type b) include, but are not
limited to, acrylamide, 2-hydroxyethyl acrylate,
2,3-dihydroxypropyl acrylate, 2,3-dihydroxypropyl methacrylate,
poly(ethyleneoxy)ethyl methacrylate (having 2 to 10 ethyleneoxy
groups), and N,N-dimethylacrylamide. A preferred monomer is
acrylamide.
[0061] Monomers identified as type c) monomers are those which are
defined herein as "hydrophobic", meaning those which, when
homopolymerized, provide homopolymers which are water-insoluble at
pH 7 or above, irrespective of the type of pendant groups they may
possess.
[0062] Representative monomers of type c) include, but are not
limited to, methacrylamide, 2-hydroxyethyl methacrylate,
N-t-butylmethacrylamide, ethyl acrylate, methyl acrylate, butyl
acrylate, methyl methacrylate, styrene, vinyltoluene and other
vinyl aromatics and others which would be readily apparent to one
skilled in the art. A preferred monomer is 2-hydroxyethyl
methacrylate.
[0063] The monomers of types a), b) and c) which are not novel are
generally readily available from commercial sources, or prepared
using conventional procedures and starting materials.
[0064] The novel monomers of structure (II) can be prepared
generally by condensation of a 1-(aminoalkyl)imidazole with a
(meth)acryloyl chloride using appropriate conditions which would be
readily apparent to one skilled in the art. A representative
preparation of a preferred monomer is provided below preceeding the
examples. More details about such monomers can be obtained from
commonly assigned U.S. Pat. No. 5,434,270, Ponticello et al.,
entitled "Weakly Basic Polymerizable Monomers and Polymers Prepared
Therefrom".
[0065] The homopolymers and copolymers described herein can be
prepared using conventional solution polymerization techniques
which are well known in the art, although there are certain
preferred conditions which are illustrated in the preparatory
methods provided below preceding the Examples. The ratio of various
monomers can be adjusted, as one skilled in the art would know, to
provide polymers which are either water-soluble or water-insoluble
at basic pH, as long as such polymers remain water-soluble at
acidic pH.
[0066] Solution polymerization generally involves dissolving the
monomers in a suitable solvent (including water or various
water-miscible organic solvents) and polymerizing in the presence
of a suitable free radical initiator. The resulting polymer is
water-soluble at acidic pH, so it is precipitated using a solvent
such as acetone, purified and redissolved in water for future
use.
[0067] Particularly useful polymers described herein include, but
are not limited to, poly[N-(3-imidazolylpropyl)methacrylamide
hydrochloride-co-acrylamide],
poly[N-(3-imidazolylpropyl)methacrylamide
hydrochloride-co-2-hydroxyethyl methacrylate],
poly(1-vinylimidazole), poly(2-aminoethyl methacrylate
hydrochloride-co-2-hydroxyethyl methacrylate),
poly(1-vinylimidazole hydrochloride-co-2-hydroxyethyl
methacrylate),
poly[N-(1,1-dimethyl-3-imidazolylpropyl)acrylamide]poly(N-2-methyl-1-viny-
l imidazole) and acid addition salts of the free base polymers.
[0068] In preferred embodiments, the polymers used are
water-insoluble at basic pH. Such polymers are prepared using type
a) monomers as well as type c) monomers but with limited amounts
(less than 15 weight of type b) monomers to prevent solubilization
of the polymer at basic pH. Representative polymers of this type
include, but are not limited to,
poly[N-(3-imidazolylpropyl)-methacrylamide
hydrochloride-co-2-hydroxyethyl methacrylate],
poly(1-vinylimidazole), poly(2-aminoethyl methacrylate
hydrochloride-co-2-hydroxyethyl methacrylate) and
poly(1-vinylimidazole hydrochloride-co-2-hydroxyethyl
methacrylate).
[0069] The present invention is also directed to the amplification
or detection of one or more specific nucleic acid sequences present
in one or more target nucleic acids released as noted above.
Moreover, a plurality of target nucleic acids can be amplified and
detected simultaneously by using a corresponding set of primers and
detection means for each specific nucleic acid. Multiple sequences
in the same nucleic acid can also be amplified and detected.
[0070] A "PCR reagent" refers to any of the reagents generally
considered useful in PCR, namely a set of primers for each target
nucleic acid, a DNA polymerase, a DNA polymerase cofactor and two
or more deoxyribonucleoside-5'-triphosphates (dNTP's).
[0071] As used herein in referring to primers or probes, the term
"oligonucleotide" refers to a molecule comprised of four or more
deoxyribonucleotides or ribonucleotides, and preferably more than
ten. Its exact size is not critical but depends upon many factors
including the ultimate use or function of the oligonucleotide. The
oligonucleotide may be derived by any method known in the art.
[0072] The term "primer" refers to an oligonucleotide, whether
naturally occurring or synthetically produced, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product
complementary to a nucleic acid strand (that is, template) is
induced. Such conditions include the presence of nucleotides (such
as the four standard deoxyribonucleoside-5'-triphosphates), a DNA
polymerase and a DNA polymerase cofactor, and suitable temperature
and pH. Normally, such conditions are what are known in the art as
"high stringency" conditions so that nonspecific amplification is
minimized. The primer must be long enough to initiate the synthesis
of extension products in the presence of the DNA polymerase. The
exact size of each primer will vary depending upon the use
contemplated, the complexity of the targeted sequence, reaction
temperature and the source of the primer. Generally, the primers
used in this invention will have from 10 to 60 nucleotides.
[0073] Primers useful herein can be obtained from a number of
sources or prepared using known techniques and equipment, including
for example, an ABI DNA Synthesizer (available from Applied
Biosystems) or a Biosearch 8600 Series or 8800 Series Synthesizer
(available from Milligen-Biosearch, Inc.) and known methods for
their use (for example as described in U.S. Pat. No. 4,965,188).
Naturally occurring primers isolated from biological sources are
also useful (such as restriction endonuclease digests). As used
herein, the term "primer" also refers to a mixture of primers.
Thus, each set of primers for a given target nucleic acid may
include two or more primers for each opposing target strand.
[0074] One or both primers can be labeled with the same or
different label for detection or capture of amplified product.
Procedures for attaching labels and preparing primers are well
known in the art, for example, as described by Agrawal et al,
Nucleic Acid Res., 14, pp. 6227-45 (1986), U.S. Pat. No. 4,914,210
(Levenson et al) relating to biotin labels, U.S. Pat. No. 4,962,029
(Levenson et al) relating to enzyme labels, and the references
noted therein. Useful labels also include radioisotopes,
electron-dense reagents, chromogens, fluorogens, phosphorescent
moieties, ferritin and other magnetic particles (see U.S. Pat. No.
4,795,698 of Owen et al and U.S. Pat. No. 4,920,061 of Poynton et
al), chemiluminescent moieties (such as luminol), and other
specific binding species (avidin, streptavidin, biotin, sugars or
leetins). Preferred labels are enzymes, radioisotopes and specific
binding species (such as biotin). Useful enzymes include, glucose
oxidase, peroxidases, uricase, alkaline phosphatase and others
known in the art and can be attached to oligonucleotides using
known procedures. Reagents to provide a colorimetric or
chemiluminescent signal with such enzymes are well known.
[0075] Where the label is an enzyme such as a peroxidase, at some
point in the assay, hydrogen peroxide and suitable dye-forming
compositions are added to provide a detectable dye. For example,
useful dye-providing reagents include tetramethylbenzidine and
derivatives thereof, and leuco dyes, such as water-insoluble
triarylimidazole leuco dyes (as described in U.S. Pat. No.
4,089,747 of Bruschi), or other compounds which react to provide a
dye in the presence of peroxidase and hydrogen peroxide.
Particularly useful dye-providing compositions are described in
EP-A-0 308 236 (published Mar. 22, 1989). Chemiluminescent signals
in response to a peroxidase label can also be generated using the
appropriate reagents.
[0076] If one or both primers are biotinylated, the amplified
nucleic acid can be detected using detectably labeled avidin or an
equivalent thereof (such as streptavidin). For example, avidin can
be conjugated with an enzyme, or have a radioisotope using known
technology. Biotin on the amplified product complexes with the
avidin, and appropriate detection techniques to detect a
radioactive, calorimetric or chemiluminescent signal are used.
[0077] As used herein, a capture "probe" is an oligonucleotide
which is substantially complementary to a nucleic acid sequence of
one or more strands of the target nucleic acid, and which is used
to insolubilize the amplified nucleic acid. The probe
oligonucleotide is generally attached to a suitable water-insoluble
substrate such as polymeric or glass beads, microtiter plate well,
thin polymeric or cellulosic film or other materials readily
apparent to one skilled in the art. The oligonucleotide is
generally from about 12 to about 40 nucleotides in length, although
the length is not critical.
[0078] A DNA polymerase is an enzyme which will add deoxynucleoside
monophosphate molecules to the 3+-hydroxy end of the primer in a
complex of primer and template, but this addition is in a template
dependent manner (that is, dependent upon the specific nucleotides
in the template). Many useful DNA polymerases are known in the art.
Preferably, the polymerase is "thermostable", meaning that it is
stable to heat, especially the high temperatures used for
denaturation of DNA strands. More particularly, the thermostable
DNA polymerases are not substantially inactivated by the high
temperatures used in PCR as described herein.
[0079] A number of thermostable DNA polymerases have been reported
in the art, including those mentioned in detail in U.S. Pat. No.
4,965,188 (noted above) and U.S. Pat. No. 4,889,818 (Gelfand et
al), incorporated herein by reference.
[0080] Particularly useful polymerases are those obtained from
various Thermus bacterial species, such as Thermus aquaticus,
Thermus thermophilus, Thermus filiformis or Thermus flavus. Other
useful thermostable polymerases are obtained from a variety of
other microbial sources including Thermococcus literalis,
Pyrococcus furiosus, Thermotoga sp. and those described in
WO-A-89/06691 (published Jul. 27, 1989). Some useful polymerases
are commercially available. A number of techniques are known for
isolating naturally-occurring polymerases from organisms, and for
producing genetically engineered enzymes using recombinant
techniques, as noted in the art cited in this paragraph.
[0081] A DNA polymerase cofactor refers to a nonprotein compound on
which the enzyme depends for activity. A number of such materials
are known cofactors including manganese and magnesium salts. Useful
cofactors include, but are not limited to, manganese and magnesium
chlorides, sulfates, acetates and fatty acid salts (for example,
butyric, caproic, caprylic, capric and lauric acid salts). The
smaller salts, that is chlorides, sulfates and acetates, are
preferred.
[0082] Also needed for PCR are two or more
deoxyribonucleotide-5'-triphosphates, such as dATP, dCTP, dGTP,
dUTP or dTTP. Usually, dATP, dCTP, dGTP and dTTP are all used in
PCR. Analogues such as dITP and 7-deaza-dGTP are also useful.
[0083] Also useful in the practice of the invention is an antibody
specific to the DNA polymerase, which antibody inhibits its
enzymatic activity at temperatures below about 50.degree. C., but
which antibody is deactivated at higher temperatures.
Representative monoclonal antibodies having these properties are
described in U.S. Pat. No. 5,338,671 (Scalice et al), incorporated
herein by reference. Antibody fragments can be used in place of the
whole molecule if they have equivalent properties.
[0084] The PCR reagents described herein are provided and used in
PCR in suitable concentrations to provide amplification of the
target nucleic acid. The minimal amounts of DNA polymerase is
generally at least about 1 unit/100 .mu.l of solution, with from
about 4 to about 25 units/100 .mu.l being preferred. A "unit" is
defined herein as the amount of enzyme activity required to
incorporate 10 nmoles of total nucleotides (dNTP's) into an
extending nucleic acid chain in 30 minutes at 74.degree. C. The
concentration of each primer is at least about 0.075.mu. molar with
from about 0.2 to about 1.mu. molar being preferred. All primers
are present in about the same amount (within a variation of 10% of
each). The cofactor is generally present in an amount of from about
1 to about 15 mmolar, and each dNTP is generally present at from
about 0.1 to about 3.5 mmolar in the reaction mixture. As used in
this paragraph, the modifier "about" refers to a variance of +/-10%
of the noted value.
[0085] The PCR reagents can be supplied individually, or in a
buffered solution having a pH in the range of from about 7 to about
9 using any suitable buffer.
[0086] Since the target nucleic acid to be amplified and detected
is usually in double strand form, the two strands must be separated
(that is, denatured) before priming can take place. This can occur
during the extraction process, but preferably, it occurs in a
separate step afterwards. Heating to a suitable temperature
(identified as "first temperature" or T.sub.1 herein) is a
preferred means for denaturation. Generally, this first temperature
is in the range of from about 85.degree. to about 100.degree. C.
for a suitable time, for example from 1 to about 240 seconds
(preferably 1 to about 40 seconds). This initial denaturation step
can also be included in the first amplification cycle. In such
instances, denaturation may be longer in the first cycle (for
example, up to 240 seconds) whereas later cycles can have much
shorter denaturation steps (for example, up to 30 seconds).
[0087] The denatured strands are then primed with the appropriate
sets of primers by cooling the reaction mixture to a second
temperature, T.sub.2, which is generally within the range of from
about 55.degree. to about 70.degree. C. It is desired that cooling
is done as quickly as possible, but with presently known equipment,
it generally takes place over a time period of from about 5 to
about 40 seconds, and more preferably for from about 5 to about 20
seconds.
[0088] Once the denatured strands are cooled, the reaction mixture
containing the PCR reagents is incubated at a third temperature,
T.sub.3, generally for from 1 to about 120 seconds, and preferably
for from 1 to about 80 seconds, to effect formation of primer
extension products. Generally, the third temperature is within the
range of from about 55.degree. to about 74.degree. C. Preferably,
it is within the range of from about 62.degree. to about 70.degree.
C.
[0089] In a most preferred embodiment, the second and third
temperatures are the same and are within the range of from about
62.degree. to about 70.degree. C. Thus, priming and primer
extension are preferably carried out in the same step.
[0090] Thus, an amplification cycle comprises the denaturation,
priming (or annealing) and primer extension steps described above.
Generally, at least 15 of such amplification cycles are carried out
in the practice of this invention with the maximum number of cycles
being within the discretion of the particular user. In most
instances, 15 to 50 amplification cycles are used in the method
with 15 to 40 cycles being preferred. Each amplification cycle is
generally from about 20 to about 360 seconds, with a cycle time of
from about 30 to about 120 seconds being preferred and from about
30 to about 90 seconds being more preferred. However, longer or
shorter cycle times can be used if desired.
[0091] When used in reference to time for a given step in the
amplification procedure, the term "about" refers to +/-10% of that
time limit. Moreover, when used in reference to temperatures, the
term "about" refers to +/-0.5.degree. C.
[0092] Detection of amplified products can be accomplished using
any known procedure, including Southern blotting techniques, as
described in U.S. Pat. No. 4,965,188 (noted above), or by use of
labeled probes or primers, as is known in the art.
[0093] Alternatively to the embodiments described above, the
amplified products can be detected using a labeled oligonucleotide
which is complementary to one of the primer extension products.
[0094] All reagents for performing the TaqMan assay were purchased
from Applied Biosystems, a Division of Perkin-Elmer Co., Foster
City, Calif., including: .beta.-Actin detection reagents (cat. no.
401846), DNA template reagents (cat. no. 401970) and TaqMan PCR
Core Reagent Kit (cat. no. N808-0228). Assays were performed using
the PCR Master mix and thermal cycling profiles for the
.beta.-Actin TaqMan assay provided by the manufacturer. One
microliter of DNA template reagent was added to 49 .mu.L of PCR
.beta.-Actin Master mix in an ABI Prism 7700 Sequence Detection
System (Applied Biosystems) and fluorescence was measured during
the 40 PCR cycles.
[0095] FIG. 1 shows a calibration curve for different starting
levels of DNA versus Threshold cycle count, which is a value
determined by the instrument and represents the estimated number of
PCR cycles at which a preselected fluorescence signal will be
obtained. Thus, the TaqMan assay for a .beta.-Actin gene fragment
provides a good analytical tool for measuring DNA concentration
present in a sample.
[0096] In the examples that follow, DNA from the single copy (per
cell) .beta.-Actin gene was extracted from the indicated samples
according to the method of the invention or using the indicated
prior art method which utilizes a cell lysing reagent. .beta.-Actin
DNA extracted thereby was amplified using the PCR Master Mix and
thermal cycling profiles and TaqMan detection as per the
manufacturer's recommended procedures.
[0097] In the heterogeneous detection systems of this invention,
the amplified products are captured on a water-insoluble substrate
of some kind, and the other materials in the reaction mixture are
removed in a suitable manner, such as by filtration,
centrifugation, washing or another separation technique.
[0098] Capture probes can be attached to water-insoluble supports
using known attachment techniques (including absorption and
covalent reactions). One such technique is described in EP-A-0 439
222 (published Sep. 18, 1991). Other techniques are described, for
example, in U.S. Pat. No. 4,713,326 (Dattagupta et al), U.S. Pat.
No. 4,914,210 (Levenson et al) and EP-B-0 070 687 (published Jan.
26, 1983). Useful separation means include filtration through
membranes such as polyamide microporous membranes commercially
available from Pall Corporation.
[0099] However, any useful solid support can be used to anchor the
capture probe and eventual hybridization product, including
microtiter plates, test tubes, beakers, magnetic or polymeric
particles, metals, ceramics, and glass wool to name a few.
Particularly useful materials are magnetic or polymeric particles
having reactive groups useful for covalently attaching the capture
probe. Such particles are generally from about 0.001 to about
10.mu. meters. Further details about examples of such materials are
provided in U.S. Pat. No. 4,997,772 (Sutton et al), U.S. Pat. No.
5,147,777 (Sutton et al), U.S. Pat. No. 5,155,166 (Danielson et al)
and U.S. Pat. No. 4,795,698 (Owen et al), all incorporated herein
by reference.
[0100] The capture probe can be affixed to a flat support such as a
polymeric film, membranes, filter papers, or resin-coated or
uncoated paper. Capture probe affixed to polymeric particles can
also be immobilized on such flat supports in a suitable manner, for
example, as dried deposits, or adhered by heat fusion or with
adhesives. The capture probe can be affixed, for example, to a flat
support in the self-contained test device of this invention. Other
details of such materials are provided in EP-A-0 408 738 (published
Jan. 23, 1991), WO 92/16659 (published Oct. 1, 1992) and U.S. Pat.
No. 5,173,260 (Sutton et al).
[0101] The capture probes can be arranged on a suitable support in
any configuration, for example rows of round deposits or
stripes.
[0102] The present invention can also be used in what are known as
"homogeneous" amplification procedures in which target nucleic
acids are detected without the need for capture reagents. The
details of such assays are known in the art, such as in EP-A-0 487
218 (published May 27, 1992) and EP-A-0 512 334 (published Nov. 11,
1992).
[0103] The amplification reaction composition can be included as
one individually packaged component of a test kit useful for
various amplification assays. The kit can include other reagents,
solutions, equipment and instructions useful in the method of this
invention, including capture reagents immobilized on a
water-insoluble substrate, wash solutions, detection reagents and
other materials readily apparent to one skilled in the art. In
addition, the test kit can include a separately packaged weakly
basic polymer as described above, buffers, weak or strong bases and
other reagents needed for either or both amplification and specimen
sample preparation. The test kit can also include a test device
containing one or more other kit components. This test device is
preferably "self-contained" as that term is understood in the art.
Other kits can include the weakly basic polymer described herein
and one or more reagents (such as detection or capture probes) used
in hybridization assays.
[0104] The following examples are included to illustrate the
practice of this invention, and are not meant to be limiting in any
way. All percentages are by weight unless otherwise noted.
Materials and Methods for Examples
Preparation of N-(3-Imidazolylpropyl)-methacrylamide
[0105] This procedure shows the preparation of a novel monomer of
structure (I), identified above, but the preparation is
representative of how other monomers within the scope of this
invention could readily be prepared.
[0106] A solvent mixture was prepared by mixing water (100 ml)
containing sodium hydroxide (12.8 g, 0.32 mole) and dichloromethane
(200 ml) containing 1-(3-aminopropyl)imidazole (37.5 g, 0.3 mole),
and cooled in an ice bath. To this cooled mixture was added all at
once, methacryloyl chloride (34.8 g, 0.3 mole) in dichloromethane
(100 ml) with vigorous stirring under a nitrogen atmosphere. Heat
was evolved with the temperature of the mixture rising to about
60.degree. C., and the mixture was vigorously stirred for another
10 minutes, and then the organic layer was allowed to separate. The
water layer was extracted twice with dichloromethane (100 ml each
time). The combined organic solution (the organic solvent layer and
extracts) was washed with saturated sodium chloride (100 ml), dried
over anhydrous sodium sulfate, filtered, and the solvent was
removed. The residue was dissolved in chloroform (50 ml), followed
by the addition of ethyl ether (50 ml) to the cloud point.
[0107] The resulting reaction product crystallized at about
0.degree. C., and was filtered to give a white solid having a
melting point of 45.degree.-46.degree. C. The yield was 70%.
Analytical data included: m/e (M-193),
[0108] .sup.1H NMR (DMSO d6) 1.8 (m, 2H,C--CH.sub.2--C,CH.sub.3),
3.02 (m, 2H,N--CH.sub.2), 3.95 (t, 2H, im-CH.sub.2), 5.25 and 5.6
(AB, 2H, vinyl-CH.sub.2), 6.82 and 7.15 (AB, 2H, 4,5-H of im), 7.6
(s, 1H, 2-H of im), 7.95 (m, 1H, NH).
Preparation of Homopolymer
[0109] A preferred homopolymer prepared from a novel monomer
described herein was prepared by adding
2,2'-azobis(2-methylpropionitrile) (300 mg) to a solution of
N-(3-imidazolylpropyl)methacrylamide (12.5 g, 0.065 mole) in water
(90 ml) and isopropanol (10 ml), maintained under a nitrogen
atmosphere. The resulting solution was heated, while being stirred,
to 65.degree.-70.degree. C. in a water bath for 3 hours. After
about 1.5 hours of that time, concentrated HCl (3 ml) was added,
and the stirring was continued under nitrogen for the remaining
time. The solution was then concentrated on a rotary evaporator to
about 25 ml, and the resulting polymer product was precipitated in
acetone (over 4 liters), filtered and dissolved in deionized water
(80 ml). The solution contained 12% solids.
Preparation of First Copolymer
[0110] Poly[N-(3-imidazolylpropyl)methacrylamide
hydrochloride-co-acrylamide] (90:10 weight ratio) was prepared by
adding 2,2'-azobis(2-methylpropionitrile) (400 mg) to a solution of
N-(3-imidazolylpropyl)methacrylamide (18 g, 0.09 mole) and
acrylamide (2 g, 0.028 mole) in deionized water (120 ml) and
isopropanol (15 ml), maintained under a nitrogen atmosphere. The
solution was heated to 65.degree.-70.degree. C. with stirring for 4
hours, followed by addition of dilute HCl to lower the pH to about
2. Stirring and heating were continued for another hour, and the
solution was then allowed to reach room temperature overnight.
[0111] The solution was concentrated to about 75 ml using a rotary
evaporator, and the resulting polymer was precipitated in acetone
(about 4 liters), filtered and dissolved in deionized water (150
ml). Further concentration to about 125 ml was carried out to
remove residual acetone. The polymer was present at 15.5%
solids.
Preparation of Second Copolymer
[0112] Poly[2-aminoethyl methacrylate
hydrochloride-co-2-hydroxyethyl methacrylate] (20:80 weight ratio)
was prepared by adding 2,2'-azobis(2-methylpropionitrile) (400 mg)
to a solution of 2-aminoethyl methacrylate hydrochloride (4 g, 0.02
mole) and 2-hydroxyethyl methacrylate (16 g, 0.12 mole) in
deionized water (180 ml) and ethanol (20 ml), maintained under a
nitrogen atmosphere. The solution was heated to
65.degree.-70.degree. C. with stirring for 4 hours. Stirring and
heating were continued for another hour, and the solution was then
allowed to reach room temperature overnight.
[0113] The resulting polymer was precipitated in acetone (about 4
liters), filtered and dissolved in deionized water (150 ml).
Further concentration to about 125 ml was carried out to remove
residual acetone. The polymer was present at 5.6% solids.
Preparation of Third Copolymer
[0114] Poly[1-vinylimidazole-co-2-hydroxyethyl methacrylate] (50:50
weight ratio) was prepared by adding
2,2'-azobis(2-methylpropionitrile) (350 mg) to a solution of
1-vinylimidazole (10 g, 0.1 mole) and 2-hydroxyethyl methacrylate
(10 g, 0.077 mole) in N,N-dimethylformamide (160 ml), maintained
under a nitrogen atmosphere. The solution was heated to
65.degree.-70.degree. C. with stirring for 7 hours.
[0115] After sitting at room temperature overnight, the polymer was
precipitated in acetone (about 4 liters), filtered and dissolved in
deionized water (200 ml) containing concentrated HCl (8.5 ml).
Further concentration was carried out to remove residual acetone.
The polymer was present at 12.4% solids.
Preparation of Fourth Copolymer
[0116] Poly(1-vinylimidazole-co-2-hydroxyethyl methacrylate) (25:75
weight ratio) was prepared in a fashion like the "Third Copolymer".
The resulting solution contained 13.7% solids.
[0117] Deoxyribonucleotides (dNTP's),
tris(hydroxymethyl)aminomethane buffer and lyophilized calf thumus
DNA were obtained from Sigma Chemical Co.
[0118] Gel electrophoresis was carried out by adding the
amplification product mixture (6.75 .mu.l) to agarose gels (2.5%)
which had been prestained with ethidium bromide (0.4 mg/ml final
concentration). The gels were electrophoresed at about 8 volts/cm
for about 1 hour using an electrophoresis buffer (600 ml)
containing ethidium bromide (0.4 mg/ml final concentration). The
buffer was a mixture of tris(hydroxymethyl)aminomethane, borate and
ethylenediaminetetraacetic acid. The resulting bands were compared
to conventional molecular weight markers, and the product band
intensity was scored (115-mer for HIV1 and 383-mer for M.
tuberculosis) on a 0 to 5 scale with 0 representing no detectable
signal and 5 representing the highest signal.
[0119] Other reagents and materials were obtained either from
commercial sources or prepared using readily available starting
materials and conventional procedures.
[0120] The invention has been described in detail with particular
reference to preferred embodiments thereof, but it will be
understood that variations and modifications can be effected within
the spirit and scope of the invention.
Example 1
Capture and Release of DNA Using Weakly Basic Homopolymer
[0121] This example illustrates the practice of the present
invention to capture and release a nucleic acid using
poly(1-vinylimidazole).
[0122] Various volumes of poly(1-vinylimidazole) [of a 1:10
dilution of 2.4% stock solution (pH 2.3)] were mixed with calf
thymus DNA (100 .mu.l, 0.5 .mu.g/.mu.l) and vortexed to form a
precipitate of nucleic acid and polymer. Centrifuging for 1 minute
was then carried out. An additional amount of polymer (10 .mu.l of
the 2.4% stock solution) was added to each supernatant and the
resulting mixtures were vortexed and centrifuged to determine if
the first precipitation was quantitative. Table I below shows the
amount of polymer used and the type of precipitation observed for
each sample.
TABLE-US-00001 TABLE I Polymer First Second Volume Precipitation
Precipitation (.mu.l) Pellet Pellet 5 Barely Visible Large 10 Small
to medium Small 25 Large Not visible 50 Very large Not visible
[0123] It was observed that precipitation occurred under acidic
conditions (pH 2.3), and that 50 .mu.l of the 1:10 dilution of
polymer stock solution could be used to precipitate 100 .mu.l of
the calf thymus DNA solution (0.5 .mu.g/.mu.l) Sigma Chemical Co.,
St. Louis, Mo., in a nearly quantitative fashion. This observation
was also confirmed using conventional gel electrophoretic
methods.
[0124] Experiments were conducted to determine how to solubilize
the precipitate, thereby releasing the nucleic acid for later use.
Table II below shows the various pellet solubilization conditions
attempted and the resulting pellet size. The most useful technique
was the use of heat in combination with basic pH (no pellet).
Conventional gel electrophoresis clearly indicated that at basic
pH, the polymer and nucleic acids were present as free materials.
Thus, the nucleic acids were available for later use, such as in
PCR.
TABLE-US-00002 TABLE II Solubilizing Conditions Pellet Size 50
.mu.l NaCl (4 molar) None 50 .mu.l NaOH (50 mmolar) with heating at
Small 55.degree. C. for 5 minutes 50 .mu.l NaOH (100 mmolar) with
heating at None 55.degree. C. for 5 minutes 50 .mu.l NaOH (50
mmolar) with heating at None 100.degree. C. for 10 minutes 50 .mu.l
NaOH (25 mmolar) with heating at None 100.degree. C. for 10 minutes
50 .mu.l "TE" buffer* with heating at 100.degree. C. Large for 10
minutes 50 .mu.l water with heating at 100.degree. C. for 10 Large
minutes *"TE" buffer includes ethylenediaminetetraacetic acid (1
mmolar) in tris(hydroxymethyl))aminomethane hydrochloride buffer
(10 mmolar, pH 8)
[0125] Table III below shows the affect of pH on the formation of a
precipitate between the polymer (50 .mu.l of 1:10 dilution of stock
solution) and calf thymus DNA (100 .mu.l of 0.5 .mu.g/.mu.l
solution). Acidic pH was clearly required for effective capture of
the nucleic acid by formation of a precipitate (pellet).
TABLE-US-00003 TABLE III pH Pellet Size 2.3 Large 3 Large 4 Large 7
Clear, thick mass 12 Barely visible
Example 2
Comparison of Polymer Capture of DNA with and Without a Lysing
Reagent
[0126] The amount of DNA released from white blood cells contacted
with a lysing reagent (control) is compared with the amount
released from cells not contacted with a lysing reagent (method of
invention) but otherwise treated identically.
[0127] In this example, 10 mL of blood was drawn into a VACUTAINER
CPT cell Preparation Tube (Becton Dickinson Co., Franklin Lakes,
N.J.), and the white blood cells (WBC) were separated by means of
centrifugation according to the manufacturer's recommended
protocol. Final WBC concentration was determined to be
3.5.times.105/mL, based on microscopy.
[0128] Two hundred microliters of the WBC suspension was placed in
each of eight 1.5 mL microcentrifuge tubes (Eppendorf North
America, Inc., Madison, Wis.). The white blood cells were
centrifuged, and washed 3 times with phosphate buffered saline,
(PBS, 0.15 M NaCl, and 0.05 M potassium phosphate buffer, pH
7.5).
Control--Use of Lysing Reagent
[0129] For samples contacted with lysing reagent, the pellet in
each of four separate tubes was treated as follows: Eighty
microliters of lysis buffer (10 mM Tris HCl, pH 8.0, and 0.5% TWEEN
20) was added, followed by 10 .mu.L of the thermostable protease
Pre-Taq, (1 U/.mu.L, Boehringer Mannheim Biochemicals,
Indianapolis, Ind.), and the tubes were heated at 100.degree. C.
for 5 min. After heat treatment, 10 .mu.L of 250 mM NaOH was added,
and the tubes were again heated at 105.degree. C. for 10 min,
followed by centrifugation at 14,000 rpm for 2 min.
Method of Invention--No Use of Lysing Reagent
[0130] Samples not contacted with lysis reagent were treated as
follows: the pellet from each of four separate tubes was
resuspended in 100 .mu.L of PBS.
[0131] Samples prepared using both above-methods were processed
identically: the tubes were centrifuged at 14,000 rpm for 2 min,
the supernatant fluid from each tube was carefully decanted into
new tubes and stored at room temperature prior to analysis. DNA
content for each tube was analyzed using the TaqMan .beta.-actin
assay and an ABI Prism 7700 Sequence Detector as described above,
with calibration based on DNA standards purchased from Perkin
Elmer. The results are summarized in Table IV.
TABLE-US-00004 TABLE IV COMPARISON OF DNA RELEASED FROM WHITE BLOOD
CELLS WITH AND WITHOUT TREATMENT WITH LYSIS REAGENT # Cell
Treatment DNA ng/.mu.l Average 1 control 2.2 2.93 2 control 3.0 3
control 3.6 4 control 2.4 5 invention 0.006 0.01 6 invention 0.016
7 invention 0.011 8 invention 0.007
[0132] These data indicate that in the presence of lysis reagent,
there is approximately a 300-fold greater amount of DNA released
from the white blood cells. Since DNA released from white blood
cells is not expected to harbor mutations, deletions or other
specific cancer markers circulating in blood from a primary tumor,
such non-target related DNA increases non-specific background, and
therefore, has a deleterious effect on an assay for either free
circulating DNA in body fluids based on the detection of specific
alterations in DNA associated with cancer.
Example 3
Comparison of Invention with Qiagen Kit Method for Extracting DNA
from Serum
[0133] The following example demonstrates a comparison of the
commercial Qiagen kit and the method of the present invention for
extracting DNA from the same serum pool.
[0134] For the isolation of DNA from serum or plasma based on the
method of the invention, all initial steps were performed on ice to
minimize possible degradation of DNA by serum nucleases. ACES
buffer (N-(2-acetamido)-2-aminoethanesulfonic acid) from Sigma
Chemical Co., St. Louis, Mo. was prepared as a 250 mM stock
solution, pH 6.8. DNA capture polymer, poly(1-vinylimidazole
hydrochloride-co-2-hydroxyethylmethacrylate) at a 76:24 monomer
weight ratio and at 2.4% solids, was synthesized by protocols
described in U.S. Pat. No. 5,582,988. It is a random linear vinyl
addition co-polymer made using conventional solution
co-polymerization in N,N-dimethylformamide with an azo initiator.
The copolymer (or simply polymer) was mixed with an excess of water
and concentrated HCl was added until a clear solution was obtained.
The solution was then diafiltered.
[0135] Two hundred microliters of serum or plasma were added to a
1.5 mL microfuge-tube followed by the addition of 100 uL of the
ACES buffer stock. After mixing by means of a vortex mixer, 15 uL
of the aqueous capture polymer solution was added to the tube and
the sample was again mixed for 5 sec. using a vortex mixer. The
tube was centrifuged by means of a Eppendorf Microcentrifuge model
5415 (Brinkman Instruments, Westbury, N.Y.) at maximum speed for 2
min, and the supernatant fluid was decanted. One hundred
microliters of 20 mM NaOH was added to the tube containing the
pellet, and the tube was mixed by means of a Vortex mixer, followed
by heating at 100.degree. C. for 5 min. Samples were either
maintained at 4.degree. C. and assayed immediately following
extraction or stored frozen prior to use.
[0136] For comparison, DNA was also extracted from serum or plasma
using a QIAmp Blood Kit (cat 29104) from Qiagen Corp., Chatsworth,
Calif. according to the manufacturer's recommended procedure.
Buffers AL, AW and AE were provided in the kit. Two hundred
microliters of serum were combined with 200 uL of 0.05M potassium
phosphate buffer, pH 7.5, and 200 uL of Buffer AL and 25 uL of
Proteinase K solution (lysing reagent) provided in the kit and the
contents were immediately mixed for 15 seconds using a vortex
mixer. Following incubation at 70.degree. C. for 10 min, 210 uL of
ethanol was added, and the sample was again mixed using the vortex
mixer. DNA was extracted by means of a QIAamp spin column into a 2
mL collection tube. After applying the sample, the tube was
centrifuged at 6,000.times.g for 1 min. The tube containing the
filtrate was discarded. Five hundred microliters of Buffer AW was
added, and the column was again centrifuged for 1 min, and the tube
containing the filtrate was discarded. The column was washed an
additional time with buffer AW and DNA was then eluted from the
column with 200 uL of Buffer AE or distilled water preheated to
70.degree. C. After addition of the buffer or water, the tube was
incubated at room temperature for 1 min and then centrifuged at
6,000.times.g for 1 min.
[0137] A comparison of the steps in the Quiagen kit method and the
method of the present invention are shown in Table V. The Quiagen
kit requires at least 8 steps as compared with the method of the
invention, which requires 3 steps.
TABLE-US-00005 TABLE V COMPARISON OF STEPS INVOLVED IN DNA
EXTRACTION USING THE METHOD OF THE INVENTION AND QIAGEN METHOD
IzMn(76/24) Polymer Capture QIAGEN Kit 1 ACES Buffer addition 1 PBS
buffer addition 2 Polymer addition 2 QIAGEN Protease Treatment 3
DNA release by NaOH 3 Incubation at 70.degree. C. for 10 min. 4
Ethanol addition 5 Load QIAamp spin column and spin 6 Buffer wash
the column, 1 min spin 7 Buffer wash the column, 3 min spin 8
Buffer elute the column
[0138] A comparison of amplifiable .beta.-actin DNA as measured by
the TaqMan .beta.-actin protocol (8 replicates) is shown in Table
VI and indicates a 58% improvement in recoverable amplifiable DNA
using the method of the invention (60.6 ng/mL serum) as compared to
the Quiagen method (25.3 ng/mL serum).
TABLE-US-00006 TABLE VI COMPARISON OF .beta.-ACTIN DNA EXTRACTION
USING THE METHOD OF THE INVENTION AND QIAGEN METHOD Serum ngDNA/
DNA Vol DNA ngDNA/.mu.l ml Sample # Prep (.mu.l) ng/.mu. AVEG SDTEV
serum serum 1 Polymer 200 0.038 0.060625 0.016475 0.0606 60.63 2
Polymer 200 0.06 3 Polymer 200 0.06 4 Polymer 200 0.095 5 Polymer
200 0.051 6 Polymer 200 0.06 7 Polymer 200 0.068 8 Polymer 200
0.053 1 QIAGEN 200 0.032 0.02525 0.014945 0.0253 25.25 2 QIAGEN 200
0.013 3 QIAGEN 200 0.022 4 QIAGEN 200 0.025 5 QIAGEN 200 0.059 6
QIAGEN 200 0.016 7 QIAGEN 200 0.015 8 QIAGEN 200 0.02
Example 4
Recovery of .beta.-Actin DNA from Serum of Individuals Diagnosed
with Pancreatic Cancer and Controls Using the Method of the
Invention
[0139] This example provides the results of experiments providing a
quantitative measure of the amount of .beta.-Actin DNA recovered
from the serum of normal and pancreatic cancer patients using the
method of the invention in accordance with the materials and
procedures of Example 8 herein. .beta.-Actin DNA so recovered from
each sample was quantified using the TaqMan .beta.-actin protocol
described earlier.
[0140] As shown in Table VII, using the method of the invention a
total of 8 replicates from the same human serum pool yielded an
average of 12 ng of .beta.-Actin DNA/mL of serum, whereas
.beta.-Actin DNA in the serum of 10 different pancreatic cancer
patients recovered using the method of the invention was greatly
elevated (average=146 ng/mL). These findings of elevated
.beta.-Actin DNA in the serum of individuals having pancreatic
cancer as compared with that of normals are supported by several
reports in the literature (4,9).
TABLE-US-00007 TABLE VII .beta.-ACTIN DNA EXTRACTION FROM NORMAL
SERUM POOL AND SERUM FROM INDIVIDUALS AFFLICTED WITH PANCREATIC
CANCER USING THE METHOD OF THE INVENTION ngDNA/ Sample source
Quantity ngDNA/.mu.l ml Sample # 300 .mu.l ng/.mu.l AVEG SDTEV in
serum in serum 1 Human Serum 0.045 0.072875 0.049453 0.0121 12 2
Pool 0.056 3 0.19 4 0.056 5 0.072 6 0.032 7 0.078 8 0.054 1
Pancreatic 1 0.881875 1.07686 0.1470 146 2 Cancer Patient 0.33 3
Serum 0.16 4 0.88 5 0.69 6 0.075 7 0.32 8 0.43 9 3.4 10 1.1
Example 5
Comparison of the Method of the Invention and Qiagen Method for
Recovery of .beta.-Actin DNA from Serum of Individuals Diagnosed
with Pancreatic Cancer
[0141] In this example, .beta.-Actin DNA recovery from 6 patients
with confirmed pancreatic cancer was compared using the method of
the invention and the Qiagen method in accordance with the
materials and procedures of Example 3 herein. In general, as shown
in Table VIII, the method of the invention yielded either higher or
comparable levels of .beta.-Actin DNA as assayed by the TaqMan
.beta.-Actin assay. Depending upon the sample, measurable DNA
concentrations ranged from 31 to 310 ng/mL.
TABLE-US-00008 TABLE VIII COMPARISON OF .beta.-ACTIN DNA EXTRACTION
FROM SERUM OF INDIVIDUALS AFFLICTED WITH PANCREATIC CANCER USING
THE METHOD OF THE INVENTION AND QIAGEN METHOD Sample # Method ng
DNA/mL serum 1 Qiagen 263 1 Polymer Capture 260 2 Qiagen 95 2
Polymer Capture 102 3 Qiagen 88 3 Polymer Capture 235 4 Qiagen 310
4 Polymer Capture 275 5 Qiagen 31 5 Polymer Capture 38 6 Qiagen 57
6 Polymer Capture 65
Results are the average of 4 replicates per sample, except for
sample 1 and 2 which are the average of 6 replicates. Sample 2
evaluated with the Qiagen protocol is the average of 2
replicates.
Example 6
Isolation of Circulating DNA from Serum of Normal and Cancer
Patients Using the Method of the Invention
[0142] This example illustrates the utility of the method of the
invention for isolating circulating DNA from serum of 20 normals
and 30 individuals having a confirmed cancer diagnosis. Cancer
patient sera included 10 confirmed pancreatic cancer patients, and
20 colon cancer samples (8 Dukes B, 5 Dukes C, and 7 Dukes D). The
DNA was isolated according to the method of the invention as in the
procedure described in Example 2 herein. DNA was quantified using
the TaqMan .beta.-actin assay. Polymer capture without use of a
lysing reagent enabled circulating DNA to be concentrated with
minimal or no contamination with DNA from undesirable cell lysis
and removal of PCR interferences that may be present in serum. DNA
in each serum was quantified by means of the TaqMan assay for the
.beta.-actin gene using the standard curve shown herein in FIG.
1.
[0143] The results of analyses for free circulating DNA in each
sample are shown in Tables IX A and IX B and indicate that DNA
levels are elevated in serum from cancer patients compared with the
serum from normal individuals.
TABLE-US-00009 TABLE IX A DNA CONTENT IN THE SERUM OF CANCER
PATIENTS Average # D.S support # Diagnosis DNA ng/.mu.l ng/ml ng/ml
1 139980708 Pancreatic 0.035 17.5 26.4 2 310980084 Pancreatic 0.024
12 3 310980107 Pancreatic 0.02 10 4 310980130 Pancreatic 0.017 8.5
5 310980153 Pancreatic 0.029 14.5 6 310980176 Pancreatic 0.037 18.5
7 1111980333 Pancreatic 0.22 110 8 2510980006 Pancreatic 0.043 21.5
9 2510980012 Pancreatic 0.054 27 10 2510980017 Pancreatic 0.049
24.5 11 139980709 Dukes B 0.17 85 135.5 12 1110980326 Dukes B 0.14
70 13 1110980328 Dukes B 0.1 50 14 1110980332 Dukes B 0.11 55 15
2410980054 Dukes B 0.03 15 16 2410980059 Dukes B 0.048 24 17
2611980009 Dukes B 1.4 700 18 2611980018 Dukes B 0.17 85 19
139980701 Dukes C 0.011 5.5 100.13 20 1110980312 Dukes C 0.21 105
21 1110980319 Dukes C 0.39 195 22 1110980324 Dukes C 0.19 95 23
2411980086 Dukes C N.D. N.D. 24 310980121 Dukes D 0.05 25 66.08 25
310980144 Dukes D 0.055 27.5 26 310980190 Dukes D 0.051 25.5 27
2411980074 Dukes D 0.057 28.5 28 2611980003 Dukes D 0.093 46.5 29
2611980004 Dukes D 0.05 25 30 2611980012 Dukes D 0.61 305
TABLE-US-00010 TABLE IX B DNA CONTENT IN SERUM OF NORMALS DNA #
Unit # ng/2 .mu.l ng/ml 1 M58234 ND* ND 2 M58088 ND ND 3 M58089
0.0510 6.38 4 M58090 ND ND 5 M58091 ND ND 6 M58092 0.0300 3.75 7
M58093 ND ND 8 M58094 0.0190 2.38 9* M58095 0.0360 4.50 10 M58111
0.0400 5.00 11 M58112 0.0370 4.63 12 M58113 ND ND 13 M58115 0.0310
3.88 14 M58116 ND ND 15 M58118 0.0230 2.88 16 M58120 0.1200 15.00
17 M58121 0.0390 4.88 18 M58124 0.0190 2.38 19 M58126 0.0590 7.38
20* M58128 0.0290 3.63 *ND = below detection limit
Example 7
Detection of K-ras Mutations in the Serum of Pancreatic Cancer
Patients
[0144] In this example, an embodiment of the invention involving
polymer capture of DNA from the serum of pancreatic cancer patients
was employed. Restriction endonuclease mediated selective PCR (REMS
PCR)was performed (Roberts, N. J. et al., 1999, BioTechniques
27:(3)418-422, Ward, R. et al., 1998, Am. J. Pathol.
153(2):373-379, and WO96/32500) followed by gel analysis was used
to detect the presence of a K-ras mutation at codon 12
(K12-ras).
[0145] Serum or plasma (300 uL) from each of 3 pancreatic cancer
patients was added to separate microfuge tubes, followed by
addition of 100 uL of 250 mM ACES
(N-(2-Acetamido)-2-aminoethanesulfonic acid) buffer (pH 6.8 at
23.degree. C.). Fifteen microliters (15 uL) of polymer poly
(1-vinylimidazole-co-2-hydroxyethyl methycrylate (weight ratio
77/23) was added (see U.S. Pat. Nos. 5,434,270; 5,523,368. and
5,582,988) and the tubes were mixed by means of a Mini Vortexer
(VWR Scientific, Rochester, N.Y.) for 10 seconds. The tubes were
then centrifuged in an Eppendorf Microcentrifuge, Model 5415, at
maximum speed for 2 min. The supernatant fluid was decanted and 100
uL of 20 mM sodium hydroxide was added to each tube, and the pellet
was resuspended by mixing and heated to 100.degree. C. for 10
min.
[0146] Each PCR admixture contained three sets of primers. The
diagnostic primers induce a Bstnl restriction site in wild-type
ras, but not in a mutation at ras codon 12. Thus, ras wild-type DNA
is selectively cleaved during PCR thermocycling, and mutant
sequences of ras at codon 12 are enriched. The PCR control primer
pair is used to confirm that PCR amplifiable DNA has been
extracted, and the enzyme control primer pair confirms that the
restriction enzyme functioned during thermocycling. Reaction
admixtures contained 12 units/100RL of recombinant Taq polymerase,
and a 5-fold excess by weight (0.842FLL) of Taq inhibiting antibody
TP4-9.2 (see U.S. 15 U.S. Pat. Nos. 5,338,671 and 5,587,287) over
the polymerase, 1 mM HT50 buffer (100 mM sodium chloride, and 50 mM
Tris (tris(hydroxymethyl)amino methane), pH 8.3, 0.3ELM of
diagnostic primers (see below), 5K15S (SEQ ID: NO 1) and 5K37 (SEQ
ID: NO 2), 0.05 pM of PCR control primer pairs, 3K42 (SEQ ID: NO 3)
and 5BK5 (SEQ ID: NO 4), 0.1.about.LM of enzyme control primer
pairs, 5N 12A (SEQ ID: NO 5) and 3N13A (SEQ ID: NO 6), 0.2 mM total
dinucleoside triphosphates (dNTPs), 0.3 units/.about.LL of Bsll
(New England BioLabs, Beverly Mass.), 1 mm dithiothreitol (DTT), 5
mM magnesium chloride, sample (typically 3.about.tL) and deionized
water up to a final volume of 100 gL. The Taq polymerase and
anti-Taq antibodies were combined and incubated for 10-15 minutes
prior to the addition of the other PCR components. Thermocycling
parameters were as follows: 1 cycle at 94.degree. C. for 100 sec.,
and 36 cycles at 92.degree. C. for 15 sec, and 60.degree. C. for 60
sec. The primer sequences are as follows:
Sequence CWU 1
1
6128DNAHomo sapiens 1tgaatataaa cttgtggtac ctggagct 28228DNAHomo
sapiens 2atataaactt gtggtagttc cagctggt 28326DNAHomo sapiens
3gaattagctg tatcgtcaag gcactc 26421DNAHomo sapiens 4tcagcaaaga
caagacaggt a 21526DNAHomo sapiens 5tatagatggt gaaacctgtt tgttgg
26629DNAHomo sapiens 6cttgctatta ttgatggcaa ccacacaga 29
* * * * *