U.S. patent application number 10/232971 was filed with the patent office on 2003-03-20 for isolation of nucleic acids.
Invention is credited to Baker, Matthew John.
Application Number | 20030054395 10/232971 |
Document ID | / |
Family ID | 24960633 |
Filed Date | 2003-03-20 |
United States Patent
Application |
20030054395 |
Kind Code |
A1 |
Baker, Matthew John |
March 20, 2003 |
Isolation of nucleic acids
Abstract
A method for extracting nucleic acids from a biological material
such as blood comprises contacting the mixture with a material at a
pH such that the material is positively charged and will bind
negatively charged nucleic acids and then eluting the nucleic acids
at a pH when the said materials possess a neutral or negative
charge to release the nucleic acids The nucleic acids can be
removed under mildly alkaline conditions to the maintain integrity
of the nucleic acids and to allow retrieval of the nucleic acids in
reagents that are immediately compatible with either storage or
analytical testing.
Inventors: |
Baker, Matthew John;
(Maidstone, GB) |
Correspondence
Address: |
DANN DORFMAN HERRELL & SKILLMAN
SUITE 720
1601 MARKET STREET
PHILADELPHIA
PA
19103-2307
US
|
Family ID: |
24960633 |
Appl. No.: |
10/232971 |
Filed: |
August 30, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10232971 |
Aug 30, 2002 |
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09736632 |
Dec 14, 2000 |
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09736632 |
Dec 14, 2000 |
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09586009 |
Jun 2, 2000 |
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09586009 |
Jun 2, 2000 |
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PCT/GB98/03602 |
Dec 4, 1998 |
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Current U.S.
Class: |
435/6.16 ;
435/270; 536/25.4 |
Current CPC
Class: |
C12N 15/1013 20130101;
C12N 15/1003 20130101; C12N 15/1006 20130101; C12N 15/101
20130101 |
Class at
Publication: |
435/6 ; 435/270;
536/25.4 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 001/08 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 6, 1997 |
GB |
9725839.6 |
Jul 17, 1998 |
GB |
9815541.9 |
Claims
1. A method for extracting nucleic acid from a sample containing
nucleic acid, which method comprises: at a first pH, bringing the
sample into contact with a material which comprises an ionisable
group, wherein the material has a positive charge at said first pH,
such that nucleic acid is bound to the material; and releasing the
nucleic acid at a second, higher, pH at which the charge on the
material is negative, neutral or less positive, wherein the release
of the nucleic acid occurs under mild conditions.
2. A method according to claim 1, wherein the mild conditions are
conditions at which said nucleic acid is not denatured and/or not
degraded and/or not depurinated and/or substantially physiological
conditions.
3. A method according to claim 1, wherein the releasing step occurs
at a pH of no more than about 10.5, preferably no more than about
9.0.
4. A method according to claim 1, wherein the releasing step occurs
at an ionic strength of no more than about 500 mM, preferably no
more than about 100 mM.
5. A method according to claim 1, wherein the releasing step occurs
at a temperature of no more than about 70.degree. C., preferably no
more than about 50.degree. C.
6. A method according to claim 5, wherein the releasing step occurs
at about room temperature.
7. A method according to claim 1, wherein the releasing step
comprises contacting the bound nucleic acid with a buffer solution
to release the nucleic acid, the buffer solution being suitable for
the storage or further processing of the released nucleic acid.
8. A method according to claim 7, wherein the buffer solution is a
buffer solution suitable for PCR.
9. A method according to claim 1, wherein the pKa of said ionisable
group is between about 3.0 and 9.0, preferably between about 4.0
and 9.0.
10. A method according to claim 9, wherein the material comprises a
positively ionisable group, the pKa of which is between about 5.0
and 8.0, preferably between about 6.0 and 7.0.
11. A method according to claim 10, wherein the material comprises
a weak base.
12. A method according to claim 10, wherein the material comprises
a biological buffer.
13. A method according to claim 10, wherein the material comprises
a positively ionisable nitrogen atom and one or more
electronegative groups capable of lowering the pKa of the
positively ionisable nitrogen atom.
14. A method according to claim 10, wherein the material comprises
a chemical species selected from the group consisting of:
N-2-acetamido-2-aminoethanesulfonic acid (ACES);
N-2-acetamido-2-iminodia- cetic acid (ADA); amino methyl
propanediol (AMP); 3-1,1-dimethyl-2-hydroxy- ethylamino-2-hydroxy
propanesulfonic acid (AMPSO); N,N-bis-2-hydroxyethyl--
2-aminoethanesulfonic acid (BES); N,N-bis-2-hydroxyethylglycine
(BICINE); bis-2-hydroxyethyliminotrishydroxymethylmethane
(Bis-Tris); 1,3-bistrishydroxymethylmethylaminopropane (Bis-Tris
Propane); 4-cyclohexylamino-1-butane sulfonic acid (CABS);
3-cyclohexylamino-1-prop- ane sulfonic acid (CAPS);
3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO);
2-N-cyclohexylaminoethanesulfonic acid (CHES);
3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid
(DIPSO); N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid
(EPPS); N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS);
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES);
N-2-hydroxyethylpiperazine-N-2-propanesulfonic acid (HEPPSO);
2-N-morpholinoethanesulfonic acid (MES);
4-N-morpholinobutanesulfonic acid (MOBS);
3-N-morpholinopropanesulfonic acid (MOPS);
3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO);
piperazine-N-N-bis-2-ethanesulfonic acid (PIPES);
piperazine-N-N-bis-2-hy- droxypropanesulfonic acid (POPSO);
N-trishydroxymethyl-methyl-4-aminobutan- esulfonic acid (TABS);
N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS);
3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid
(TAPSO); N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid
(TES); N-trishydroxymethylmethylglycine (TRICINE);
trishydroxymethylaminomethane (Tris); polyhydroxylated amines;
histidine, and polyhistidine; imidazole, and derivatives thereof
(i.e. imidazoles), especially derivatives containing hydroxyl
groups; triethanolamine dimers and polymers; and di/tri/oligo amino
acids, for example; Ala-Ala; Gly-Gly, pKa 8.2; Ser-Ser;
Gly-Gly-Gly, Ser-Gly; a detergent, such as decylmethylimidazole or
dodecyl-Bis-Tris; a carbohydrate containing nitrogen and
electronegative groups, such as a glucosamine, a polyglucosamine
(e.g. a chitosan), a kanamycin or derivative thereof; a nucleic
acid base, such as cytidine; and a monomeric, oligomeric or
polymeric compound containing an aliphatic or aromatic
nitrogen-containing heterocyclic ring, such as morpholine-,
pyrrole-, pyrrolidine-, pyridine-, pyridinol-, pyridone-,
pyrroline-, pyrazole-, pyridazine-, pyrazine-, piperidone-,
piperidine-, or piperazine-containing compounds, e.g.
polyvinylpyridlne, said ring optionally being substituted with one
or more electronegative groups.
15. A method according to claim 14, wherein the chemical species is
selected from the group consisting of: Tris; Bis-Tris; Bis-Tris
Propane; Tricine; Bicine; polyhydroxylated amines; and
polyhistidine.
16. A method according to claim 9, wherein the material comprises:
a negatively ionisable group, the pKa of which is between about 3.0
and 7.0; and a group which is positively charged at said first pH,
and optionally also at said second pH.
17. A method according to claim 16 wherein said negatively
ionisable group is a carboxy group.
18. A method according to claim 16 wherein said group which is
positively charged is a metal oxide, such as iron II,III oxide.
19. A method according to claim 1, wherein the material comprises
an ionisable group having a pKa value, said pKa value being between
the first and second pH, or within about 1.0 pH unit, preferably
within about 0.5 pH unit, below said first pH.
20. A method according to claim 19, wherein said second pH is
within about 3 pH units, preferably within about 2 pH units, above
the pKa value.
21. A method according to claim 1, wherein the method is for
separating single stranded nucleic acid from double stranded
nucleic acid.
22. A method according to claim 1, wherein the method is for
extracting single stranded nucleic acid, said method comprising a
prior step of converting double stranded nucleic acid into single
stranded nucleic acid.
23. A method according to claim 1, wherein the material is a solid
phase material.
24. A method according to claim 1, wherein the binding step occurs
in a solution having a concentration of 1M or less.
25. A solid phase product for use in a method of extracting nucleic
acid from a sample, the product comprising a plurality of
positively ionisable groups, the ionisable groups being provided by
a chemical species selected from the list consisting of: biological
buffers, polyhydroxylated amines; histidine; and polyhistidine.
26. A product according to claim 25 wherein the biological buffer
is selected from the group consisting
N-2-acetamido-2-aminoethanesulfonic acid (ACES);
N-2-acetamido-2-iminodiacetic acid (ADA); amino methyl propanediol
(AMP); 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic
acid (AMPSO); N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid
(BES); N,N-bis-2-hydroxyethylglycine (BICINE);
bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris);
1,3-bistrishydroxymethylmethylaminopropane (Bis-Tris Propane);
4-cyclohexylamino-1-butane sulfonic acid (CABS);
3-cyclohexylamino-1-prop- ane sulfonic acid (CAPS);
3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO);
2-N-cyclohexylaminoethanesulfonic acid (CHES);
3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid
(DIPSO); N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid
(EPPS); N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS);
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES);
N-2-hydroxyethylpiperazine-N-2-propanesulfonic acid (HEPPSO);
2-N-morpholinoethanesulfonic acid (MES);
4-N-morpholinobutanesulfonic acid (MOBS);
3-N-morpholinopropanesulfonic acid (MOPS);
3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO);
piperazine-N-N-bis-2-ethanesulfonic acid (PIPES);
piperazine-N-N-bis-2-hy- droxypropanesulfonic acid (POPSO);
N-trishydroxymethyl-methyl-4-aminobutan- esulfonic acid (TABS);
N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS);
3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid
(TAPSO); N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid
(TES); N-trishydroxymethylmethylglycine (TRICINE);
trishydroxymethylaminomethane (Tris); polyhistidine;
polyhydroxylated imidazoles; triethanolamine dimers and polymers;
and di/tri/oligo amino acids, for example Gly-Gly, Ser-Ser,
Gly-Gly-Gly, and Ser-Gly.
27. A product according to claim 25, wherein the plurality of
ionisable groups are separately immobilised on a solid support by
covalent or ionic bonding or by adsorption.
28. A product according to claim 25, wherein the plurality of
ionisable groups are separately attached to a polymer, said polymer
being immobilised on a solid support by covalent or ionic bonding
or by adsorption.
29. A product according to claim 25, wherein the ionisable groups
are polymerized, optionally by means of cross-linking reagents.
30. A product according to claim 29, wherein the polymer is
immobilised on a solid support by covalent or ionic bonding or by
adsorption.
31. A product according to claim 29, wherein the polymer is a
solid.
32. A product according to claim 29 which is a container.
33. A container according to claim 32 which is a PCR or storage
tube or well, or a pipette tip.
34. A water soluble product for use in a method of extracting
nucleic acid from a sample, the product comprising a plurality of
positively ionisable groups, the ionisable groups being provided by
a chemical species selected from the list consisting of: biological
buffers; polyhydroxylated amines; histidine; and polyhistidine.
35. A product according to claim 34 wherein the biological buffer
is selected from the group consisting of:
N-2-acetamido-2-aminoethanesulfoni- c acid (ACES);
N-2-acetamido-2-iminodiacetic acid (ADA); amino methyl propanediol
(AMP); 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic
acid (AMPSO); N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid
(BES); N,N-bis-2-hydroxyethylglycine (BICINE);
bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris);
1,3-bistrishydroxymethylmethylaminopropane (Bis-Tris Propane);
4-cyclohexylamino-1-butane sulfonic acid (CABS);
3-cyclohexylamino-1-prop- ane sulfonic acid (CAPS);
3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO);
2-N-cyclobexylaminoethanesulfonic acid (CHES);
3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid
(DIPSO); N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid
(EPPS); N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS);
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES);
N-2-hydroxyethylpiperazine-N-2-propanesulfonic acid (HEPPSO);
2-N-morpholinoethanesulfonic acid (MES);
4-N-morpholinobutanesulfonic acid (MOBS);
3-N-morpholinopropanesulfonic acid (MOPS);
3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO);
piperazine-N-N-bis-2-ethanesulfonic acid (PIPES);
piperazine-N-N-bis-2-hy- droxypropanesulfonic acid (POPSO);
N-trishydroxymethyl-methyl-4-aminobutan- esulfonic acid (TABS);
N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS);
3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid
(TAPSO); N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid
(TES); N-trishydroxymethylmethylglycine (TRICINE);
trishydroxymethylaminomethane (Tris); polyhistidine;
polyhydroxylated imidazoles; triethanolamine dimers and polymers;
and di/tri/oligo amino acids, for example Gly-Gly, Ser-Ser,
Gly-Gly-Gly, and Ser-Gly.
36. A product according to claim 34, wherein the plurality of
ionisable groups are separately attached to a polymer.
37. A product according to claim 34, wherein the ionisable groups
are polymerised, optionally by means of cross-linking reagents.
38. A product for use in a method of extracting nucleic acid from a
sample, wherein the product possesses a positive charge at both a
first pH at which it is desired to bind nucleic acid and a second
higher pH at which it is desired to release nucleic acid, the
product comprising a plurality of negatively ionisable groups, the
combined charge of which becomes more negative between said first
pH and said second pH, such that the product is capable of binding
nucleic acid at said first pH, which bound nucleic acid is released
from the product at said second pH.
39. A product according to claim 38, wherein the negatively
ionisable group has a pKa between about 3 and 7, preferably between
about 4 and 7.
40. A product according to claim 38, wherein the negatively
ionisable is a carboxy group.
41. A product according to claim 38 wherein said positive charge is
provided by a metal or metal oxide, preferably iron II,III oxide.
Description
[0001] This application is a continuation-in-part of U.S. Ser. No.
09/586,009, filed Jun. 2, 2000, which derives from PCT/GB98/03602,
filed Dec. 4, 1998, which claims priority from UK patent
application numbers 9725839.6, filed Dec. 6, 1997, and 9815541.9,
filed Jul. 17, 1998. The entire disclosure of the '009 application
is incorporated by reference herein. The present invention relates
to a method for extracting nucleic acids and other biomolecules
from biological materials, particularly blood and other liquid
samples.
[0002] There is a very large demand for DNA analysis for a range of
purposes and this has lead to the requirement for quick, safe, high
throughput methods for the isolation and purification of DNA and
other nucleic acids.
[0003] Samples for use for DNA identification or analysis can be
taken from a wide range of sources such as biological material such
as animal and plant cells, faeces, tissue etc. also samples can be
taken from soil, foodstuffs, water etc.
[0004] Existing methods for the extraction of DNA include the use
of phenol/chloroform, salting out, the use of chaotropic salts and
silica resins, the use of affinity resins, ion exchange
chromatography and the use of magnetic beads. Methods are described
in U.S. Pat. Nos. 5,057,426, 4,923,978, EP Patents 0512767 A1 and
EP 0515484B and WO 95/13368, WO 97/10331 and WO 96/18731. These
patents and patent applications disclose methods of adsorbing
nucleic acids on to a solid support and then isolating the nucleic
acids. The previously used methods use some type of solvent to
isolate the nucleic acids and these solvents are often flammable,
combustible or toxic.
[0005] EP0707077A2 describes a synthetic water soluble polymer to
precipitate nucleic acids at acid pH and release at alkaline pH.
The re-dissolving of the nucleic acids is performed at extremes of
pH, temperature and/or high salt concentrations where the nucleic
acids, especially RNA, can become denatured, degraded or require
further purification or adjustments before storage and
analysis.
[0006] WO 96/09116 discloses mixed mode resins for recovering a
target compounds especially a protein, from aqueous solution at
high or low ionic strength, using changes in pH. The resins have a
hydrophobic character at the pH of binding of the target compound
and a hydrophilic and/or electrostatic character at the pH of
desorption of the target compound.
[0007] Blood is one of the most abundant sample sources for DNA
analysis as blood samples are routinely taken for a wide range of
reasons. However because of the viscous and proteinaceous nature of
blood using known DNA extraction methods it has proved difficult to
accomplish using automation due to the difficulties of handling
large volumes containing relatively small amounts of DNA. Hitherto
nucleic acid extraction has been partially automated only by using
hazardous reagents and slow processing times.
[0008] I have now devised an improved method for the extraction of
nucleic acids and other biomolecules from blood and other
biological materials, and other samples containing nucleic acid
[0009] According to the invention there is provided a method for
the extraction of biomolecules from biological material which
method comprises contacting the biological material with a solid
phase which is able to bind the biomolecules to it at a first pH
and then extracting the biomolecules bound to the solid phase by
elution using an elution solvent at a second pH.
[0010] In particular there is provided a method for extracting
nucleic acid from a sample containing nucleic acid, which method
comprises: contacting the sample with said solid phase at a first
pH at which the solid phase has a positive charge and will bind
negatively charged nucleic acid; and then releasing the nucleic
acid at a higher pH at which the solid phase possesses a neutral,
negative or less positive charge than at the first pH.
[0011] Generally the solid phase will possess an overall positive
charge, that is the sum of all positive and negative charges on the
solid phase as a whole is positive. It is possible (though not
preferred), however, that the solid phase as a whole could be
negatively charged, but have areas of predominantly positive charge
to which the nucleic acid can bind. Such solid phases are within
the scope of the invention.
[0012] The change in the charge of the solid phase is referred to
herein as "charge switching" and is accomplished by the use of a
"charge switch material" in, on or as the solid phase.
[0013] The charge switch material comprises an ionisable group,
which changes charge to according to the ambient conditions. The
charge switch material is chosen so that the pKa of the ionisable
group is appropriate to the conditions at which it is desired to
bind nucleic acid to and release nucleic acid from the solid phase.
Generally, nucleic acid will be bound to the charge switch material
at a pH below or roughly equal to the pKa, when the charge switch
material is positively charged, and will be released at a higher pH
(usually above the pKa), when the charge switch material is less
positively charged, neutral, or negatively charged.
[0014] The present invention is more particularly directed to the
use of charge switch materials which allow binding and/or releasing
(especially releasing) of the nucleic acid to occur under mild
conditions of temperature and/or pH and/or ionic strength.
[0015] Generally the charge switch material will change charge
because of a change in charge on a positively ionisable group from
positive to less positive or neutral, as the pH is increased in a
range spanning or close to the pKa of the positively ionizable
group. This may also be combined with a change of charge on a
negatively ionisable group from neutral or less negative to more
negative. In an alternative embodiment (described below), however,
the charge switch material comprises a material which is positively
charged at both pH values (such as a metal oxide) and a negatively
ionisable group, the charge of which becomes more negative as the
pH is increased in a range spanning or close to its pKa.
[0016] The charge switch material may comprise an ionisable group
having a pKa between about 3 and 9. For positively ionisable
groups, the pKa is more preferably at least about 4.5, 5.0, 5.5,
6.0 or 6.5 and/or at most about 8.5, 8.0, 7.5 or 7.0. A
particularly preferred pKa for a positively ionisable group is
between about 5 and 8; even more preferred is a pKa between about
6.0 and 7.0, more preferably between about 6.5 and 7.0. The pKa for
negatively ionisable groups is preferably between about 3 and 7,
still more preferably between about 4 and 6, further preferably
approximately at the pH at which it is desired to bind nucleic
acid.
[0017] Materials having more than one pKa value (e.g. having
different ionisable groups), or combinations of materials having
different pKa values, may also be suitable for use as charge switch
materials in accordance with the invention, provided that at a
first (lower) pH the material(s) possess(es) a positive charge and
that at a higher pH the charge is less positive, neutral or
negative.
[0018] Generally a charge switch will be achieved by changing the
pH from a value below to a value above the pKa of the or an
ionisable group. However, it will be appreciated that when the pH
is the same as the pKa value of a particular ionisahle group, 50%
of the individual ionisable groups will be charged and 50% neutral.
Therefore, charge switch effects can also be achieved by changing
the pH in a range close to, but not spanning, the pKa of an
ionisable group. For example, at the pKa of a negatively ionisable
group, such as a carboxy group (pKa typically around 4), 50% of
such groups will be in the ionised form (e.g. COO.sup.-) and 50% in
the neutral form (e.g. COOH). As the pH increases, an increasing
proportion of the groups will be in the negative form.
[0019] Preferably the binding step is carried out at a pH of below
the pKa of the ionisable group, or (though this is not preferred)
within about 1 pH unit above the pKa. Generally the releasing step
is carried out at a pH above the pKa of the ionisable group,
preferably at a pH between 1 and 3 pH units above the pKa.
[0020] Prior art methods, such as those disclosed in EP0707077,
often use high pH to release the nucleic acid, for example using
strong bases such as NaOH. Such high pH can cause depurination of
nucleic acid, leading to the problems of imperfect replication,
which can impede subsequent use of the nucleic acid, e.g. in
detection and/or amplification techniques such as Southern or
northern blotting or PCR.
[0021] The use of strong bases, or weak bases in combination with
heating, again as in EP0707077, can also lead to degradation of RNA
(especially at pH values of 10 or above), and denaturation of
double stranded DNA (i.e. irreversible conversion of DNA from the
double stranded form at least partially into the single stranded
form), which can lead to a lack of specific binding in PCR.
[0022] The appropriate choice of pKa value(s) in accordance with
the invention allows the step of releasing DNA from the solid phase
to be performed under mild conditions, unlike in the prior art. As
used herein, the term "mild conditions" generally means conditions
under which nucleic acid is not denatured and/or not degraded
and/or not depurinated, and/or conditions which are substantially
physiological.
[0023] Preferably the releasing step is performed at a pH of no
greater than about pH 10.5, more preferably no greater than about
pH 10.0, 9.8, 9.6, 9.4, 9.2, 9.0, 8.9, 8.8, 8.7, 8.6 or 8.5.
Depending on the pKa(s) of the charge switch material, the
releasing step may even be performed at lower pH values, such as
8.0, 7.5 or 7.0. Preferably the releasing step is carried out in
the substantial absence of NaOH, preferably also the substantial
absence of other alkali metal hydroxides, more preferably the
substantial absence of strong mineral bases. Substantial absence
may mean that the concentration is less than 25 mM, preferably less
than 20 mM, more preferably, less than 15 mM or 10 mM.
[0024] The desired change in pH can be achieved by altering the
ionic strength of the solution and/or the temperature, since pH is
dependent on both these factors. However, neither high temperature
nor high ionic strength are generally compatible with the desired
mild conditions, and accordingly, the change in pH is preferably
not achieved by large changes in ionic strength or temperature.
Moreover, increasing ionic strength increases competition of
charged species with the nucleic acid for binding to the solid
phase, so can assist in releasing the nucleic acid. Small changes
of ionic strength are therefore acceptable and may be used in
conjunction with the change in pH to release the nucleic acid,
preferably within the limits and ranges given below.
[0025] Preferably the temperature at which the releasing step
performed is no greater than about 70.degree. C., more preferably
no greater than about 65.degree. C., 60.degree. C., 55.degree. C.,
50.degree. C., 45.degree. C. or 40.degree. C. More preferably, such
temperatures apply to the entire process. The releasing step, or
the entire process, may even be performed at lower temperatures,
such as 35.degree. C., 30.degree. C. or 25.degree. C.
[0026] Furthermore, the releasing step preferably occurs under
conditions of low ionic strength, suitably less than 1M or 500 mM,
preferably less than 400 mM, 300 mM, 200 mM, 100 mM, 75 mM, 50 mM,
40 mM, 30 mM, 25 mM, 20 mM or 15 mM. It may even be below 10 mM.
The ionic strength may be at least about 5 mM, more preferably at
least about 10 mM. More preferably, these ionic strengths also
apply to the binding step.
[0027] PCR is sensitive to pH and the presence of charged
contaminants. In particularly preferred embodiments, the releasing
step is performed using reagents suitable for storing nucleic acid
(such as a commercially available storage buffer, e.g. 10 mM
Tris.HCl, pH8.0-8.5, optionally in the presence of 1 mM EDTA), or
using reagents suitable for use in a procedure to which the nucleic
acid is to be subjected (such as a PCR buffer, e.g. 10 mM Tris.HCl,
50 mM KCl, pH 8.5).
[0028] Common previously known nucleic acid extraction processes
require a step of diluting the elution product containing nucleic
acid, to make the solution suitable for e.g. PCR. Preferably the
present invention substantially avoids diluting the released
nucleic acid.
[0029] Preferably the step of binding DNA occurs under mild
conditions, suitably at a pH of no less than 3.0, preferably no
less than 3.5, 4.0, 4.5 or 5.0. Previous methods have used high
concentrations of chaotropic agents, such as 8M guanidine. Such
conditions may not be necessary in the practice of the present
invention, in which the binding step preferably occurs in solution
having a total concentration of 1M or less. More preferred
temperatures and ionic strengths are as detailed above for the
releasing step.
[0030] The use of such mild conditions for the release of nucleic
acid is especially useful for extracting small quantities of
nucleic acid, as the extracted DNA or RNA can be added directly to
a reaction or storage tube without further purification steps (e.g.
steps necessitated by the use of high ion concentrations in prior
art methods), and without the need to dilute high ionic strength
(as is the case with prior art methods using high ionic strength to
elute the nucleic acid). Therefore loss of nucleic acid through
changing the container, imperfect recovery during purification
steps, degradation, or denaturation, and dilution of small amounts
of nucleic acid can be avoided. This is particularly advantageous
when a nucleic acid of interest is present in a sample (or is
expected to be present) at a low copy number, such as in certain
detection and/or amplification methods.
[0031] Broadly speaking, preferred chemical species for use as
charge switch materials in accordance with the invention comprise a
positively ionisable nitrogen atom, and at least one, but
preferably more than one, electronegative group (such as a hydroxy,
carboxy, carbonyl, phosphate or sulphonic acid group) or double
bond (e.g. C.dbd.C double bond), which is sufficiently close to the
nitrogen atom to lower its pKa. It has been found that such
molecules tend to have suitable pKa values for the extraction of
nucleic acid under mild conditions according to the present
invention. Preferably at least one (but more preferably more than
one) electronegative group is separated from the ionisable nitrogen
by no more than two atoms (usually carbon atoms). Hydroxyl groups
are particularly preferred electronegative groups (particularly
when several hydroxyl groups are present, e.g. in polyhydroxyl
amines, such as Tris (C(CH.sub.2OH).sub.3--NH.sub.2) or Bis-Tris
(see below)), as they (1) lower the pKa of the nitrogen atom (e.g
amine group, e.g. from about 10 or 11) to a suitable value around
neutral (i.e. pKa of about 7), (2) allow the species to remain
soluble/hydrophilic above the pKa, when the nitrogen atom of the
amine group loses its positive charge, (3) provide a site for
covalent linkage to a solid substrate, e.g. a polycarboxylated
polymer (such as polyacrylic acid), and (4) are uncharged at pH
values suitable for the releasing step and at which procedures such
as PCR are performed (typically pH 8.5); the presence of charged
species can interfere with PCR especially. Especially preferred are
chemical species having an ionisable nitrogen atom and at least 2,
3, 4, 5 or 6 hydroxyl groups.
[0032] Many standard, weakly basic, buffers are ideal chemical
species to provide the ionisable groups of charge switch materials,
as they have pKa values close to neutral (i.e. 7).
[0033] For use as a charge switch material, chemical species
comprising ionisable groups can be immobilised onto solid supports
(e.g. beads, particles, tubes, wells, probes, dipsticks, pipette
tips, slides, fibers, membranes, papers, celluloses, agaroses,
glass or plastics) in a monomeric or polymeric form via adsorption,
ionic or covalent interactions, or by covalent attachment to a
polymer backbone which is in turn immobilised onto the solid
support. Alternatively, they can be incorporated into solid,
insoluble forms (with or without attachment to a polymer backbone)
which inherently exhibit charge switching, e.g. beads, particles,
tubes, wells, probes, dipsticks, pipette tips, slides, fibers,
membranes or plastics.
[0034] Solid phase materials, especially beads and particles, may
be magnetisable, magnetic or paramagnetic. This can aid removal of
the solid phase from a solution containing the released nucleic
acid, prior to further processing or storage of the nucleic
acid.
[0035] Preferably the weakly basic buffers are biological buffers,
i.e. buffers from the class of buffers commonly used in biological
buffer solutions. Examples of biological buffers may be found in
commercial chemical catalogues, such as the Sigma catalogue.
[0036] Leaching (i.e. transfer from the solid phase into solution
in the liquid phase) of chemical species used to provide ionisable
groups in ion exchange resins is a virtually inevitable phenomenon
to some extent, especially when the species are attached to the
solid phase by adsorption. Such leaching typically causes impurity
in the resultant product, which can lead to significant problems,
particularly if the resultant product is intended to be used in PCR
(and especially when the species are charged). The use of
biological buffers to provide the ionisable groups in charge switch
materials can avoid this problem, since leaching of such buffers
into the liquid phase will generally not significantly affect the
nucleic acid, nor any downstream processes such as PCR to which it
might be subjected. Indeed, many biological buffers are routinely
used in PCR buffers, storage buffers and other buffer
solutions.
[0037] In a particularly preferred embodiment, the releasing step
takes place in a buffer solution containing the same biological
buffer that is used in, as or on the charge switch material.
[0038] Examples of suitable biological buffers for use in charge
switch materials in accordance with the invention, and their pKa
values, are as follows:
[0039] N-2-acetamido-2-aminoethanesulfonic acid
.dagger-dbl..dagger-dbl. (ACES), pKa 6.8;
[0040] N-2-acetamido-2-iminodiacetic acid .dagger-dbl..dagger-dbl.
(ADA), pKa 6.6;
[0041] amino methyl propanediol .dagger. (AMP), pKa 8.8;
[0042] 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic
acid .dagger. (AMPSO), pKa 9.0;
[0043] N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid
.dagger..dagger. (DES), pKa 7.1;
[0044] N,N-bis-2-hydroxyethylglycine .dagger. (BICINE), pKa
8.3;
[0045] bis-2-hydroxyethyliminotrishydroxymethylmethane
.dagger-dbl..dagger-dbl. (Bis-Tris), pKa 6.5;
[0046] 1,3-bistrishydroxymethylmethylaminopropane
.dagger-dbl..dagger-dbl. (BIS-TRIS Propane), pKa 6.8;
[0047] 4-cyclohexylamino-1-butane sulfonic acid (CABS), pKa
10.7;
[0048] 3-cyclohexylamino-1-propane sulfonic acid (CAPS), pKa
10.4;
[0049] 3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO),
pKa 9.6;
[0050] 2-N-cyclohexylaminoethanesultonic acid (CHES) pKa 9.6;
[0051] 3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid
.dagger..dagger. (DIPSO), pKa 7.6;
[0052] N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid
.dagger..dagger. (EPPS or HEPPS), pKa 8.0;
[0053] N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid .dagger.
(HEPBS), pKa 8.3;
[0054] N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
.dagger..dagger. (HEPES), pKa 7.5;
[0055] N-2-hydroxyethylpiperazine-N-2-propanesulfonic acid
.dagger..dagger. (HEPPSO), pKa 7.8;
[0056] 2-N-morpholinoethanesulfonic acid .dagger-dbl. (MES), pKa
6.1;
[0057] 4-N-morpholinobutanesulfonic acid .dagger..dagger. (MOBS),
pKa 7.6;
[0058] 3-N-morpholinopropanesulfonic acid .dagger..dagger. (MOPS),
pKa 7.2;
[0059] 3-N-morpholino-2-hydroxypropanesulfonic acid
.dagger-dbl..dagger-dbl. (MOPSO), pKa 6.9;
[0060] piperazine-N-N-bis-2-ethanesultonic acid
.dagger-dbl..dagger-dbl. (PIPES), pKa 6.8;
[0061] piperazine-N-N-bis-2-hydroxypropanesulfonic acid
.dagger..dagger. (POPSO), pKa 7.8;
[0062] N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid
.dagger. (TABS), pKA 8.9;
[0063] N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid
.dagger..dagger. (TAPS), pKa 8.4;
[0064] 3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic
acid .dagger..dagger. (TAPSO), pKa 7.4;
[0065] N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid
.dagger..dagger. (TES), pKa 7.4;
[0066] N-trishydroxymethylmethylglycine .dagger. (TRICINE), pKa
8.1; and
[0067] trishydroxymethylaminomethane .dagger. (TRIS), pKa 8.1;
[0068] histidine*, pKa 6.0, and polyhistidine
.dagger-dbl..dagger-dbl.;
[0069] imidazole*, pKa 6.9, and derivatives* thereof (i.e.
imidazoles), especially derivatives containing hydroxyl
groups**;
[0070] triethanolamine dimers**, oligomers** and polymers**;
and
[0071] di/tri/oligo amino acids**, for example Gly-Gly, pKa 8.2;
and Ser-Ser, Gly-Gly-Gly, and Ser-Gly, the latter three having pKa
values in the range 7-9.
[0072] In a preferred embodiment, the buffers marked above with an
asterisk (*) are not considered to be biological buffers for the
purposes of the invention (whether or not they are designated as
such in any chemical catalogue). In a more preferred embodiment,
those marked with two asterisks (**) are also not considered to be
biological buffers. Preferred biological buffers are marked with a
dagger (.dagger.), more preferred buffers are marked with two
daggers (.dagger..dagger.), still more preferred buffers are marked
with a double dagger (.dagger-dbl.) and most preferred buffers are
marked with two double daggers (.dagger-dbl..dagger-dbl.).
[0073] These and other chemical species comprising ionisable groups
may be coated as monomers onto a solid phase support using
covalent, ionic or adsorption interactions. Additionally or
alternatively, they may be coated onto such solid phase supports in
polymeric form (preferably following condensation polymerization),
for example by adsorption onto a negatively charged surface (e.g. a
surface having exposed COOH or SO.sub.3 groups), or by covalent
attachment. Additionally or alternatively, the chemical species
containing ionisable groups may be attached to a polymer (see
below) which is then attached to a solid support, e.g. by
adsorption or covalent attachment.
[0074] Preferably the chemical species or polymer backbones are
covalently coupled to the solid support via a hydroxyl is group or
other group so that the ionisable group having the desired pKa
value (usually, but not limited to, a nitrogen atom) remains
capable of binding and releasing nucleic acid.
[0075] Biological buffers and other chemical species comprising
positively ionisable groups may be used in conjunction with a
chemical species containing a negatively ionisable group which has
a suitable pKa, preferably in the ranges described above. For
example a biological buffer (having one or more positively
ionisable nitrogen atoms) may be attached to a polymer or other
solid phase material which has exposed carboxy groups even after
attachment of the biological buffer. Such a material may bind
nucleic acids at a low pH when few of the carboxy groups are
negatively charged (i.e. few are in the COO.sup.- form, most being
in the COOH form) and most of the ionisable nitrogen atoms are
positively charged. At higher pH the negative charge is stronger
(i.e. a greater proportion of carboxy groups are in the COO.sup.-
form) and/or the positive charge is weaker, and the nucleic acid is
repelled from the solid phase.
[0076] Chemical species containing ionisable groups (such as the
biological buffers listed above) can be attached to a polymer
backbone using known chemistries. For example a chemical species
containing a hydroxyl group can be attached using carbodiimide
chemistry to a carboxylated polymer backbones. Other chemistries
include can be employed by someone skilled in the art using other
polymer backbones (e.g. based on polyethylene glycol (PEG) or
carbohydrate) using a range of standard coupling chemistries (see
e.g. Immobilised Affinity Ligand Techniques, Greg T. Hermanson, A.
Krishna Mallia and Paul K. Smith, Academic Press, Inc., San Diego,
Calif., 1992, ISBN 0123423309, which is incorporated herein by
reference in its entirety.)
[0077] Alternatively, the chemical species containing ionisable
groups can be polymerised without a backbone polymer, using
cross-linking agents, for example reagents that couple via a
hydroxy group (e.g. carbonyldiimidazole, butanediol diglycidyl
ether, dialdehydes, diisothiocyanates). Polymers may also be formed
by simple condensation chemistries to generate polymeric amino
acids with the appropriate pKa e.g. Gly-Gly.
[0078] Preferably such immobilisation, attachment and/or
polymerisation of the chemical species containing the ionisable
group does not affect the pKa of the ionisable group, or leaves it
in the desired ranges given above. For example it is generally
preferred not to couple or polymerise the chemical species via a
positively ionisable nitrogen atom (in constrast for example to
WO97/2982). In the practice of the invention, it is especially
preferred to immobilise, attach and/or polymerise the chemical
species via an hydroxyl group.
[0079] A preferred polymeric material is a dimer or oligomer of
Bis-Tris, or a material formed by attaching a plurality of Bis-Tris
molecules to a polyacrylic acid backbone, e.g. by reacting Bis-Tris
monomer with polyacrylic acid using 1-ethyl-3-dimethylaminopropyl
carbodiimide (EDC), The polymer can then be easily separated from
the reactants using dialysis against a suitable reagent or water.
Preferably the polyacrylic acid has molecular weight of between
about 500 and 5 million or more. More preferably it has a molecular
weight of between 100,000 and 500,000.
[0080] The nature of the resultant Bis-Tris/polyacrylic acid
molecule will depend on the ratio of the coupled components, since
the polymer will have different properties depending on the
proportion of the acrylic acid groups that are modified with
Bis-Tris, for example it is desirable for some carboxy groups to
remain unmodified, as the presence of these will not prevent the
Bis-Tris from binding nucleic acid at low pH (especially if the
Bis-Tris is in excess), but their negative charge at higher pHs
will assist with release of the nucleic acid. For use in the
present invention, the molar ratio of Bis-Tris:carboxy groups
(before attachment) is preferably between 5:1 and 1:5, more
preferably between 3:1 and 1:3, still more preferably between 2:1
and 1:2, further preferably between 1.5:1 and 1:1.5, and most
preferably about 1:1.
[0081] The presence of high residual charge (i.e. charged species
present in solution along with the extracted nucleic acid) may
adversely affect the analysis of nucleic acids by PCR, or interfere
with the binding of primers, dNTPs or polymerase to the nucleic
acid, or to the sequestration of Mg.sup.2+ ions, which are
essential to PCR. It is particularly preferable to avoid residual
positive charge.
[0082] Preferred materials for use in the invention, such as the
biological buffers described above, possess minimal residual
positive charge (preferably minimal residual charge) at the pH at
which the nucleic acid is released, and/or at pHs 8-8.5, making
interference with or inhibition of downstream processes
unlikely.
[0083] Patent application PCT/GB00/02211, of the same inventor,
discloses certain methods within the scope of the present invention
and is incorporated herein by reference in its entirety as
exemplification of the present invention (in all its aspects--see
below for other aspects of the invention). In particular, it
discloses a method for the extraction of biomolecules from
biological material which method comprises contacting the
biological material with a solid phase which incorporates histidine
or a polyhistidine which will tend to bind nucleic acids at low pH
and then extracting the biomolecules bound to the solid phase by
elution using an elution solvent which will then release the bound
nucleic acids when the pH is increased.
[0084] An alternative embodiment of the present invention uses a
material which is positively charged across a wide pH range, such
as 0-12 or 0-14 (e.g. an electropositive substance such as a metal
oxide, metal, strong or weak base, which lacks a pKa value, or for
which the pKa value is at an extreme of high pH. Such a positively
charged material is combined with negatively ionisable material
having a pKa intermediate between the pH values at which it is
desired to bind and release nucleic acid, or slightly below the pH
at which it is desired to bind nucleic acid. This combination of
materials allows nucleic acid to be bound at certain pH values,
around and below the pKa of the negatively ionisable material, when
there are fewer negatively charged groups, but allows the nucleic
acid to be released when the pH is increased and a greater number
of the ionisable groups are negatively charged. For example, the
combination of iron II, III oxide and polycarboxylates (see
Examples) binds nucleic acid at pH 4, when a relative scarcity of
negative charges allowing the positively charged iron oxides to
bind the nucleic acid. When the pH is increased to around 8, a
large proportion of the carboxy groups become negatively charged
and, despite the remaining presence of positive charges on the iron
oxides, the reduction in overall positive charge allows the nucleic
acid to be released.
[0085] Further examples of charge switching molecules for nucleic
acid purification are based on detergents or surfactants that have
a hydrophobic portion and a hydrophilic portion which comprises a
positively ionisable group with a suitable pKa, e.g. decyl methyl
imidazole or dodecyl-Bis-Tris. These detergents/surfactants can be
adsorbed onto surfaces e.g. plastic via their hydrophobic portions
and the hydrophilic (ionisable) portions can be used to capture
nucleic acid.
[0086] Another family of suitable materials for capture and easy
release of nucleic acids are carbohydrates e.g. glucosamine,
polyglucosamine (including chitosans), kanamycins and their
derivatives i.e. sugar ring based structures containing one or more
nitrogen atoms surrounded by hydroxyl groups which may also contain
other groups such as acetate or sulphate groups to provide a
suitable pKa for binding and release of nucleic acids.
[0087] Another group of materials with suitable pKa values are
nucleic acid bases, e.g cytidine (pKa 4.2). These can be
immobilised via hydroxy groups to a polymer or solid phase carboxy
group using carbodiimides.
[0088] A still further group of materials having members with
suitable pKa values are heterocyclic nitrogen-containing compounds.
Such compounds may be aromatic or aliphatic and may be monomers,
oligomers or polymers, such as morpholine-, pyrrole-, pyrrolidine-,
pyridine-, pyridinol-, pyridone-, pyrroline-, pyrazole-,
pyridazine-, pyrazine-, piperidone-, piperidine-, or
piperazine-containing compounds, e.g. polyvinylpyridine. Such
compounds may be substituted with electronegative groups to bring
the pKa value(s) of the ionisable nitrogen atom(s) into an
acceptable range, e.g. as defined above. However, in some compounds
this may not be necessary, the pKa already being in such a
range.
[0089] Preferred materials for use in accordance with the invention
are hydrophilic, for example comprising charge switch materials
which are (or which comprise chemical species which before
immobilisation or polymerisation are) water soluble.
[0090] Once a suitable solid phase has been prepared, comprising a
charge switch material, repeated capture and release of nucleic
acids can be performed by adjusting the pH up or down. Thus
sequential reactions or analysis can be performed on the nucleic
acids using the same solid phase. For example, DNA can be isolated
from a biological sample using a PCR tube comprising a charge
switch material. Then, following PCR, the amplified DNA product may
be isolated from the buffer constituents or primers by adjusting
the pH in the same tube.
[0091] Particularly preferred solid phase materials are non-porous.
Porous supports are commonly used for isolating proteins, which can
be trapped in the pores of the support. However, nucleic acids tend
to be too big to enter into pores of commonly used such supports,
and will therefore become bound to the surface of the support,
potentially trapping impurities in the pores.
[0092] The method can be used to separate single stranded RNA or
DNA from double stranded DNA, because of the different charge
densities on single and double stranded molecules, by appropriate
manipulation of the pH or salt concentration. Typically, single
stranded molecules will be released from binding to the solid phase
at a lower pH than double stranded molecules.
[0093] In some circumstances, for example for the construction of
gene chips, and for the preparation of probes, it may be desirable
to produce single stranded DNA. Manipulation of pH and/or ionic
strength can assist in purification and release of single stranded
nucleic acid. The method of the invention may comprise a prior step
of converting double stranded nucleic acid in the sample to single
stranded nucleic acid (preferably using a strong base, e.g. 100 mM
NaOH, or a weak base at high temperature, e.g. 60-100.degree. C.).
The solid phase material is preferably then added simultaneously
with a buffer which changes the pH of the sample to the pH for
binding single stranded nucleic acid (typically a pH of 4-7).
[0094] The materials described herein may also be employed to
capture nucleic acids in the liquid phase where binding leads to a
cross-linked lattice large enough to separated from the liquid
phase, e.g. by filtration or centrifugation.
[0095] Accordingly, in a second aspect, the present invention
provides a method for extracting nucleic acid from a sample
containing nucleic acids, which method comprises: contacting the
sample with a charge switch material at a first pH at which the
charge switch material has a positive charge and will bind
negatively charged nucleic acid; and then releasing the nucleic
acid at a second, higher pH at which the charge is neutral,
negative or less positive than at the first pH, wherein the charge
switch material is soluble at said first pH, and wherein the
combination of the charge switch material and the bound nucleic
acid is insoluble at or above said first pH and below said second
pH.
[0096] Preferred features of the method are as set out above, with
the exception of the charge switch material being formed into,
immobilised on, or attached to, a solid phase material.
[0097] Usually the charge switch materials will be soluble at the
second pH, and will remain in solution with the nucleic acid upon
release of the nucleic acid; the use of a weakly basic buffer
(optionally bound to a soluble backbone, e.g. polyacrylic acid) as
the charge switch material can avoid problems of contamination as
described above.
[0098] The methods of the invention preferably include one or more
washing steps between the binding and releasing steps. Such (a)
washing step(s) will generally be carried out at said first pH, or
a pH above said first pH but lower then said second pH, such that
the nucleic acid is substantially not released during the washing
step(s).
[0099] As has been indicated previously, the methods of the
invention are particularly suitable for extracting nucleic acid
which is then stored or further processed (e.g. by PCR),
particularly when the charge switch material is in the form of e.g.
a tube or well in which such storage and/or processing can occur.
For the avoidance of doubt, however, it is emphasised that the
releasing step and any subsequent storage or processing need not be
carried out as discrete steps, but can coincide, when said storage
or processing occurs at a pH at which release of the nucleic acid
occurs. For example, the method of the invention includes binding
nucleic acid to a charge switch material coated on or otherwise
provided by a PCR tube, washing the bound nucleic acid, and then
without a separate releasing step commencing the PCR reaction using
a PCR buffer which causes release of the nucleic acid.
[0100] In a further aspect, the present invention provides novel
charge switch materials for use in the methods of the receding
aspects. It further comprises the use of such charge switch
materials in such methods. All preferred features of the charge
switch materials described in above in the context of the methods
apply equally and independently to the present aspect of the
invention (i.e. preferred combinations of features may be different
in relation to this aspect from the preferred combinations in
relation to the method aspects).
[0101] In a further aspect, the present invention provides a
container (preferably a PCR or storage tube or well, or a pipette
tip) coated with, comprising or formed from a charge switch
material, preferably a charge switch material comprising a
biological buffer.
[0102] The following description is directed particularly to the
extraction of nucleic acid from blood, but applies also to the
extraction of nucleic acid from any liquid sample, particularly
biological samples or samples produced during laboratory
techniques, such as PCR.
[0103] The method is particularly useful if the biological material
is blood, but the method can be used for a range of applications
substances such as plasmid and vector isolation and plant DNA
extraction.
[0104] Preferably the cells in the blood are lysed to release
nucleic acids and known lysing agents and methods can be used, such
as contacting with ionic and non ionic detergents, hypotonic
solutions of salts, proteases, chaotropic agents, solvents, using
pH changes or heat. A method of lysing cells to isolate nucleic
acid is described in WO 96/00228.
[0105] When the biological material consists of blood the samples
can optionally be diluted with water or other diluent in order to
make it easier to manipulate and to process.
[0106] Dilutions up to ten times can be used and in general more
dilution can be better and it is a feature of the present invention
that it allows low dilution of blood to be possible.
[0107] The solid phase with which the blood is contacted, can be a
formed of a material which has a natural affinity for nucleic acids
or it can be formed of a material which has its surface treated
with an agent which will cause nucleic acids to bind to it or
increase its affinity for nucleic acids. Suitable materials include
controlled pore glass, polysaccharide (agarose or cellulose), other
types of silica/glass, ceramic materials, porous plastic materials
such as porous plastic plugs which in a single moulded part or as
an insert in a standard tube, polystyrene beads para magnetic beads
etc. The size and porosity is not critical and can vary and be
selected for particular applications.
[0108] Suitable means for treating the surface of the solid phase
or for derivatising it include treating it with a substance which
can introduce a charge e.g. a positive charge on the surface or a
hydrophilic or hydrophobic surface on the solid phase e.g. hydroxyl
groups, nitrate groups, autoreactive groups, dyes and other
aromatic compounds.
[0109] In a preferred embodiment of the invention the solid phase
will cause DNA to be bound to it at one pH in preference to
contaminants in the blood sample and will allow the bound nucleic
acid to be released when it is contacted with an eluant at a
different pH. This system can be used with a solid phase which
incorporates histidine or a polyhistidine which will tend to bind
nucleic acids at low pH e.g. less than 6 and will then release the
bound nucleic acids when the pH is increased e.g. to greater than
8. Alternatively the nucleic acids are bound at substantially
neutral pH to an aminated surface and released at very high pH.
[0110] In another embodiment of the invention a plastic moulding
can incorporate a binding agent e.g. in a well in a plate etc. so
that the binding agent is incorporated in the surface, the blood
sample is then contacted with the surface so as to cause nucleic
acids to be bound to the surface. The blood sample is then removed
and the surface treated with an eluting agent to release the bound
nucleic acids. When the surface is part of a well in a multi-well
plate, the total system can be readily adapted for rapid large
scale sampling and extraction techniques.
[0111] Binding agents which can be used include charge switchable
ion exchange resins using a positively charged solid phase that can
be reversed or made neutral by changing the pH above its pKa. e.g.
nucleotides, polyamines, imidazole groups and other similar
reagents with a suitable pKa value.
[0112] Also, nucleic acids can be bound by intercalation using a
variety of intercalating compounds incorporated into the solid
phase e.g. actinomycin D, ethidium bromide etc.
[0113] In a further embodiment of the invention a plastic surface
can be modified to include functional groups. The plastic can be
any plastic used for containing samples e.g. polypropylene. The
functional groups can be positively or negatively charged so as to
bind the nucleic acids in the correct buffer solution.
[0114] Alternatively the functional groups can be chemical groups
capable of covalent coupling to other ligands or polymers.
[0115] When the plastic is used in a plastic moulding e.g. in a
well in a plate, or as a polymerase chain reaction (PCR) tube, the
surface characteristics of the plastic can be suitably modified for
use in the present invention by including or adding the appropriate
chemicals in the moulding compound e.g. as in an injection moulding
compound.
[0116] When this is used in a PCR tube or in a deep well plate the
tubes or wells can be used to isolate and immobilise small
quantities of DNA or RNA generating a pure template for subsequent
PCR or other genetic analysis and manipulation.
[0117] When the plastic is polypropylene e.g. it is in the form of
a thin walled PCR tube the polypropylene surface can be modified by
oxidising the surface with an oxidising agent such as potassium
permanganate and sulphuric acid to create a carboxylated surface
(COOH groups). This tube can then be used to improve the isolation
of DNA from solutions or from crude samples e.g. blood. By
adjusting the pH, di-electric constant, solubility or ionic
strength the DNA or RNA can be immobilised on the walls of the
tube, washed free of contaminants, ready for PCR or other
analytical techniques.
[0118] The carboxy groups can be further modified by covalently
coupling an anionic group such as imidazole or polyhistidine or any
strong or weak ion exchanger, to allow binding of nucleic acids by
a charge interaction. This tube could then be used to improve the
isolation of DNA from solutions or from crude samples e.g. of
blood. Again by adjusting the pH, di-electric constant, or ionic
strength the DNA or RNA can be immobilized on the walls of the
tube, washed free of contaminants, ready for PCR or other
analytical techniques.
[0119] The nucleic acids can be eluted with in a low salt buffer so
that it is ready for PCR or other analysis.
[0120] The solid phase can be contacted with a blood sample by
mixing with the solid phase in a mixing/stirring device, by passing
the blood sample over the solid phase or the solid phase can be
paramagnetic and manipulated by a magnetic field. Although the
invention is particularly suitable for the separation or isolation
of nucleic acids from blood it can be used with a range of
biomolecules particularly those that require removal of cell wall
debris or insoluble particles.
[0121] In a preferred embodiment of the invention the solid phase
is in granular form in a column and the blood sample is drawn up
through the column by means of a pressure differential being
applied through the column, the blood sample is drawn up with air
and the granular solid material can become fluidised thus
increasing the mixing and contacting rates and minimising
clogging.
[0122] The method of the invention is suitable for use in a
multi-well format when a series of extractions from different
samples can take place substantially simultaneously and this will
facilitate the automation of the extraction process allowing rapid
high throughput extraction to take place and to allow combinational
chemistry to be performed. This will enable there to be a high
throughput in a standard well array e.g. an eight by twelve array
so that a large number of sample types can be treated automatically
at the same time.
[0123] The invention, in its various aspects, will now be described
in detail, by way of example only.
EXAMPLE 1
[0124] Extraction of Nucleic Acids from Whole Blood
[0125] A charge switchable ion-exchanger was prepared by covalently
coupling polyhistidine to 100 (m glass beads using glutaldehyde by
mixing 1 gram of the aminated glass beads with 0.01% (v/v)
glutaldehyde in 0.1M sodium bicarbonate at pH8 containing 20 mg
polyhistidine. After overnight incubation the beads were washed
exhaustively to remove non-covalently bound material and stored in
10 mM HES, pH5 containing 0.1% (v/v) Tween 20.
[0126] About 300 mg of the 100 (m derivitised glass beads were
added to a 1 ml plastic column enclosed at both ends.
[0127] A blood sample was incubated with an equal volume of 10 mM
MES pH5, containing 1% Tween 20, proteases (200(g/ml) and 1 mM
EUTA. After digestion is complete the blood was sucked up the
column containing the glass beads and the DNA became immobilised
allowing the contaminating proteins to pass through to waste.
[0128] The glass beads containing the immobilised DNA were washed
with a buffer comprising 10 mM MES pH5, containing 1% Tween 20, and
1 mM EDTA and this was repeated until the wash solution was
colourless.
[0129] After washing, the beads were dried with air and DNA eluted
with a small quantity of 10 mM Tris HCl, pH 8.5 and collected in a
sterile tube ready for analysis. Thus the DNA were separated from
the blood.
[0130] For different biomolecules, the buffer etc. can be suitably
modified.
EXAMPLE 2
[0131] One gram of carboxylated paramagnetic beads were washed in
50 mM Imidazole buffer pH6 and then mixed with 100 mg of
polyhistidine in 50 ml of 50 mM Imidazole buffer pH 6. A chemical
coupling agent was added (EDC) at a final concentration of 5 mg per
ml and mixed overnight. The beads were washed in water, 0.5M sodium
chloride, 1% Tween 20, 100 mM Tris HCl pH 8 and stored in 10 mM
MES, 0.1% Tween 20 pH5.
[0132] To extract DNA from blood, 1 mg of beads were mixed with
blood diluted in 10% Tween 20 with 25 mM MES, 1M EDTA pH 5. The
beads were separated with a magnet and washed by resuspending in 1
mM MES, 0.1% Tween 20. To elute the DNA the beads were resuspended
in 10 mM Tris HCl pH 8.5 and separated with magnet leaving the DNA
in solution.
EXAMPLE 3
[0133] Bis-Tris Solid Phase Magnetic Beads
[0134] 200 mg of carboxylated 1 .mu.m magnetic particles were
reacted in a one step procedure with 100 mg of Bis-Tris and 100 mg
of the carbodiimide, EDC, in 50 mM imidazole buffer pH6.0.
Following an overnight incubation, the magnetic particles were
washed and used to isolate Plasmid DNA.
[0135] An alkaline lysis method was used to prepare a cleared 5 ml
bacterial lysate generating a supernatant containing the plasmid in
0.5M potassium acetate, pH5. To the supernatant, 2.5 mg of magnetic
particles were added and mixed for 1 minute. After magnetic
separation and washing with water pH5, the pure plasmid DNA was
eluted off in 200 .mu.l of 10 mM Tris.HCl pH 8.5.
[0136] The magnetic beads were also used to extract DNA directly
from whole blood using a detergent based digestion reagent
containing proteinase K.
EXAMPLE 4
[0137] Tricine on Solid Phase Magnetic Beads
[0138] 50 mg of carboxylated 1 .mu.m magnetic particles were
reacted in a one step procedure with 50 mg of Tricine and 100 mg of
the carbodiimide, EDC, in 50 mM imidazole buffer pH6.0. Following
an overnight incubation, the magnetic particles were washed and
used to isolate Plasmid DNA. An alkaline lysis method was used to
prepare a cleared 5 ml bacterial lysate generating a supernatant
containing the plasmid in 0.5M potassium acetate, pH5. To the
supernatant, 2.5 mg of magnetic particles were added and mixed for
1 minute. After magnetic separation and washing with water pH5, the
pure nucleic acids were eluted off in 200 .mu.l of 10 mM Tris.HCl
pH 8.5.
EXAMPLE 5
[0139] Bis-Tris Solid Phase Polystyrene Beads
[0140] 1 gram of carboxylated 60 .mu.m polystyrene particles were
reacted in a one step procedure with 500 mg of Bis-Tris and 500 mg
of the carbodiimide, EDC, in 50 mM imidazole buffer pH6.0.
Following an overnight incubation, the particles were washed and
used to isolate plasmid nucleic acids as described above.
EXAMPLE 6
[0141] Bis-Tris Polymer
[0142] Bis-Tris monomer was converted into a polymer by mixing
together 160 mg of polyacrylic acid with a molecular weight of
240,000, 1.6 g of Bis-Tris and 1.6 g of EDC in 50 mM imidazole
pH6.0. Following an overnight incubation, the mixture was dialysed
in water. The purified polymer was then coated onto magnetic COOH
beads or used in the liquid phase to bind genomic DNA from blood A
5 ml blood sample was centrifuged to obtain the nuclei and WBC
population and the resulting pellet digested with 1% SDS. Following
precipitation with potassium acetate the cleared supernatant was
mixed with either 25 mg of magnetic-Bis-Tris or about 250 .mu.g of
poly-Bis-Tris as a liquid. In both cases the captured DNA could be
separated, washed in water and then redissolved in 10 mM Tris HCl
pH8.5 in a pure form.
EXAMPLE 7
[0143] Insoluble Tris HCl Polymer
[0144] In this example an insoluble polymer was made with inherent
charge switching properties by mixing 80 mg of polyacrylic acid
with 800 mg of Tris HCl and 800 mg of EDC in 50 mM Imidazole pH6.
The insoluble precipitate that formed generated a particulate solid
phase that was used to capture DNA and release it in a similar
manner to that described in example 4 for genomic DNA.
EXAMPLE 8
[0145] Immobilised Poly Bis-Tris on Tips
[0146] A solution of poly Bis-Tris at 1 mg/ml, prepared as in
Example 2, in 0.1M sodium bicarbonate pH8 incubated at 60.degree.
C. for 8 hours with twenty 200 .mu.l polyproplylene pipette tips.
The tips were then rinsed and used to capture about 150 ng of
plasmid DNA from a cleared bacterial lysate by pumping up and down
ten times. After a quick wash with water pH5, the DNA was eluted in
50 .mu.l of 10 mM Tris pH 8.5.
EXAMPLE 9
[0147] Immobilised Poly Bis-Tris on PCR Tubes
[0148] A solution of poly Bis-Tris at 1 mg/ml, prepared as in
Example 2, in 0.1M sodium bicarbonate pH8 incubated at 60.degree.
C. for 8 hours in a 200 .mu.l PCR plate of 8.times.12 tubes. After
rinsing, the tubes were used to bind genomic DNA from a sample
prepared according to example 4. About 50 ng of DNA was
subsequently eluted off per tube using 10 mM Tris HCl pH8.5.
EXAMPLE 10
[0149] Charge Switch Detergents in Liquid Phase
[0150] A blood sample was prepared as described in Example 4 and to
the resulting supernatant decyl imidazole was added at pH 4 causing
precipitation of the DNA. The DNA pellet was collected by
centrifugation and redissolved in 10 mM Tris pH 8.5.
EXAMPLE 11
[0151] Charge Switch Detergents on Solid Phase
[0152] Decyl imidazole was adsorbed onto a 200 ul plastic pipette
tip by soaking in a 1% solution at pH4 in 0.1M sodium acetate. A
blood sample was prepared as described in Example 3 and the tips
were used to bind the DNA by repeated pumping and sucking. After a
wash with water, about 50 ng of DNA was recovered in water at
pH10.
EXAMPLE 12
[0153] Polyglucosamines
[0154] 10 mg of low molecular weight Chitosan was dissolved in
acidified water and then 50 mM imidazole pH5.5, this was mixed with
100 mg of carboxy 1 .mu.m magnetic beads and with 20 mg of the
carbodiimide EDC in 50 mM imidazole pH5.5. Following an overnight
incubation, the beads were washed and resuspended in 10 mM MES pH5.
To bind genomic DNA, 2 mg of magnetic particles were added to a
supernatant prepared by methods described earlier in Example 1,
after magnetic separation, the DNA was eluted using 100 mM Tris-HCl
pH 9.5.
EXAMPLE 13
[0155] Kanamycin
[0156] A solution of genomic DNA was prepared as described in
example 3. To this sample 2 mg of Kanmycin was added at a
concentration of 10 mg/ml. The resulting precipitate of DNA was
filtered, washed in water at pH5 and re-dissolved in water at
pH10.
EXAMPLE 14
[0157] Magnetisable Iron Oxides in Carboxylated Polystrene
[0158] A 5 ml Plasmid mini-prep was prepared using standard
alkaline lysis reagents to generate a cleared lysate with a
potassium acetate composition of 0.5M pH4. To this cleared
supernatant, 2.5 mg of commercially available 1 .mu.m carboxylated
polystyrene magnetisable particles were added to bind the plasmid
DNA. The particles were washed with water at pH4 and then the DNA
eluted using 10 mM Tris HCl at pH 8.5. Typical UV ratios at 260 and
280 nm were 1.7-2.0, indicating pure nucleic acids with a single
band observed with standard gel electrophoresis.
EXAMPLE 15
[0159] Titanium Dioxide in Polystyrene Microtitre Plates
[0160] A solution of DNA at 100 .mu.g per ml in 0.1M Potassium
Acetate pH4 was allowed to stand for 1 hour in a 300 .mu.l flat
bottomed microtitre plastic plate, the plastic plate contained
titanium oxide which was incorporated as a powder in the plastic
when the plate was formed. After washing at pH4, the DNA was
recovered with water at pH10 and 2 ml measured at 260 nm versus a
plain polystyrene plate with no titanium oxide. Approximately, 50
ng of DNA was recovered per 300 .mu.l well for the plate
incorporating the titanium oxide compared to zero for the plain
polystyrene plate.
EXAMPLE 16
[0161] Cytidine Coupled to Magnetic Beads
[0162] 1 gram of carboxylated 1 .mu.m magnetic particles were
reacted in a one step procedure with 500 mg of Cytidine and 500 mg
of the carbodiimide, EDC, in 50 mM imidazole buffer pH6.0. After
thorough washing, the beads were used to bind nucleic acids from a
plasmid preparation as described in example 1 and recovering the
pure nucleic acids in water at pH1.0.
EXAMPLE 17
[0163] Polyvinyl Pyridine (PVP)
[0164] 20 mg of commercially available PVP beads was mixed with the
supernatant containing genomic DNA from a 5 ml blood extraction
described in example 4. After allowing the DNA to bind, the beads
were washed with water at pH5 and the DNA recovered using water at
pH10. Ultra violet analyisis at 260 and 280 nm indicated a purity
ratio of 1.65.
EXAMPLE 18
[0165] Separation of RNA and DNA
[0166] A solution of tRNA and sheared genomic DNA was prepared at
30 kg per ml in 50 mM Potassium acetate buffer pH6.5 with 1M sodium
chloride. Approximately 4 mg of magnetic polyhistidine heads were
mixed with 1 ml of the nucleic acid solution for one minute until
binding was complete. The beads were then thoroughly washed with
water at pH5. To elute the bound material, the beads were mixed
with 300 .mu.l of 10 mM Tris.HCl, 10 mM NaCl, pH8.5. Gel analysis
showed that most of the tRNA remained in solution and was not bound
to the beads. The eluted material contained mostly genomic DNA with
little or no tRNA.
EXAMPLE 19
[0167] DNA Analysis
[0168] In all previous examples, extracted DNA was analysed by one
or more of the following:
[0169] (1): ultra violet (UV) analysis at 260 nm and 280 nm, to
provide a measure of nucleic acid concentration;
[0170] (2): Gel electrophoresis using 1% agarose in TBE buffer run
at 60V for 20 minutes vs a commercial preparation of DNA as
control, with ethidium bromide staining to measure molecular size
and to provide an estimate of quantity of the nucleic acid; or
[0171] (3): PCR using primers specific for actin or other
ubiquitous genes, to test integrity of the nucleic acid.
[0172] The results are presented as (1): direct readings from the
instrument; and (2): and (3): gel pictures.
[0173] In all cases, the examples demonstrated effective extraction
of nucleic acid which was not significantly damaged.
* * * * *