U.S. patent application number 14/717724 was filed with the patent office on 2015-09-10 for methods and compositions for amplification of nucleic acids.
The applicant listed for this patent is Affymetrix, Inc.. Invention is credited to Anthony D. Barone, Christopher J. Kubu, Glenn H. McGall.
Application Number | 20150252351 14/717724 |
Document ID | / |
Family ID | 53270547 |
Filed Date | 2015-09-10 |
United States Patent
Application |
20150252351 |
Kind Code |
A1 |
McGall; Glenn H. ; et
al. |
September 10, 2015 |
Methods and Compositions for Amplification of Nucleic Acids
Abstract
The present invention provides methods, compositions, and kits
for storing and enhancing the activity of polymerases and
particularly thermostable polymerases. The methods comprise mixing
a thermostable polymerase with at least one zwitterionic or ylide
surfactant that has at least one PEO group. In another aspect the
polymerase is mixed with a blocker such as PLURONIC.RTM. or
TETRONIC.RTM. or an amine N-oxide derivative thereof. The
thermostable polymerase may be reversibly inactivated by treatment
with 2-(Methylsulfonyl)ethyl 4-nitrophenyl carbonate. Compositions
and kits for performing the process according to the invention are
also provided.
Inventors: |
McGall; Glenn H.; (Palo
Alto, CA) ; Barone; Anthony D.; (San Jose, CA)
; Kubu; Christopher J.; (Twinsburg, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Affymetrix, Inc. |
Santa Clara |
CA |
US |
|
|
Family ID: |
53270547 |
Appl. No.: |
14/717724 |
Filed: |
May 20, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13791164 |
Mar 8, 2013 |
9085761 |
|
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14717724 |
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61659542 |
Jun 14, 2012 |
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Current U.S.
Class: |
435/6.12 ;
435/188; 435/91.2 |
Current CPC
Class: |
C12N 9/1252 20130101;
C12Q 1/6869 20130101; C12Q 2527/125 20130101; C12P 19/34 20130101;
C12N 9/1241 20130101; C12Q 1/686 20130101; C12N 9/96 20130101; C12Q
1/686 20130101 |
International
Class: |
C12N 9/96 20060101
C12N009/96; C12P 19/34 20060101 C12P019/34; C12N 9/12 20060101
C12N009/12 |
Claims
1. A composition comprising a thermostable polymerase that has been
reversibly modified with a polymerase-modifying reagent of the
following structure: ##STR00026## wherein
Y.dbd.N-hydroxysuccinimide (NHS), p-nitrophenol (pNP) or imidazole;
X.dbd.CONMe.sub.2, MeSO.sub.2, CN, NO.sub.2, CO.sub.2Me,
(CH.sub.2).sub.2SMe, N(CH.sub.3)CHO, or CH.sub.2C(O)CH.sub.3.
2. The composition of claim 1, wherein the polymerase-modifying
reagent is ##STR00027## (2-(methylsulfonyl)ethyl 4-nitrophenyl
carbonate (MSEC)).
3. The composition of claim 1, wherein the thermostable polymerase
is a Pfu DNA polymerase, a thermostable DNA polymerase fusion
protein, a Pfu DNA polymerase-Sso7 fusion polypeptide, or a Taq DNA
polymerase.
4. The composition of claim 3, wherein the thermostable polymerase
is a Taq DNA polymerase.
5. The composition of claim 1, further comprising a non-ionic or a
cationic surfactant.
6. The composition of claim 5, wherein the surfactant is a
poloxamer.
7. The composition of claim 6, wherein the poloxamer has the
following structure: ##STR00028## m (avg)=2-100 and n
(avg)=2-200.
8. The composition of claim 5, wherein the surfactant is an alkyl
diamine.
9. The composition of claim 8, wherein the alkyl diamine has the
following structure: ##STR00029## m (avg)=2-100; (avg) n=2-200
R1=C2-C.sub.6 Alkyl (preferably 2-3) R2=CH.sub.3, CH.sub.2CH.sub.3;
x=1,2.
10. A method for the amplification of a nucleic acid contained in a
sample comprising the steps of: (a) modifying a thermostable
polymerase with a polymerase-modifying reagent of the structure:
##STR00030## wherein Y.dbd.N-hydroxysuccinimide (NHS),
p-nitrophenol (pNP) or imidazole; X.dbd.CONMe.sub.2, MeSO.sub.2,
CN, NO.sub.2, CO.sub.2Me, (CH.sub.2).sub.2SMe, N(CH.sub.3)CHO, or
CH.sub.2C(O)CH.sub.3 to provide a modified thermostable polymerase;
(b) forming a mixture comprising the sample, the modified
thermostable polymerase and a primer complementary to the nucleic
acid; (c) incubating the resulting mixture of step (b) at a
temperature which is greater than about 50.degree. C. for a time
sufficient to reactivate the enzyme; and (d) generating one or more
amplification products of the nucleic acid.
11. The method of claim 10, wherein the contacting a thermostable
polymerase with a polymerase-modifying reagent results in
essentially complete inactivation of the modified polymerase at
about 25.degree. C., and wherein incubation of the modified
polymerase at a temperature greater than about 50.degree. C.
results in an increase in polymerase activity.
12. The method of claim 10, wherein the polymerase-modifying
reagent is ##STR00031## (2-(methylsulfonyl)ethyl 4-nitrophenyl
carbonate (MSEC)).
13. The method of claim 10, wherein a solution of
polymerase-modifying reagent is added to the thermostable
polymerase within about 5 minutes of preparing the solution.
14. The method of claim 10, wherein the temperature of step (c) is
about 90.degree. C. to about 95.degree. C.
15. The method of claim 14, wherein the temperature is about
95.degree. C.
16. The method of claim 10, wherein the time to reactivate the
enzyme is about 5 to 10 minutes.
17. The method of claim 10, wherein the amplification reaction
mixture further comprises betaine.
18. The method of claim 10, wherein the amplification reaction
mixture further comprises one or more surfactants selected from the
group consisting of zwitterionic surfactants, cationic surfactants,
non-ionic surfactants and mixtures thereof.
19. The method of claim 18, wherein the surfactant is an
amine-N-oxide.
20. The method of claim 10, wherein the thermostable enzyme is
present in an amount of about 0.25 units per 4.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/791,164 filed Mar. 8, 2013, which claims
the benefit of U.S. Provisional Patent Application Ser. No.
61/659,542 filed Jun. 14, 2012, the contents of each of which are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention is generally in the field of nucleic
acid amplification.
BACKGROUND OF THE INVENTION
[0003] Compositions of thermostable nucleic acid polymerases are
useful for amplification of nucleic acids by multiple cycles of the
polymerase chain reaction. Various compositions for stabilizing
polymerases using surfactants have been disclosed. In an early
study it was observed that viral DNA polymerase activity was
stimulated and stabilized against thermal inactivation by nonionic
detergent (see, Wu and Cetta, Biochemistry (1975) 14(4):789-795).
U.S. Pat. No. 6,127,155 discloses stabilization of thermostable DNA
polymerases in a composition containing non-ionic polymeric
detergents. U.S. Pat. No. 6,242,235 discloses cationic
polyethoxylated amine surfactants as polymerase stabilization
agents, WO 2008152102 (Liu et al.) and US Pat. Pub. No. 20100099150
(Fang et al.) disclose polymerase stabilization by anionic
detergents. US Pat. Pub. No. 20080064071 (Hogrefe et al.) discloses
zwitterionic detergents for storage and use of DNA polymerases and
US Pat. Pub. No. 2008145910 (Ward et al.) discloses stabilization
of DNA polymerase using anionic or zwitterionic detergents during
thermal cycling.
[0004] Amplification of nucleic acids involves the thermal cycling
of a reaction mixture containing a nucleic acid polymerase to
generate an amplified target nucleic acid. An example of this
thermal cycling process is that which occurs in Polymerase Chain
Reaction (PCR), a laboratory technique that can theoretically take
one molecule of DNA and produce measurable amounts of identical DNA
in a short period of time. PCR is a widely used method in the
fields of biotechnology, forensics, medicine, and genetic research.
In this method, oligonucleotides are used as primers for a series
of synthetic reactions that are catalyzed by a DNA polymerase. The
reaction mixture is subjected to multiple cycles of denaturation,
annealing, and synthesis performed at different temperatures.
Thermostable polymerases are generally used to amplify the target
nucleic acid sequences in these thermal cycling reactions because
they are not inactivated by the heat denaturation step and,
therefore, do not need to be replaced in every round of the
amplification cycle. Although efficient, exponential amplification
of target sequences is not an unlimited process. Under normal
reaction conditions, the amount of DNA polymerase becomes limiting
after a certain number of cycles of amplification.
[0005] Attempts have been made to improve the PCR amplification
process by employing detergents and/or surfactants. For example,
U.S. Pat. No. 6,127,155 discloses that the non-ionic detergents
NP-40 and Tween stabilize Taq DNA polymerase. However, this patent
does not disclose the use of non-detergent surfactants or
zwitterionic detergents for the stability of thermostable
polymerases in PCR reactions. As another example, US Pat. Pub. No.
20030017567 discloses a method for performing an amplification
reaction utilizing a dye that converts electromagnetic energy into
thermal energy to heat the reaction mixture. A zwitterionic
surfactant is added to the reaction mixture to reduce interference
of the dye with the functioning of the nucleic acid polymerase.
Additionally, U.S. Pat. Pub. No. 20020168658 discloses the use of
zwitterions in combination with a compound that disrupts base
pairing, e.g., DMSO, to improve the amplification of nucleic acids
that are G+C rich. However, this publication does not disclose the
use of zwitterionic detergents alone in improving the amplification
of nucleic acids and actually teaches that the zwitterionic
detergents used should be selected carefully so as not to inhibit
the activity of the DNA polymerase in the reaction.
[0006] Given the widespread use and importance of thermal cycling
processes, there is a need in the art for ways to improve the
stability and/or enhance the activity of thermostable enzymes used
in DNA amplification.
SUMMARY OF THE INVENTION
[0007] In one aspect, stabilization of nucleic acid polymerases by
zwitterionic derivatives of poly-alkoxylated alkyl derivatives and
particularly amine-N-oxide derivatives is disclosed. Amine-N-oxide
surfactants containing a fatty alkyl group, that is preferably
linear, and one or more polyoxyethylene groups are particularly
preferred for stabilizing polymerase activity.
[0008] In another aspect, stabilization of nucleic acid polymerases
by nonionic poly(propylene oxide)-poly(ethylene oxide) block
copolymer surfactants is disclosed.
[0009] In another aspect, stabilization of nucleic acid polymerases
by cationic poly(propylene oxide)-poly(ethylene oxide) block
copolymer surfactants based on ethylenediamine is disclosed.
[0010] In another aspect, a thermostable polymerase that has been
modified with 2-(Methylsulfonyl)ethyl 4-nitrophenyl carbonate
(MSEC) is disclosed. This modification inactivates the polymerase
so that it is inactive until incubated at temperatures greater than
about 50.degree. C. for at least 10 minutes.
[0011] In another aspect, compositions including a thermostable
polymerase that has been modified with MSEC and an ylide surfactant
are disclosed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows a gel image of products of PCR in the presence
of different non-ionic and zwitterionic detergents.
[0013] FIG. 2 shows a gel image of products of PCR in the presence
of varying amounts of a PEO.sub.n detergent or non-ionic detergents
Tween-20 and NP-40 with detergent-free Taq DNA Polymerase.
[0014] FIG. 3 shows a gel image of PEO.sub.n length and zwitterion
variations with "detergent-free" Taq DNA Polymerase.
[0015] FIG. 4 shows a gel image of various zwitterionic detergents
without PEO.sub.n groups and a non-ionic, non-polymeric detergent
(lauryl maltoside) with "detergent-free" Taq DNA Polymerase.
[0016] FIG. 5 shows a gel image of PCR products from reactions that
include various alkyl chains with PEO.sub.n groups and a
polyethylene glycol.
[0017] FIG. 6 shows a gel image of PCR products from reactions that
included blocked co-polymers under extreme conditions with
detergent-free Taq DNA Polymerase.
DETAILED DESCRIPTION
[0018] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. The
following definitions supplement those in the art and are directed
to the current application and are not to be imputed to any related
or unrelated case, e.g., to any commonly owned patent or
application. Although any methods and materials similar or
equivalent to those described herein can be used in the practice
for testing of the present invention, the preferred materials and
methods are described herein. Accordingly, the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting.
[0019] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a molecule" includes a plurality of such molecules,
and the like.
[0020] The term "about" as used herein indicates the value of a
given quantity varies by +/-10% of the value, or optionally +/-5%
of the value, or in some embodiments, by +/-1% of the value so
described.
[0021] Throughout this disclosure, various aspects of this
invention can be presented in a range format. It should be
understood that when a description is provided in range format,
this is merely for convenience and brevity and should not be
construed as an inflexible limitation on the scope of the
invention. Accordingly, the description of a range should be
considered to have specifically disclosed all the possible
sub-ranges as well as individual numerical values within that
range. For example, description of a range such as from 1 to 6
should be considered to have specifically disclosed sub-ranges such
as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6,
from 3 to 6, for example, as well as individual numbers within that
range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless
of the breadth of the range.
[0022] Many of the methods and systems disclosed herein utilize
enzyme activities. A variety of enzymes are well known, have been
characterized and many are commercially available from one or more
supplier. For a review of enzyme activities commonly used in
molecular biology see, for example, Rittie and Perbal, J. Cell
Commun. Signal. (2008) 2:25-45, incorporated herein by reference in
its entirety.
[0023] There are a variety of methods for amplification that may be
used in combination with the methods disclosed herein include.
Amplification of nucleic acids is widely used in research,
forensics, medicine and agriculture. One of the best-known
amplification methods is the polymerase chain reaction (PCR), (See
for example, U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159). A
PCR reaction typically utilizes two oligonucleotide primers, which
are hybridized to the 5' and 3' borders of the target sequence and
a DNA polymerase, which can extend the annealed primers by adding
on deoxynucleoside-triphosphates (dNTPs) to generate
double-stranded products. By raising and lowering the temperature
of the reaction mixture, the two strands of the DNA product are
separated and can serve as templates for the next round of
annealing and extension, and the process is repeated.
[0024] The invention provides compositions, kits and methods that
include a polymerase and a zwitterionic or non-ionic surfactant
that may be a detergent or non-detergent. Such compositions and
methods are useful in, among other things, the storage and use of
DNA polymerases in thermal cycling reactions, including, but not
limited to PCR and all of its variants (e.g., real-time PCR or
quantitative PCR). The present invention identifies novel
surfactants that increase stability and enhance activity of
thermostable DNA polymerases.
[0025] It has been previously observed that product yields are
dramatically higher when PCR amplification reactions are conducted
in buffers containing one or more non-ionic detergents (NP-40,
TWEEN-20), zwitterionic detergents (e.g., CHAPS, CHAPSO, Anzergent
3-10, and Anzergent 3-12) or non-detergent surfactants (e.g.,
Surfynol 465). Similarly, detergents and non-detergent surfactants
are known to produce higher amplification efficiencies, higher
total fluorescence, and earlier Ct values in QPCR reactions
employing thermostable DNA polymerase and SYBR Green to monitor
duplex DNA formation.
[0026] In general, the invention is directed to storage and
reaction compositions having a polymerase and at least one
surfactant. In one embodiment, the storage and reaction
compositions comprise a polymerase and two or more surfactants.
Generally, a reaction mixture will include some or all of the
necessary components to perform a nucleic acid synthesis reaction,
such as primers, dNTPs, and buffers. A storage mixture may or may
not include all the components necessary to perform a nucleic acid
synthesis reaction.
[0027] The polymerases may be stored in a storage buffer comprising
a zwitterionic detergent, which may be an ylide, a non-detergent
surfactant, or both. The polymerases of the invention, described
herein, may be obtained commercially or produced by methods well
known to one of skill in the art. The storage buffer and reaction
buffers may include from about 0.001% to 5% volume/volume of each
zwitterionic detergent or non-detergent surfactant employed.
[0028] The terms "surfactant" as used herein refers to compounds
that are amphiphilic, meaning they contain both hydrophobic groups
(their tails) and hydrophilic groups (their heads). Surfactants
lower the surface tension of a liquid, the interfacial tension
between two liquids, or that between a liquid and a solid.
Surfactants may act as detergents, wetting agents, emulsifiers,
foaming agents, and dispersants. The terms surfactant and detergent
may be used interchangeably herein, but detergents typically have
the additional quality of having cleaning properties in dilute
solution. Surfactants or detergents may be anionic, (e.g.
alkylbenzenesulfonates), cationic, non-ionic (e.g. Tween, Triton
and Brij series detergents) and zwitterionic (e.g. CHAPS).
[0029] The term "ylide" or "ylides" refers to a subset of
zwitterionic compounds in which an anionic site Y- is attached
directly to a heteroatom X+ (usually nitrogen, phosphorus or
sulfur) carrying a formal positive charge. They are thus
1,2-dipolar species of the type RmX+-Y-Rn. If X is a saturated atom
of an element from the first row of the periodic system, the ylide
is commonly represented by a charge-separated form. If X is a
second, third, etc. row element uncharged canonical forms are
available RmX.dbd.YRn. If X is an unsaturated atom, doubly bonded
to another first row element Z, the negative charge on Y may be
stabilized by p-conjugation, Z.dbd.X+-Y-Rn<<Z-X+.dbd.YRn.
Such ylides belong to the class 1,3-dipolar compounds. Note that
ylide is a complete word, not to be confused with the suffix
-ylide, used for some radical anions. Subclasses of ylides: Ylides
RmX+-C-R2 having the negative charge on carbon are classified by
citing the name of the element X before the word ylide. For
example, nitrogen ylide, phosphorus ylide, oxygen ylide, and sulfur
ylide. A further specification may be achieved by citing the class
name of RmX before the word ylide. Thus nitrogen ylides include
amine ylides, R3N+-C-R2, azomethine ylides, R2C.dbd.N+R-C-R2, and
nitrile ylides, RC.degree.N+-C-R2.
[0030] A "zwitterion" is a neutral compound having formal unit
electrical charges of opposite sign, for example,
H.sub.3N+CH.sub.2C(.dbd.O)O.sup.- ammonioacetate (glycine). The
compound betaine
((CH.sub.3).sub.3N.sup.+--CH.sub.2C(.dbd.O)O.sup.-N,N,N-trimethylammonioa-
cetate) and other "betaines" (neutral molecules having
charge-separated forms with an onium atom which bears no hydrogen
atoms and that is not adjacent to the anionic atom) are also
zwitterionic compounds. As used herein the term refers to compounds
with the charges on either adjacent or non-adjacent atoms.
Zwitterionic compounds with charges on adjacent atoms may also be
referred to herein as ylides. Such compounds include, but are not
limited to, CHAPS and sulfobetaines sold under the brand names
ZWITTERGENT.RTM. (Calbiochem, San Diego, Calif.) and ANZERGENT.RTM.
(Anatrace, Inc., Maumee, Ohio).
[0031] An "amine oxide" or "amine N-oxide" is a compound derived
from tertiary amines by the attachment of one oxygen atom to the
nitrogen atom R.sub.3N.sup.+--O.sup.-. By extension the term
includes the analogous derivatives of primary and secondary amines.
Because the charges are on adjacent atoms, amine oxides are
considered to be ylides herein.
[0032] In some aspects, zwitterionic or ylide derivatives of
poly-alkoxylated alkyl derivatives, particularly the amine N-oxides
are used to stabilize polymerases. These surfactants have the
following general characteristics: a hydrophobic group comprising a
linear alkyl or poly(propylene oxide) chain; a hydrophilic
polyether group comprising a poly(ethylene oxide) or a random
copolymer of polyethylene oxide/poly(propylene oxide); and an ylide
or zwitterionic group comprising an amine oxide,
carboxy-alkylammonium, or sulfo-alkylammonium.
[0033] In other aspects, the surfactant is block co-polymer that
may be a poloxamer or a poloxamine. The term "block co-polymer"
herein refers to a polymer composed of two or more different
polymers ("co-polymer") arranged in segments or "blocks" of each
constituent polymer. Both poloxamers and poloxamines are block
copolymers.
[0034] The term "poloxamer" herein refers to any di- or tri-block
copolymer composed of polypropylene oxide and polyethylene oxide
blocks arranged in a basic A-B-A structure:
PEO.sub.x-PPO.sub.y-PEO.sub.x. Polypropylene oxide (PPO or
polyoxypropylene, also (poly(propylene oxide))) has the formula
(C.sub.3H.sub.6O).sub.x, (thus a subunit mw of 58) and is a
hydrophobe. Polyethylene oxide (PEO or polyoxyethylene) is a
nonionic homopolymer of ethylene oxide and can be represented by
the formula (OCH.sub.2CH.sub.2).sub.x where x represent the average
number of oxyethylene groups, it may also be represented by the
formula (C.sub.2H.sub.4O).sub.x, (thus a subunit mw of 44) and is a
hydrophile. Poloxamers are nonionic and have a central hydrophobic
chain of PPO flanked by two hydrophilic chains of PEO. For a more
comprehensive description see U.S. Pat. No. 3,740,421. The common
chemical name for poloxamers is polyoxypropylene-polyoxyethylene
block copolymer. The CAS number is 9003-11-6.
[0035] There are many species of poloxamers differing in total
molecular weight, polyoxypropylene to polyoxyethylene ratio,
surfactant properties and physical form in undiluted solution.
Physical forms include Liquids (L), Pastes (P) and Flakable solids
(F). The manner in which poloxamers are typically synthesized
results in a population of molecules in a relatively circumscribed
range of molecular weights characterized by a hydrophobe having a
defined average molecular weight and total average percentage of
hydrophile groups. Because the lengths of the polymer blocks can be
customized, many different poloxamers exist with slightly different
properties. Properties include, for example, hydrophilic-lipophilic
balance or HLB and cloud point.
[0036] Poloxamers are also known by the trade name PLURONIC.RTM. in
the US and LUTROL.RTM. in Europe (BASF). BASF developed a
PLURONIC.RTM. grid to provide a graphic representation of the
relationship between copolymer structure, physical form and
surfactant characteristics. On the PLURONIC.RTM. surfactant grid
the molecular weight ranges of the PPO are plotted against the
weight-percent of the PEO present in each molecule. Poloxamer
species defined by their location on the PLURONIC.RTM. grid can be
expected to have shared properties that are a function of their
total molecular weight and relative hydrophobicity. For a
description of the PLURONIC.RTM. grid and an explanation of the
nomenclature used by BASF in naming PLURONICS.RTM. see US Pat. Pub.
No. 20110044929.
[0037] Copolymers with a short hydrophilic poly-PEO block or/and an
extended lipophilic poly-PPO block (such as PLURONIC.RTM. L121 and
L101) are highly lipophilic and are characterized by a relatively
low CMC and low HLB. In contrast, copolymers with an extended
hydrophilic poly-PEO block or/and short lipophilic poly-PPO block
(such as PLURONIC.RTM. F108 and F88) are hydrophilic and are
characterized by relatively high CMC and high HLB.
PLURONIC.RTM.compositions such as P85 or P103 are intermediate in
their lipophilicity and have CMC and HLB values that fall between
the two extremes identified above.
[0038] As used herein, the term "poloxamine" refers to
poly(oxyethylene)-poly(oxypropylene) (PEO-PPO) block copolymers
where a PEO-PPO unit is linked to another PEO-PPO unit by an amine
and having the general structure
(PEO.sub.n-PPO.sub.m).sub.2-N--C.sub.2H.sub.4--N-(PPO.sub.m-PEO.sub.n)-.s-
ub.2. TETRONIC.RTM. and TETRONIC.RTM. R nonionic surfactants
produced by BASF are exemplary poloxamines.
[0039] Poloxamines are in the alkoxylated amine chemical family and
have a hydrophobic center consisting of two tertiary amino groups
carrying both two hydrophobic PPO chains of equal length each
followed by a hydrophilic PEO chain. Poloxamines can still be
described as a tri-block copolymer although bulkier than
poloxamers. Poloxamines of the BASF TETRONIC.RTM. type have the
chemical name: 1,2-Ethanediamine, polymer with the and the CAS
number: 11111-34-5. Reverse TETRONICS.RTM. have the formula
(PPO.sub.n-PEO.sub.m).sub.2-N--C2H4-N-(PEO.sub.m-PPO.sub.n).sub.2
and the CAS number: 26316-40-5.
[0040] In another aspect polymerases are stabilized by inclusion of
poly-alkoxylated alkylamine derivatives, particularly amine
N-oxides. Although alkyl (C.sub.8-C.sub.18)-dimethylamine N-oxides
are commonly available, we did not observe them to be effective for
polymerase stabilization. On the other hand amine oxide detergents
containing a fatty alkyl group (preferably linear) and one or more
polyoxyethylene groups were observed to be particularly effective
for stabilizing polymerase activity. The following general
structures are exemplary of the compounds found to be effective for
polymerase stabilization:
##STR00001## [0041] where Q=-O.sup..crclbar. or --X--Y; where
X.dbd.C.sub.1-C.sub.3 alkyl; and Y.dbd.CO.sub.2.sup..crclbar.,
SO.sub.3.sup..crclbar.; [0042] m+n=2-200 and more preferably 5-50;
[0043] R1=C.sub.1-C.sub.30 alkyl and more preferably
C.sub.10-C.sub.20; [0044] R2=CH.sub.3, CH.sub.2CH.sub.3; x=1,2; and
[0045] R3=H, CH.sub.3, or CH.sub.2OH.
[0046] In another aspect polymerases are stabilized using ylide or
zwitterionic surfactants derived from alkanediamine block
copolymers (for example TETRONIC.RTM. surfactants). The following
structures are exemplary:
##STR00002## [0047] Q=-O.sup..crclbar.; or --X--Y; where
X.dbd.C.sub.1-C.sub.3 alkyl; and Y.dbd.CO.sub.2.sup..crclbar.,
SO.sub.3.sup..crclbar. [0048] m (avg)=2-100; n (avg)=2-200 [0049]
R1=C.sub.2-C.sub.6 Alkyl (preferably 2-3) [0050] R2=CH.sub.3,
CH.sub.2CH.sub.3; x=1,2
[0051] In another embodiment polymerases are stabilized using
cationic polypropylene oxide/polyethylene oxide block copolymer
surfactants based on alkyl diamines (for example TETRONIC.RTM.
surfactants). The following general structures are exemplary:
##STR00003## [0052] m (avg)=2-100; (avg) n=2-200 [0053]
R1=C.sub.2-C.sub.6 Alkyl (preferably 2-3) [0054] R2=CH.sub.3,
CH.sub.2CH.sub.3; x=1,2.
[0055] In another aspect, the TETRONIC.RTM. surfactants are
modified to an amine oxide and the amine oxide form is used for
stabilization. In particular TETRONIC.RTM. 1107 amine oxide was
found to be particularly preferred. (TETRONIC.RTM. 1107 is
tetrafunctional, ethoxylated and propoxylated ethylenediamine block
copolymer surfactant, which has a cloud point (10% aqueous
solution) greater than 100.degree. C., an average molecular weight
of 15000, a specific gravity of 1.04 at 25.degree. C., a viscosity
of 1100 cps at 77.degree. C. and a melt point of 51.degree.
C.).
[0056] In another embodiment polymerases are stabilized using
nonionic polypropylene poloxamers, for example, the PLURONIC.RTM.
surfactants. The following general structure is exemplary:
##STR00004## [0057] m (avg)=2-100 and n (avg)=2-200.
[0058] In one aspect the general structure of the surfactant
additive is an amine oxide ylide or zwitterion derived from
polyethoxylated alkylamines having the general structure:
##STR00005## [0059] where Q=-O.sup..crclbar. or
--(CH.sub.2).sub.pY, where p=1-6 and Y=CO.sub.2--, SO.sub.3--,
PO.sub.3H--; OSO.sub.3--, or OPO.sub.3H--; [0060] m+n=2-200
(preferably 5-50): [0061] x=1 or 2; [0062] R1=C.sub.8-C.sub.20
Alkyl (eg., --(CH.sub.2).sub.nCH.sub.3) [0063] R2 =CH.sub.3,
CH.sub.2CH.sub.3, CH.sub.2OCH.sub.3, CH.sub.2CH.sub.2OCH.sub.3, or
CH.sub.2CH.sub.2OH, and [0064] R3=CH3, CH.sub.2OH ,
CH.sub.2CH.sub.3, CH.sub.2OCH.sub.3, CH.sub.2CH.sub.2OCH.sub.3, or
CH.sub.2CH.sub.2OH.
[0065] In another aspect, the surfactant has the following general
structure:
##STR00006## [0066] where m+n=2-200 (preferably 5-50); x=1-2 [0067]
R1=C.sub.8-C.sub.24 Alkyl (e.g. --(CH.sub.2).sub.nCH.sub.3) [0068]
R2=CH.sub.3, CH.sub.2CH.sub.3, CH.sub.2OCH.sub.3,
CH.sub.2CH.sub.2OCH.sub.3 or CH.sub.2CH.sub.2OH [0069] R3=CH.sub.3,
CH.sub.2CH.sub.3, CH.sub.2OCH.sub.3, CH.sub.2CH.sub.2OCH.sub.3 or
CH.sub.2CH.sub.2OH [0070] R4=CH.sub.3, CH.sub.2OH,
CH.sub.2CH.sub.3, CH.sub.2OCH.sub.3, or
CH.sub.2CH.sub.2OCH.sub.3.
[0071] In another aspect, the surfactant has the following general
structure:
##STR00007## [0072] where m+n=2-200 (preferably 5-50); x=1-2 [0073]
R1=C.sub.8-C.sub.24 Alkyl (e.g. --(CH.sub.2).sub.nCH.sub.3) [0074]
R2=CH.sub.3, CH.sub.2CH.sub.3, CH.sub.2OCH.sub.3,
CH.sub.2CH.sub.2OCH.sub.3 or CH.sub.2CH.sub.2OH [0075] R3=CH.sub.3,
CH.sub.2CH.sub.3, CH.sub.2OCH.sub.3, CH.sub.2CH.sub.2OCH.sub.3 or
CH.sub.2CH.sub.2OH [0076] R4=CH.sub.3, CH.sub.2OH,
CH.sub.2CH.sub.3, CH.sub.2OCH.sub.3, or
CH.sub.2CH.sub.2OCH.sub.3.
[0077] In another aspect, the surfactant has the following general
structure:
##STR00008## [0078] Q=-O.sup..crclbar.; or --(CH.sub.2).sub.pY,
where p=1-6, and Y=CO.sub.2.sup.-, SO.sub.3.sup.-, PO.sub.3H.sup.-,
OSO.sub.3.sup.-, OPO.sub.3H.sup.-;
[0079] m+n=2-200 (preferably 5-50); x=1-2 [0080]
R1=C.sub.8-C.sub.24 Alkyl (e.g. --(CH.sub.2).sub.nCH.sub.3) [0081]
R2=CH.sub.3, CH.sub.2OH, CH.sub.2CH.sub.3, CH.sub.2OCH.sub.3, or
CH.sub.2CH.sub.2OCH.sub.3.
EXAMPLE 1
[0082] Amine oxide derivatives of selected surfactants were
prepared as follows: amine containing surfactants (10 mmole in
amine equivalents) were dissolved in .about.3-4 volumes of ethanol.
H.sub.2O.sub.2 (30%; 2.3 ml; 20 mmole) was added, and the solution
stirred at 55.degree. C. for 24-48 hrs. After cooling to room
temperature, .about.20 mg of 10% Pt.degree. on C (platinum on
activated carbon) was added, and stirring continued for another 4 h
to decompose the excess peroxide. The solution was filtered through
celite and evaporated under vacuum. The .sup.1H-NMR was recorded in
MeOH-d.sub.4.
[0083] The procedure above was used to prepare amine N-oxides from
the following polyalkoxylated amine surfactants: PEO(15)
Laurylamine (DeThoxamine C-15, Deforest); PEO(5)
Isodecyloxypropylamine (Tomamine E-14-5, Air Products); PEO(5)
Isotridecyloxypropylamine (Tomamine E-17-5, Air Products); PEO(5)
Stearylamine; PEO(10) Stearylamine; PEO(15) Stearylamine; PEO(50)
Stearylamine; TETRONIC.RTM. 304 (MW 1650); TETRONIC.RTM. 904; and
TETRONIC.RTM. 1107. When using "PEO(X)" herein the (X) is the
number of moles of ethylene oxide.
[0084] For testing, several additional amine oxide surfactants were
obtained directly from commercial sources, including MACAT AO-12-2
(N,N-Bis(2-hydroxyethyl)laurylamine N-Oxide, available from Mason
Chemicals) which is an ylide lacking the PEO groups and having the
following structure:
##STR00009## [0085] TOMAMINE.RTM. AO-405 Surfactant (Air Products)
(Polyethoxylated/Polypropoxylated (isodecyloxypropyl)amine N-Oxide
(CAS #218141-38-9), falls into a series of amine oxide compounds
available from Air Products under the brand name TOMAMINE.RTM. and
having the general structure:
##STR00010##
[0085] and Lauryldimethylamine N-oxide (available from
Anatrace):
##STR00011##
[0086] PEO(10) Stearyl (3-sulfo-propyl)-ammonium was prepared as
follows: a mixture of propanesultone (0.36 g, 3 mmole) and PEO(10)
Stearylamine (1.6 g, 2 mmole) was stirred in a sealed vial at
50-55.degree. C. for 24 hr, producing a viscous yellow oil.
Ethanolamine (0.12 ml, 2 mmole) was added and stirring continued at
50-55.degree. C. for another 24 hr to quench the unreacted
propanesultone. The .sup.1H-NMR was recorded in MeOH-d.sub.4.
EXAMPLE 2
[0087] In order to test the efficacy of detergents on Taq DNA
Polymerase, a stock of Taq DNA Polymerase at a base concentration
of 111 units/ul was diluted to 5 units/ul in a storage buffer that
lacked detergents. The standard Taq DNA Polymerase storage buffer
has 0.5% Tween-20 and 0.5% NP-40. In this example 0.625 units of
Taq were added per 25 ul reaction, so that represents a change in
final detergent concentration of 0.0025% each detergent to 0.00011%
of each. A 455 bp single-copy target from the human numb gene was
PCR-amplified from 1 ng human genomic DNA in a 25 ul reaction with
0.625 units of the diluted Taq DNA polymerase. The cycling
conditions were 95.degree. C. for 2 minutes; 35 cycles of
95.degree. C. for 10 seconds, 55.degree. C. for 20 seconds, and
72.degree. C. for 30 seconds; 72.degree. C. for 5 minutes; and
10.degree. C. until required. An aliquot of 10 ul was run on a 1.5%
agarose/ethidium bromide gel.
[0088] In FIG. 1, the results of the initial screen of several
non-ionic and zwitterionic detergents are shown. In this example
there is residual Tween-20 and NP-40 in the enzyme storage buffer.
The concentrations of surfactants listed are the final
concentration of that surfactant in the assay. All conditions in
the example, including the "no detergent" condition, have 0.00011%
Tween-20 and 0.00011% NP-40 resulting from carry-over from the
enzyme storage buffer. As can be seen from the "no detergent" lanes
on both gels, the carry over is not sufficient to promote the
production of a visible PCR product of the expected size. As
expected Tween-20 and NP-40 worked when present at 0.01% and 0.1%.
PMAL-C8, -C10, -C12 or -C16, which are zwitterions but do not
contain PEO did not. A0-12-2 worked at 0.01% and PEO(10)SAP worked
at 0.01, 0.1 and 1%. It is clear that in addition to the standard
non-ionic detergents with PEO.sub.n, a zwitterionic or ylide with
that polymer will also contribute to PCR product generation.
[0089] AO-12-2 is Dihydroxyethyl Cocamine Oxide; AO-405 is
Polyethoxylated/Polypropoxylated(isodecyloxypropyl)amine N-Oxide;
and PEO(10)SAO is Polyethoxylated (10) Stearylamine N-Oxide. The
AO-12-2 resulted in a product at 0.01% but not at 0.1 or 1%. AO-405
results in no visible product. The PEO(10)SAO at between 0.01% and
1% showed similar amounts of product as the inclusion of 0.1%
Tween-20. The number in parenthesis (X) refers to the average total
number of ethylene oxide units, so if there are two PEO groups and
X is 5 then the average length (n) is 2.5.
[0090] PMAL.RTM.-C12 is Poly (Maleic Anhydride-alt-1-Tetradecene)
substituted with 3-(Dimethylamino)Propylamine and has the following
structure:
##STR00012## [0091] PMAL.RTM.-C8 is Poly (Maleic
Anhydride-alt-1-Decene) substituted with
3-(Dimethylamino)Propylamine and has the following structure:
[0091] ##STR00013## [0092] PMAL.RTM.-C16 is Poly (Maleic
Anhydride-Alt-1-Octadecene) substituted with
3-(Dimethylamino)Propylamine and has the following structure:
##STR00014##
[0093] Because a small amount of Tween-20 and NP-40 was carried
over in the previous experiment, Taq DNA Polymerase was purified
without the addition of detergents after the lysis step to provide
a "detergent-free" Taq for use in the next example. The previous
numb target and reaction conditions were used with 0.625 units of
Taq per reaction. As can be seen in FIG. 2, without detergent no
PCR product is generated while both non-ionic and zwitterionic
surfactants with PEO.sub.n groups generate product.
[0094] The results shown in FIG. 3, demonstrate that PEO.sub.n of
different lengths and using amine-sulfoxide as the zwitterion
rather than amine-oxide also led to positive results using
detergent-free Taq DNA Polymerase.
[0095] PEO(X)SA0 is polyethyoxylated (X) stearylamine N-oxide,
where X is the sum of the number of PEO groups, and has the general
structure:
##STR00015##
and PEO(X)SAPS is polyethyoxylated (X)
Stearylammoniumpropylsulfonic acid, inner salt and has the general
structure:
##STR00016##
[0096] Because the results demonstrate that PEO polymeric groups
are useful in the methods, detergent-free Taq was tested using the
same assay against various other detergents without PEO.sub.n
groups. The results are shown in FIG. 4 and demonstrate the need
for a specific type of polymer surfactant. Note that the band in
the Amphipol lane is background fluorescence. FIG. 4 shows the
results of testing other zwitterionic detergents without PEO.sub.n
groups and a non-ionic, non-polymeric detergent, lauryl maltoside,
with detergent-free Taq DNA Polymerase. LAPAO is
3-Dodecylamido-N,N'-dimethylpropyl amine oxide
##STR00017## [0097] DDDAO is n-dodecyl-n,n-dimethylamine-n-oxide;
[0098] TDDAO is n-tetradecyl-n,n-dimethylamine-n-oxide;
[0098] ##STR00018## [0099] FOS-CHOLINE.RTM. is
n-Dodecylphosphocholine;
##STR00019##
[0099] and [0100] LM is Lauryl maltoside
##STR00020##
[0100] As can be seen from the results, PCR product was observed in
the presence of the non-ionic detergent Tween-20 and in the
presence of the PEO(10)SAO, but not when the other detergents
lacking PEO.sub.n groups were tested.
[0101] FIG. 5 shows the effect of different alkyl groups on
zwitterionic amine-oxide derivatives as well as polyethylene glycol
(chain length 1000) "PEG-1000". The data indicates that PEG-1000
does not function as a stabilizer, suggesting that micelle
formation may be important for stabilization.
[0102] To further test the effectiveness of PEO.sub.n-derivatives,
the denaturation temperature was changed from 95.degree. C. to
98.degree. C. in the cycling protocol. This increase in temperature
adds additional stress to the function of Taq DNA Polymerase. A
selection of PLURONIC.RTM. and TETRONIC.RTM. surfactants were
tested (labels in the figures are abbreviated, for example, "T908"
refers to TETRONIC.RTM. 908 and "P F87" refers to PLURONIC.RTM.
F87). In addition, TETRONIC.RTM. 904 and 1107 were modified to
amine-oxide, forms and then tested. FIG. 6 shows that not only are
blocked co-polymers effective, the amine-oxide TETRONIC.RTM.
derivatives also function as thermal stabilizers of DNA
polymerases.
[0103] In some aspects surfactants are selected for use in the
methods because they have a cloud point (temperature at which the
solid begins to precipitate and give the fluid a cloudy appearance)
greater than or equal to 80.degree. C., greater than or equal to
90.degree. C. and in particularly preferred aspects the cloud point
is greater than about 100.degree. C. (in each case when measured at
about 1% aqueous). The cloud point is the temperature above which a
surfactant solution separates into a detergent rich phase and a
detergent poor phase (see, Rosen et al. Surfactants and interfacial
Phenomena, .sup.3rd ed. 2004, Hoboken; John Wiley & Sons, Inc.
and Neugebauer, J.M. Detergents: an overview, Methods Enzymol,
1990, 182, p. 239-53). The separation is visualized as turbidity
within the solution. An increase in temperature favors micelle
formation; the rapid growth of micelles along with intermicellar
attraction likely results in the formation of large particles that
can precipitate out of solution, thus causing turbidity. This phase
separation is reversible upon cooling. Nonpolar additives (i.e.,
hydrocarbons) tend to increase the cloud point whereas polar
compounds (i.e., alcohols) and salts tend to decrease the cloud
point. For stabilization of thermal stable enzymes that are used
for PCR, which typically includes a denaturation step above
90.degree. C., higher cloud points are desirable.
[0104] The amine oxide detergents of the present invention have
higher cloud points than their non-amine oxide counterparts. For
example, PEO(10)stearlyamine has a cloud point of 60.degree. C.
compared to PEO(10)stearylamine N-oxide's cloud point of greater
than 100.degree. C., in both cases when measured at about 1%
detergent concentration in 6.times. SSPE buffer. In some aspects
detergents with cloud points above 90.degree. C. are preferred.
[0105] Table 1 summarizes the surfactants or blocked co-polymers
that worked in the assay used and those that did not.
TABLE-US-00001 Surfactant Function AO Y/Z/NI PEO groups Tween 20
YES NO NI NO NP 40 YES NO NI NO PEO (5)SAO YES YES Y YES PEO
(10)SAO YES YES Y YES PEO (15)SAO YES YES Y YES PEO(10)SAPS YES NO
Z YES PEO(5)LAO YES YES YES TETRONIC .RTM. 304 YES NO Y/Z YES
TETRONIC .RTM. 1107 YES NO Y/Z YES TETRONIC .RTM. 904 AO YES YES
Y/Z YES TETRONIC .RTM. 1107 AO YES YES Y/Z YES TETRONIC .RTM. 908
YES NO Y/Z YES TETRONIC .RTM. 1307 YES NO Y/Z YES PLURONIC .RTM.
P85 YES NO NI YES PLURONIC .RTM. 10R5 YES NO NI YES PLURONIC .RTM.
L35 YES NO NI YES PLURONIC .RTM. L64 YES NO NI YES PLURONIC .RTM.
P65 YES NO NI YES PLURONIC .RTM. P84 YES NO NI YES PLURONIC .RTM.
P1036 YES NO NI YES PLURONIC .RTM. P103 YES NO NI YES PLURONIC
.RTM. P104 YES NO NI YES PLURONIC .RTM. P105 YES NO NI YES PLURONIC
.RTM. P123 YES NO NI YES PLURONIC .RTM. F68 YES NO NI YES PLURONIC
.RTM. F77 YES NO NI YES PLURONIC .RTM. F87 YES NO NI YES PLURONIC
.RTM. F127 YES NO NI YES AO-12-2 NO YES Y NO AO-405 NO YES Y YES
LAPAO NO YES Y NO DDDAO NO YES Y NO TDDAO NO YES Y NO Amphipol
A8-35 NO NO Z NO Lauryl Maltoside NO NO NI NO PEG-1000 NO NO NI YES
PEO (5) E17 AO NO YES Z YES TETRONIC .RTM. 304-AO NO YES Z YES
FOS-CHOLINE .RTM.-12 NO NO z NO PMAL .RTM.-C8 NO NO Z NO PMAL
.RTM.-C10 NO NO Z NO PMAL .RTM.-C12 NO NO Z NO PMAL .RTM.-C16 NO NO
Z NO
[0106] In some aspects, the stabilization of thermostable
polymerases using the compounds disclosed herein are combined with
methods for modifying thermostable polymerases to be inactive until
activated by heating. Specifically, methods for inactivating
polymerases until they are heated for at least 10 minutes at a
temperature at or above 50.degree. C. are disclosed. Inhibiting the
activity of thermostable polymerases at lower temperature is known
to mitigate non-specific amplification that can generate
non-specific primer extension products that compete with
amplification of the desired target sequences. This non-specific
amplification can decrease the efficiency of the amplification of
the desired target. Problems caused by non-specific amplification
are discussed in Chou et al., 1992, Nucleic Acids Research
20(7):1717-1723, incorporated herein by reference. See also, Birch
et al. U.S. Pat. No. 5,677,152, which is incorporated herein by
reference.
[0107] Non-specific amplification can be reduced by reducing the
formation of extension products from primers bound to non-target
sequences prior to the start of the reaction. Several strategies
have been developed for minimizing non-specific extension. These
methods, referred to as a "hot-start" approaches, generally rely on
withholding one or more reagent until the temperature is raised
sufficiently to provide the necessary hybridization specificity.
Some hot-start methods use a heat labile material, such as wax, to
separate or sequester reaction components (see, U.S. Pat. No.
5,411,876). Another method of reducing the formation of
non-specific extension products relies on inhibition of the DNA
polymerase using a compound which non-covalently binds to the DNA
polymerase in a heat-reversible manner. U.S. Pat. No. 5,338,671,
describes polymerase inactivation by the use of antibodies specific
for a thermostable DNA polymerase. The antibodies are incubated
with the DNA polymerase to allow formation of the antibody-DNA
polymerase complex. Antibody inhibition of DNA polymerase activity
is inactivated by a high temperature pre-reaction incubation.
[0108] Another hot-start method uses the non-covalently binding of
a compound to the primers in a heat-reversible manner, thereby
preventing the primers from hybridization to any sequence, target
or otherwise. For example, single-stranded binding protein added to
a reaction mixture will bind the primers, thereby preventing primer
hybridization and inhibiting primer extension. Improvements in the
yield of PCR products using gene 32 protein are described in
Schwarz et al., 1990, Nucleic Acids Research 18(4):10.
[0109] Other hot-start methods rely on a reaction between the
enzyme and a modifier reagent, which results in a reversible
chemical modification of the enzyme, resulting in the loss of all,
or nearly all, of the enzyme activity. The modification consists of
the covalent attachment of the modifier group to the protein. The
modifier compound is chosen such that the modification is reversed
by incubation at an elevated temperature in the amplification
reaction buffer. The use of 2,3-substituted maleic anhydrides for
such inactivation has been disclosed, see U.S. Pat. No.
5,773,258.
[0110] The methods disclosed herein are similar to those of the
methods disclosed in U.S. Pat. No. 5,773,258, but involve
incubating the polymerase in the presence of
2-(Methylsulfonyl)ethyl 4-nitrophenyl carbonate (MSEC) under
conditions where lysines in the polymerase are modified reversibly.
The modification is reversed, activating the enzyme, by heating for
about 5-10 min at a temperature above 50.degree. C., preferably at
a temperature of about 90 to 95.degree. C. and more preferably
about 95.degree. C. Typical reaction conditions for PCR may be (i)
5 min at 95.degree. C., (ii) 15 sec at 95.degree. C., (iii) 60 sec
at 60.degree. C., (iv) 2 min at 68.degree. C., (v) repeat steps
(ii), (iii) and (iv) 35 times, (vi) 10 min at 68.degree. C. The
reaction conditions for the PCR using the MSEC modified enzyme may
also contain 5 mM-500 mM betaine. The dNTP concentration in the
reaction is may be about 100-400 uM of each of four dNTPs, (dATP,
dGTP, dTTP and dCTP). In one aspect the reaction conditions are 40
mM Tricine-KOH pH 8.0, 16 mM KCl, 3.5 mM MgCl2, 3.75 ug/ml BSA, 400
mM betaine, 1.4 mM dNTP (350 uM of each) and 2.5 to 30 U enzyme in
a 50 ul reaction. In one aspect the enzyme is present in the
reaction at about 0.25 units per ul. In another aspect, the
reaction contains 20 mM EPPS, pH 8.5, 50 mM NaOAc, 3.5 mM MgCl2, 5%
glycerol, 0.01% Tetronic-AO-1107, 400 uM each of dATP, dTTP, dGTP
and dCTP, and about 0.005 mg/ml modified enzyme. Primer
concentration may be 0.1 uM each primer for products greater than
500 bp in length or 0.2 uM each primer for products less than 500
bp in length. Betaine may also be added to the reaction under any
of the disclosed reaction conditions, preferably to about 400 mM.
In another aspect the PCR may be set up as follows:
TABLE-US-00002 Reagents A (.mu.l) B (.mu.l) C (.mu.l) Final Conc.
Nuclease-free water 33.75 33.75 33.0 10X PCR Reaction Buffer 1 5 1X
(71165) 10X PCR Reaction Buffer 2 5 1X (74155) PCR Nucleotide Mix
(10 mM) 1 1 1 200 .mu.M ea Taq DNA Polymerase (5 U/.mu.l) 0.25 2.5
units Exo-free Taq DNA Polymerase 0.25 2.5 units (5 U/.mu.l) MSEC
Taq DNA Polymerase 1 12.5 units (12.5 U/.mu.l) Forward/.lamda.
Primer #1 (10 .mu.M) 2.5 2.5 2.5 0.5 .mu.M Reverse/.lamda.Primer #2
(10 .mu.M) 2.5 2.5 2.5 0.5 .mu.M DNA Template (1 ng/.mu.l) 5 5 5 10
ng Total volume 50 50 50
[0111] Three classes of protecting groups were evaluated:
2,3-substituted maleic anhybrides, alpha-activated compounds that
can undergo beta-elimination and molecules that can undergo
intramolecular cyclic displacement or assisted elimination. The
experimental compounds had the general structure:
##STR00021##
[0112] Where Y could be NHS, pNP, or imidazole and X could be
CONMe.sub.2, MeSO.sub.2, CN, NO.sub.2, CO.sub.2Me,
(CH.sub.2).sub.2SMe, N(CH.sub.3)CHO, or CH.sub.2C(O)CH.sub.3. The
reactivity was tested in Tris pH 8 or Tricine pH 8.8. Buffer pH
measurements are at room temperature (.about.25.degree. C.). The
compound found to be most effective of the test compounds was
2-(Methylsulfonyl)ethyl 4-nitrophenyl carbonate or MSEC. MSEC has
the following structure:
##STR00022##
[0113] The MSEC modifies lysines in the enzyme. The enzyme to be
modified is preferably a thermostable DNA polymerase and may be a
5' exonuclease domain deletion mutant (exo-), e.g. of Taq DNA
polymerase (exo-), or may retain the 5' exonuclease domain (exo+).
In preferred aspects MSEC is used to reversibly modify Taq DNA
polymerase. To inactivate the Taq DNA polymerase an appropriate
volume of a freshly prepared 100.times. solution (120 mM) of MSEC
(SIGMA PN 69227) is added to a 1 mg/ml solution of Taq in acylation
buffer (100 mM Bicine pH 8.5, 1 mM EDTA, 100 mM KCl). The MSEC
solution should preferably be added to the Taq within about 5 min
of preparation as it degrades over time. After adding the MSEC
solution to the Taq the solution is mixed by inverting, preferably
end over end about 5 times, and then placed in a 25.degree. C.
incubator for 2 hours without rotating or stirring. The MSEC
reaction with lysines results in a change of the buffer to a yellow
color. Following this incubation the Taq is dialyzed against
transition buffer (20 mM EPPS pH 7.5, 100 mM KCl, 0.1 mM EDTA, 1%
Tetronic-AO, 10% glycerol and 1 mM DTT) and then against a final
buffer (20 mM EPPS pH 7.5, 100 mM KCl, 0.1 mM EDTA, 1% Tetronic-AO,
50% glycerol and 1 mM DTT).
[0114] In another aspect FRET-based molecules to be used as passive
reference dyes for qPCR are included in the reaction mixture. The
sample being amplified in a PCR reaction may contain one or more
types of dye molecules that serve as a "passive" reference having
some fluorescence in the same wavelength range as the DNA binding
dye. This reference dye is made up, for example, of a nucleic acid
sequence labeled with Rhodamine and Fluorescein dye derivatives.
These passive dye molecules do not take part in the PCR reaction,
so that their fluorescence is substantially without influence from
DNA and remains constant during the reaction. This fluorescence can
be used to normalize the fluorescence from the DNA binding dye with
a standard concentration of passive dye included in the ingredients
of at least one vial, preferably in every vial. Passive reference
dyes have been described, for example, in U.S. Pat. No. 6,818,437.
The dye is used as an internal reference for fluorescent signal
normalization and correction of well-to-well optical variation and
pipetting errors. The dye is preferably matched to the excitation
optics of the instrument being used. The dye may be made available
in a solution, for example a 25 um solution, and may be in a
buffer, for example, 10 mM Tris-HCl (pH 8.6), 0.1 mM EDTA and 0.01%
Tween-20.
[0115] In preferred embodiments the compounds will be compatible
with a broad range of commercial qPCR platforms, molecules will
preferably exhibit good solubility, thermal stability, and
efficient and stable 488 nm excitation and 610 nm emission
characteristics under typical conditions used in RT PCR. The
molecules of this invention consist of combinations of a
fluorescein (FAM) or FAM analog as the donor dye and a
rhodamine-101 (ROX) or ROX analog as acceptor dye, the two being
connected by a stable covalent attachment to allow efficient FRET.
The following molecules were evaluated. The first compound was
referred to as "Dye 592" and has structure:
##STR00023##
[0116] The second was FRET Dye 1 where the donor is 6FAM and the
acceptor is ROX. The structure is:
##STR00024##
[0117] FRET Dyes 2-4 where the donor is 6-FAM and the acceptor is
CalFluorRed 610 with structures:
##STR00025##
* * * * *