U.S. patent application number 10/588587 was filed with the patent office on 2010-02-11 for anti-freeze protein enhanced nucleic acid amplification.
Invention is credited to Jessica Jaclyn Greenlee, Lars-Erik Peters, Ryan Smith Westberry.
Application Number | 20100035238 10/588587 |
Document ID | / |
Family ID | 34860246 |
Filed Date | 2010-02-11 |
United States Patent
Application |
20100035238 |
Kind Code |
A1 |
Westberry; Ryan Smith ; et
al. |
February 11, 2010 |
Anti-freeze protein enhanced nucleic acid amplification
Abstract
Methods and compositions are provided for enhanced signal
intensity and storage stability of standard nucleic acid
amplification buffers, real-time PCR buffers or both. Buffers in
accordance with the present invention include anti-freeze
protein(s) (AFPs), optionally with a carrier protein, such as
BSA.
Inventors: |
Westberry; Ryan Smith;
(Westminster, CO) ; Peters; Lars-Erik; (Lafayette,
CO) ; Greenlee; Jessica Jaclyn; (Lafayette,
CO) |
Correspondence
Address: |
DORSEY & WHITNEY LLP;INTELLECTUAL PROPERTY DEPARTMENT
370 SEVENTEENTH STREET, SUITE 4700
DENVER
CO
80202-5647
US
|
Family ID: |
34860246 |
Appl. No.: |
10/588587 |
Filed: |
February 4, 2005 |
PCT Filed: |
February 4, 2005 |
PCT NO: |
PCT/US05/03502 |
371 Date: |
August 12, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60541999 |
Feb 4, 2004 |
|
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|
Current U.S.
Class: |
435/6.11 ;
435/188; 435/6.1; 435/6.18; 435/91.1 |
Current CPC
Class: |
C12Q 1/6853 20130101;
C12Q 1/6848 20130101; C12Q 1/6853 20130101; C12Q 1/6848 20130101;
C12Q 2525/101 20130101; C12Q 2527/125 20130101; C12Q 2525/121
20130101; C12Q 2527/125 20130101; C12Q 2525/121 20130101; C12Q
2525/101 20130101 |
Class at
Publication: |
435/6 ; 435/188;
435/91.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 9/96 20060101 C12N009/96; C12P 19/34 20060101
C12P019/34 |
Claims
1-98. (canceled)
99. An enzyme solution comprising an anti-freeze protein and an
enzyme; wherein said enzyme retains enzymatic activity after at
least one freeze/thaw event.
100. The enzyme solution according to claim 99, wherein said enzyme
retains activity after more than ten freeze/thaw events.
101. The enzyme solution according to claim 99, further comprising
a buffer.
102. The enzyme solution according to claim 101, wherein said
buffer is zwitterionic.
103. The enzyme solution according to claim 99, further comprising
a carrier protein.
104. The enzyme solution according to claim 103, wherein said
carrier protein is bovine serum albumin (BSA).
105. The enzyme solution according to claim 99, wherein said
anti-freeze protein comprises an alanine-rich motif.
106. The enzyme solution according to claim 99, wherein said
anti-freeze protein is an AFP Type I protein.
107. The enzyme solution according to claim 101, wherein said
enzyme solution has a pH from about 7.9 to about 8.9.
108. The enzyme solution according to claim 99, further comprising
a polyol.
109. The enzyme solution according to claim 108, wherein said
polyol is selected from the group consisting of sorbitol and
trehalose.
110. The enzyme solution according to claim 108, wherein said
polyol comprises sorbitol and trehalose.
111. The enzyme solution according to claim 99, wherein said
anti-freeze protein has a concentration of from about 10 ug/ml to
about 200 ug/ml.
112. The enzyme solution according claim 99, wherein said enzyme is
a DNA polymerase and the addition of said enzyme solution to an
amplification reaction mixture improves the sensitivity and yield
of the nucleic acid amplification reaction.
113. A reaction mixture for use in a nucleic acid amplification
reaction, comprising dNTPs and an enzyme solution according to
claim 112.
114. A method for enhancing the stability of an enzyme over the
course of two or more freeze/thaw events, comprising the addition
of an anti-freeze protein to an enzyme solution containing said
enzyme prior to said freeze thaw events.
115. A method for increasing the sensitivity and yield of a nucleic
acid amplification reaction, comprising combining a target nucleic
acid sequence with at least one primer in a reaction mixture
according to claim 113 and amplifying said target nucleic acid
sequence, wherein the inclusion of said anti-freeze protein
increases amplicon yield and sensitivity.
116. An improved method for detecting a target nucleic acid
sequence in a sample, comprising combining said sample with at
least one primer in a reaction mixture according to claim 113 and
amplifying said target nucleic acid sequence; wherein the inclusion
of said anti-freeze protein increases signal intensity and improves
the signal-to-noise ratio.
117. An improved method for quantifying a target nucleic acid
sequence in a sample, comprising combining said sample with at
least one primer in a reaction mixture according to claim 113 and
amplifying said target nucleic acid sequence; wherein the inclusion
of said anti-freeze protein increases signal intensity and improves
the signal-to-noise ratio.
118. A kit comprising: a solution comprising an anti-freeze protein
and an enzyme.
119. The kit of claim 117 wherein the solution further comprises a
carrier protein.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 60/541,999 titled "METHODS AND COMPOSITIONS TO
ENHANCE AMPLIFICATION EFFICIENCY AND SIGNAL," filed Feb. 4, 2004,
which is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] The invention generally relates to methods and compositions
for increasing amplification yields in nucleic acid amplification
reactions, for enhancing signal intensity and improving the signal
to noise ratio in nucleic acid detection and quantification
methods, such as real time polymerase chain reactions (real time
PCR), and for increasing the stability of amplification enzyme
solutions for use in such reactions over repeated freeze/thaw
cycles.
BACKGROUND OF THE INVENTION
[0003] The ability to prepare large amounts of nucleic acid
molecules is requisite to a number of protocols in molecular
biology, as well as a basic requirement in numerous downstream uses
in biotechnology and clinical research. For example, amplified
nucleic acid molecules are often used in cloning experiments, DNA
sequencing reactions, restriction digestion reactions, and
subsequent ligation reactions, and these uses are all, or to some
extent, dependent on the quality and quantity of the starting DNA
material. As such, there has been, and continues to be, a need for
reliable methods for preparing large amounts of quality,
sequence-specific nucleic acid molecules.
[0004] In addition, the ability to detect and/or quantify target
nucleic acid molecules from a mixed starting material is useful in
a number of clinical, industrial and basic research applications.
For example, sensitive and accurate detection and quantification of
viral nucleic acid sequences in a patient sample is helpful in a
clinical setting for accurate diagnosis and subsequent treatment of
a patient. Such detection and quantification processes generally
require amplification of one or more target nucleic acid molecules
present in the starting material. As such, there has been, and
continues to be, a need for facilitating the detection and
quantification of target nucleic acid sequences from a starting
material, which again requires reliable methods for preparing large
amounts of quality, sequence-specific nucleic acid molecules.
[0005] The predominant approach for amplifying nucleic acid is via
the polymerase chain reaction (PCR). PCR is a convenient in vitro
amplification process useful in the exponential increase of
template nucleic acid. Particular applications of PCR include the
detection and/or quantification of target gene expression as well
as confirmation of differential expression of target genes detected
using array techniques. In general, optimization of PCR techniques
could facilitate both the accuracy (i.e. sequence specificity) and
total levels or amounts of the amplified product (amplicon), and
optimization of real time-PCR techniques could facilitate the
sensitivity and suppress nonspecific amplification during the
procedure. Each real time assay relies upon the release of a
detectable signal upon production of a PCR product, and nonspecific
amplification is particularly problematic when the starting
material is small. In addition, there is a need for greater
stability of the constituent enzymes involved in the PCR reaction,
both during the reaction and during storage of the relevant
constituents of the reaction prior to combination with the reaction
mixture.
[0006] Conventionally PCR optimization has focused on modifying
standard PCR buffers, altering primer annealing temperatures,
providing more effective thermostable polymerase enzymes, and
designing more effective primer molecules. With regard to product
quantification in real-time PCR, optimization has focused on the
development of two relatively new assays: the TaqMan.TM. method of
real time PCR employing intercalating dyes and binary hybridization
probes (Lee et al., 1993 Nucleic Acids Res., 21(16:3761.6)). In
either situation, further reduction of non-specific amplification,
enhancement of signal intensity and facilitation of enzyme
stability is critically needed to provide more sensitive, accurate
and robust results.
[0007] Against this backdrop the present invention has been
developed.
SUMMARY OF THE RELEVANT LITERATURE
[0008] Anti-freeze proteins (AFPs) are a class of ice-binding
proteins prevalent in organisms that live at or below freezing
temperatures. The first AFP discovered was from the blood of a
species of Antarctic fish. DeVries (1969) Science 163, 1073-1075.
Since these early discoveries, a number of organism have been
identified that express these proteins, including fish, insects,
plants and microorganisms. Jia et al. (2002) TRENDS in Biochem.
Sciences, 27:2, 101-106. AFPs have been classified based on protein
sequence and structural characteristics, a number of which have
significantly different structures. A number of different AFP-based
structures have been identified within these analysis, including:
helix bundle proteins, helical proteins with Thr residues arrayed
on one side of the protein, helical proteins with Ala-Ala-Thr
repeats, and globular type proteins.
[0009] In recent years, AFP proteins, and especially AFP type I
proteins, have been used in cosmetic applications for general
product stability. In addition, AFP has been used in a limited
setting as a food additive to help protect frozen food under
freezing conditions, for cold protection in mammalian cells, and
for enhanced tumor cell destruction during cytosurgery. Fletcher et
al. (1999) CHEMTECH 30(6):17-28.
SUMMARY OF THE INVENTION
[0010] The present invention provides compositions and methods for
improving the performance of nucleic acid amplification reactions,
where the reaction typically comprises at least one cycle of a
denaturation step, an annealing step, and an extension step.
Compositions of the present invention provide the beneficial effect
of improving enzyme stability over the course of numerous
freeze/thaw events and, more surprisingly, also significantly
enhance the performance of the amplification reaction and
dramatically increased signal intensity and improve signal to noise
ratios in associated detection and quantification methods. The
present invention provides amplification reaction mixture
compositions comprising an anti-freeze protein (AFP), an AFP
combined with a carrier protein such as, e.g., BSA and the like, an
AFP combined with a polyol, and an AFP combined with a carrier
protein and a polyol.
[0011] In one aspect of the present invention, methods and
compositions are provided for improving the stability of an enzyme
in an amplification enzyme solution, where the enzyme solution is
subjected to at least one freeze/thaw cycle before use in a
subsequent amplification reaction. In one embodiment, the enzyme
solution comprises AFP, and optionally further comprises a carrier
protein and/or a polyol. In a preferred embodiment, the AFP is
added to the enzyme solution prior to, or contemporaneous with, the
freeze/thaw cycle.
[0012] In another aspect of the present invention, methods and
compositions are provided for improving the performance of nucleic
acid amplification reactions in general, and related detection and
quantification methods in particular, comprising including in the
amplification reaction mixture at least one anti-freeze protein
optionally in combination with a carrier protein in a zwitterionic
buffer. As demonstrated herein, these novel amplification reaction
mixtures improve amplicon yield and signal intensity, increase the
signal-to-noise ratio, and enhance the overall sensitivity of the
reactions.
[0013] In a particular embodiment, improved nucleic acid detection
and quantification methods are provided comprising a nucleic acid
amplification reaction in which the reaction mixture comprises at
least one anti-freeze protein to improve the signal-to-noise ratio
of the reaction. Preferred anti-freeze protein(s) have one or more
alanine-rich motifs for enhancement of signal and sensitivity. In
particularly preferred embodiments, a carrier protein such as,
e.g., BSA or other like protein, is included with the anti-freeze
protein in the reaction mixture to maximize the signal-to-noise
ratio. Surprisingly, the combination of AFP and carrier protein
provides a synergistic improvement in the signal-to-noise ratio
over AFP alone or carrier protein alone.
[0014] In a preferred embodiment, a polyol is included in
amplification reaction mixtures comprising a template nucleic acid
having a degree of secondary structure. In a particularly preferred
embodiment, the polyol is selected from the group consisting of
sorbitol, maltitol, adonitol, arabitol and mannitol. Ancillary
materials can also be included with these reaction mixtures,
including, but not limited to single-stranded binding protein,
n-propyl sulfoxide, and the like, to further facilitate the
amplification reaction.
[0015] Additional embodiments of the present invention include
compositions for maximizing signal amplification during real time
PCR where release of fluorescent signal upon 5'-3' nucleolytic
degradation is anticipated. Preferred compositions and methods of
the present invention employ reaction mixtures comprising at least
one anti-freeze protein, preferably having one or more alanine rich
motifs (for example exhibited by AFP type I), alone or in
combination with a carrier protein. In particularly preferred
embodiments, the reaction mixtures are prepared with a zwitterionic
buffer having a pH of between about 7.9 and about 8.1. In another
preferred embodiment, the nucleic acid quantification method is
real time PCR and the addition of the anti-freeze protein and
carrier protein provides for synergistic signal amplification.
[0016] These and various other features and advantages of the
invention will be apparent from a reading of the following detailed
description and a review of the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a stained 1% agarose gel showing yields of PCR
reaction products from reactions having from 0 to 200 mg/ml
anti-freeze protein 1 included within the reaction.
[0018] FIGS. 2A and 2B graphically illustrate signal amplification
synergy between anti-freeze protein 1 and BSA during PCR (A)
Average RFU and (B) Threshold Cycle (Ct).
[0019] FIGS. 3A and 3B graphically illustrate that inclusion of BSA
and AFP1 in the PCR Master Mix facilitates storage of the buffer at
-20.degree. C. FIG. 3A shows average RFU values and FIG. 3B shows
threshold cycle values. Buffer samples were either: BSA alone, AFP1
alone, BSA and AFP1 combined or no additives.
[0020] FIGS. 4A and 4B graphically illustrate PCR buffer
compositions including AFP1, BSA, and assorted combinations of
buffer and salt, (A) threshold cycle and (B) average RFU.
[0021] FIG. 5 graphically compares the threshold cycle for
Real-Time PCR products prepared in 25 mM TAPS-KOH with 15 mM KCl
pH8, 25 mM TAPS-Tris with 50 mM KCl pH 8.0, and 25 mM Bicine-Tris
with 50 mM KCl pH 8.4.
[0022] FIG. 6 graphically compares the threshold cycle for
Real-Time PCR products prepared in 25 mM Bicine-Tris with 50 mM KCl
pH 8.4, 25 mM TAPS-Tris with 50 mM KCl pH 8.0, and Invitrogen
Platinum qPCR Supermix-UDG, used according to manufacturers
standards.
[0023] FIG. 7 graphically compares the threshold cycle for
real-time PCR products prepared in the presence of no additives,
BSA, AFGP, AFGP and BSA, AFP1 and BSA, and AFGP, AFP1 and BSA.
[0024] FIGS. 8A and 8B graphically compares the RFU for long term
stability testing of an embodiment of the present invention as
compared to testing performed with the Invitrogen Platinum qPCR
Supermix UDG product, purchased and performed using Invitrogen
protocol.
[0025] FIGS. 9A and 9B graphically represent that inclusion of 100
mM sorbitol in RT-PCR Master Mix facilitates storage stability at
-20.degree. C. FIG. 9A shows Ct values and FIG. 9B shows RFU values
from PCR performed after freeze/thaw on sorbitol containing and
non-containing master mix buffers for one or three weeks. Samples
were either stored at 4, -20 or -80.degree. C.
[0026] FIGS. 10A and 10B are stained 1% agarose gels showing RT PCR
reaction products after one-week (10A) or three-week (10B) of
storage at either 4, -20 or -80.degree. C. in zero, 100 mM, or 200
mM sorbitol.
[0027] FIG. 11 graphically represents the performance consistency
of the embodiment of the present invention across 15 freeze/thaw
cycles.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The following definitions are provided to facilitate
understanding of certain terms used frequently herein and are not
meant to limit the scope of the present disclosure:
[0029] As used herein, "amplification reaction" or "nucleic acid
amplification reaction" refers to any in vitro method for
increasing the number of copies of a desired nucleic acid sequence
with the use of a DNA polymerase. Nucleic acid amplification
reactions include, for example, the polymerase chain reaction (PCR)
(as described in U.S. Pat. Nos. 4,683,195 and 4,683,202, which are
hereby incorporated by reference), Nucleic Acid Sequence-Based
Amplification (NASBA) (as described in U.S. Pat. No. 5,409,818,
which is hereby incorporated by reference) and Strand Displacement
Amplification (SDA) (as described in U.S. Pat. No. 5,455,166, which
is hereby incorporated by reference). As is well known in the art,
such reactions find advantageous use in numerous nucleic acid
detection methods for determining the presence of one or more
target nucleic acid sequences in a sample, as well as in a wide
variety of nucleic acid quantification methods for quantifying the
amount of amplicon(s) produced by the reaction.
[0030] As used herein, "antisense" refers to polynucleotide
sequences that are complementary to target "sense" polynucleotide
sequences.
[0031] As used herein, "carrier protein(s)" refers to Bovine Serum
Albumin (BSA), Prionex, Cold Water Fish Gelatin, gelatin, Gro L,
Gro S, DNAK, Heat Shock Protein 70 (HSP70), Apolipoprotein, as well
as other like serum albumins.
[0032] As used herein, "Ct shift" or "threshold cycle" refers to
the cycle at which an amplification product is detectable, a Ct
shift of 1.5 to 3 cycles is equivalent to an approximate 5 to 10
fold higher DNA.
[0033] As used herein, "nucleic acid" or "NA" refers to both a
deoxyribonucleic acid (DNA) and a ribonucleic acid (RNA), as well
as modified and/or functionalized versions thereof. Similarly, the
term "nucleotide" as used herein includes both individual units of
ribonucleic acid and deoxyribonucleic acid as well as nucleoside
and nucleotide analogs, and modified nucleotides such as labeled
nucleotides. In addition, "nucleotide" includes non-naturally
occurring analog structures, such as those in which the sugar,
phosphate, and/or base units are absent or replaced by other
chemical structures. Thus, the term "nucleotide" encompasses
individual peptide nucleic acid (PNA) (Nielsen et al., Bioconjug.
Chem. 1994; 5(1):3-7) and locked nucleic acid (LNA) (Braasch and
Corey, Chem. Biol. 2001; 8(1):1-7) units.
[0034] As used herein, "polynucleotide," "oligonucleotide" or
grammatical equivalents thereof means at least two nucleotides
covalently linked together. As will be appreciated by those of
skill in the art, various modifications of the sugar-phosphate
backbone may be done to increase the stability of such molecules in
physiological environments, including chemical modification such
as, e.g., phosphorothioate or methyl phosphonate. Further, such
molecules may be functionalized by coupling with one or more
molecules having distinct characteristic properties for purposes
of, e.g., facilitating the addition of labels.
[0035] As used herein, "nucleic acid sequence" refers to the order
or sequence of nucleotides along a strand of nucleic acids. In some
cases, the order of these nucleotides may determine the order of
the amino acids along a corresponding polypeptide chain. The
nucleic acid sequence thus codes for the amino acid sequence. The
nucleic acid sequence may be single-stranded or double-stranded, as
specified, or contain portions of both double-stranded and
single-stranded sequences. The nucleic acid sequence may be
composed of DNA, both genomic and cDNA, RNA, or a hybrid, where the
sequence comprises any combination of deoxyribo- and
ribo-nucleotides, and any combination of bases, including uracil
(U), adenine (A), thymine (T), cytosine (C), guanine (G), inosine,
xathanine hypoxathanine, isocytosine, isoguanine, etc.
[0036] As used herein, "complementary" or "complementarity" refers
to the ability of a nucleotide in a polynucleotide molecule to form
a base pair with another nucleotide in a second polynucleotide
molecule. For example, the sequence 5'-A-C-T-3' is complementary to
the sequence 3'-T-G-A-5'. Complementarity may be partial, in which
only some of the nucleotides match according to base pairing, or
complete, where all the nucleotides match according to base
pairing. For purposes of the present invention "substantially
complementary" refers to 95% or greater identity over the length of
the target base pair region.
[0037] As used herein, "expression" refers to transcription and
translation occurring within a host cell. The level of expression
of a DNA molecule in a host cell may be determined on the basis of
either the amount of corresponding mRNA that is present within the
cell or the amount of DNA molecule encoded protein produced by the
host cells. Further detail for the term "expression" within the
context of the present invention can be obtained via a review of
Sambrook et al., 1989, Molecular Cloning; A Laboratory Manual;
18.1-18.88.
[0038] As used herein, "freeze/thaw" conditions are characterized
by freezing a target material at a temperature typically below
0.degree. C. and preferably at a temperature not to go below about
-30.degree. C. to about -40.degree. C. Thaw conditions typically do
not exceed room temperature. A typical slow-freeze/thaw cycle is
where a material is frozen between about 0.degree. C. and about
-40.degree. C., stored at that temperature for some period of time,
and thawed between about 1.degree. C. to about 27.degree. C.,
dependent on the end use required.
[0039] As used herein, "host cell" or "host cells" refers to cells
expressing or capable of expressing a heterologous polynucleotide
molecule, for example a plasmid vector. Host cells of the present
invention express polynucleotides encoding polypeptides useful in
any number of uses, including biotechnological, molecular
biological and clinical settings. Examples of suitable host cells
in the present invention include, but are not limited to,
bacterial, yeast, insect and mammalian cells. Specific examples of
such cells include, E. Coli DH5a cells, as well as various other
bacterial cell sources, for example the E. Coli strains: DH10b
cells, XL1Blue cells, XL2Blue cells, Top10 cells, HB101 cells, and
DH12S cells, and yeast host cells from the genera including
Saccharomyces, Pichia, and Kluveromyces.
[0040] As used herein, "isolated" and "purified" for purposes of
the present invention are interchangeable, and refer to a
polynucleotide, for example a target nucleic acid sequence, that
has been separated from cellular debris, for example, high
molecular weight DNA, RNA and protein. This would include an
isolated RNA sample that would be separated from cellular debris,
including DNA.
[0041] As used herein, "protein," "peptide," and "polypeptide" are
used interchangeably to denote an amino acid polymer or a set of
two or more interacting or bound amino acid polymers.
[0042] As used herein, "real-time PCR" refers to quantitative PCR
techniques that typically use fluorescence probes, beacons, and/or
intercalating dyes during all cycles of the process.
[0043] As used herein, "stringency" refers to the conditions, i.e.,
temperature, ionic strength, solvents, and the like, under which
hybridization between polynucleotides occurs. Hybridization being
the process that occurs between the primer and template DNA during
the annealing step of the amplification process.
[0044] Embodiments of the present invention provide methods and
compositions for enhancing the overall performance of nucleic acid
amplification reactions and improving related detection and
quantification methods. In particular, embodiments of the present
invention are directed toward facilitating the signal intensity of
nucleic acid amplification reactions in general, enhancing signal
amplification and/or improving signal-to-noise ratios in nucleic
acid detection and quantification methods, and increasing the
stability for enzyme solutions through one or several freeze/thaw
cycles before their use in such reactions and methods.
[0045] Preferably, specific embodiments of the present invention
include the following: inclusion of anti-freeze protein(s) (AFP) to
enhance both signal amplification (RFU) and sensitivity (threshold
cycle) during real-time PCR; inclusion of AFP within enzyme
solutions to enhance storage stability during freeze/thaw
conditions; inclusion of a carrier protein, for example BSA, with
the AFP to provide an additional synergistic effect on signal size
and sensitivity, as well as on storage stability; inclusion of
sorbitol or other like polyol material with the AFP in a mixture to
both enhance the product yield and further improve stability during
freeze/thaw cycles; and inclusion of dUTP, polyol, and AFP in a
zwitterionic buffer formulation, i.e., TAPS-Tris with KCL or
TAPS-KOH with KCl based buffer, to provide a high performance
nucleic acid amplification buffer, e.g., PCR buffer (increases
sensitivity, specificity, signal intensity and storage stability),
especially where the pH is between about 7.9 and about 8.1.
Anti-Freeze Proteins (AFP)
[0046] Anti-freeze proteins (AFPs) represent a family of proteins
that contribute freeze resistance and freeze tolerance to a number
of species that thrive in freezing conditions. In general, AFP
molecules within the organism bind to the surface of seed ice
crystals and control the crystal's growth. See Jia et al., (2002)
TRENDS in Biochem. Sciences, 27(2) 101-106. A number of different
organisms have been found to express AFPs, including several
species of fish and several species of insect. Fish AFPs are
classified into five groups (see Table 1), based principally on
primary and secondary structural analysis. With regard to insect
AFPs, several insect species, including Tm or Dc (beetle) and Cf
(moth), have been classified into two groups. AFPs have been
isolated from several plant species as well Atici et al., (2003)
Phytochemistry, 64(7) 187-96.
[0047] Importantly, several different identified structural motifs
and motif repeats within the AFP family have been shown to function
similarly to inhibit ice growth, including an alanine-rich helix
having 11 amino acids (aa) (three turns of a helix) (AFP Type 1) or
a 3 aa repeat of Ala-Ala-X (side-chain disaccharide) motif (AFGP)
where X is serine, arginine, lysine, asparagine, glutamine, or
threonine. Additional structural information can be obtained from
Jia et al. and is shown in Table 1 (see also U.S. Pat. No.
6,017,574 which is incorporated herein by reference).
TABLE-US-00001 TABLE 1 Structural Characteristics of AFPs AFGP
(Anti-Freeze Characteristic Glycoprotein) AFP Type I AFP Type II
AFP Type III AFP Type IV MW ~2,600-33,000 ~3,300-4,500
~11,000-24,000 ~6,500 ~12,200 1.degree. Structure (ala-ala-thr)n
ala rich cystine rich general 17% gluramine; disaccharide repeats -
disulphide lack of disulphide typically 11 aa linked bridges
repeats Carbohydrate Yes No Generally not No No Linked 2.degree.
Structure Expanded alpha helical beta sheet beta amphipathic
amphiphilic sandwich alpha helix 3.degree. Structure NA 100% helix
NA NA Four-helix Antiparallel Bundle Biosynthesis Multi-protein
Prepro AFP Prepro-AFP Pro-AFP No Post- translational Modifications
Protein 8 7 2-6 12 1 Components Gene Copies NA 80-100 15 30-150 NA
Natural Source Antarctic Right-eyed Sea raven; Ocean pout; Longhorn
Sculpin Notothenioids; Flounders; smelt; herring Wolffish Northern
Cod Sculpins
[0048] The present invention provides compositions and methods for
enhancing signal amplification in nucleic acid detection and
quantification methods such as, e.g., real-time PCR, as well as for
increasing the stability of enzyme solutions employed in nucleic
acid amplification reactions.
[0049] In one embodiment, the improved amplification reaction
mixtures provided herein generally include primer(s), template DNA,
dNTPs and an appropriate amplification enzyme, for example Taq DNA
polymerase, and specifically include at least one AFP. In preferred
embodiments, the AFP is selected from the group having an
alanine-rich helix or a repetitive repeat of
-Ala-Ala-X-(disaccharide), derived from AFP type I or AFGP,
respectively. In addition, fragments of AFP including the
alanine-rich helix motif derived from AFP type I (See for example
U.S. Pat. No. 5,925,540, the disclosure of which is expressly
incorporated herein by reference), or repetitive repeats of
-Ala-Ala-Thr-(disaccharide) derived from AFGP, and variants,
derivatives, isoforms and fusion proteins thereof (all these
examples are referred to generally as AFP for purposes of the
invention) are useful in these regards. In preferred embodiments
the amplification enzyme solution and/or reaction mixture further
includes a carrier protein, for example BSA or gelatin, to enhance
the AFP-derived results. As demonstrated herein, the inclusion of a
carrier protein in the reaction mixture with AFP results in a
synergistic effect greater than that of AFP alone or carrier
protein alone.
[0050] Embodiments in accordance with the present invention
typically include an enzyme solution of AFP from about 10-200
.mu.g/ml, and preferably from 25-100 .mu.g/ml and most preferably
from about 40-60 .mu.g/ml. Inclusion of carrier protein with the
AFP is typically at a concentration of about 100 .mu.g/ml to about
300 .mu.g/ml, and preferably from about 150 to about 250 .mu.g/ml,
and most preferably about 200 .mu.g/ml. Inclusion of AFP and
carrier protein in standard real-time PCR buffers provide a
significant improvement in signal amplification as measured by
average RFU, and cycle of detection as measured by threshold cycle.
As shown in the Examples that follow, the combination of AFP with a
carrier protein provides a synergistic improvement in signal
strength or intensity and sensitivity of such assays.
[0051] AFP for use in the present invention can be purchased from
AFP Proteins, Inc., Waltham, Mass. In addition, AFP, and fragments,
derivatives and fusion proteins of AFP in accordance with the
invention can be expressed in insect, yeast, prokaryote, and
eukaryote cells. Suitable prokaryotic hosts to be used for
expression include but are not limited to bacteria of the genera
Escherichia, Bacillus and Salmonella. AFP sequence for design of
recombinant protein expression can be obtained from a review of Jia
et al which is and was previously incorporated herein by reference
in its entirety (also see U.S. Pat. No. 5,925,540, also
incorporated in its entirety).
[0052] Modifications of the amino acid sequence of AFP molecules
useful in the present invention can be accomplished by a number of
known techniques. For example, mutations may be introduced at
particular locations by oligonucleotide-directed mutagenesis
(Walder et al., 1986, Gene, 42:133; Bauer et al., 1985, Gene 37:73,
Craik, 1985, Biotechniques, 12-19; Smith et al., 1981, Genetic
Engineering: Principles and Methods, Plenum Press; U.S. Pat. No.
4,737,462). Modifications may be useful in the enhancement of AFP
activity as discussed herein and shown in the Examples.
[0053] AFP polypeptides of the present invention are preferably
used in an isolated or partially isolated form. The polypeptides
may be recovered and purified from recombinant cell cultures by
known methods, including, for example, ammonium sulfate or ethanol
precipitation, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography and lectin
chromatography.
[0054] AFP polypeptides can be fused to heterologous polypeptides
to facilitate purification. Many available heterologous peptides
allow selective binding of the fusion protein to a binding partner.
Non-limiting examples of peptide tags include 6-His, thioredoxin,
hemagglutinin, GST, and the OmpA signal sequence tag. A binding
partner that recognizes and binds to the heterologous peptide can
be any molecule or compound, including metal ions, antibodies,
antibody fragments, or any protein or peptide that preferentially
binds the heterologous peptide to permit purification of the fusion
protein.
[0055] The present invention further provides amplification enzyme
solutions and amplification buffers, for example real-time PCR
buffers, having enhanced stability for use after numerous
freeze/thaw cycles, and extended storage times from 4.degree. C. to
-80.degree. C. (see Examples below). As above, standard
amplification buffer ingredients are mixed with AFP or with AFP and
a carrier protein, using the concentrations discussed above, to
minimize enzyme degradation between any two slow freeze/thaw
cycles. Note that AFP is particularly effective in stabilizing
against slow freeze events, for example freezing an enzyme solution
between the temperatures of 0.degree. C. and -40.degree. C.
[0056] As such, and without being bound by theory, it is believed
that the inclusion of AFP or AFP in combination with a carrier
protein results in the dual benefit of stabilizing the
amplification enzyme during repetitive freeze/thaw cycles, and
surprisingly further enhances the sensitivity and signal strength
of the subsequent nucleic acid amplification reaction. Typically,
stability is provided for freeze conditions between 0.degree. C. to
about -40.degree. C. Kits having an amplification buffer, for
example a real-time PCR buffer, that include appropriate amounts of
AFP can be packaged to maximize the sensitivity of the assay and
stabilize the storage of the buffer under slow-freeze conditions.
In addition, AFP or AFP and carrier protein can be packaged with a
desired polymerase for addition to a PCR or other like
amplification reaction mixture.
[0057] Note that although the inclusion of AFP in PCR buffers has
been shown to enhance polymerase-based amplification reactions and
detection and quantification methods employing the same, it is also
envisioned to be useful in the storage of enzymes having other
desirable activities. For example, AFP may find use in any enzyme
solution that will undergo slow freezing conditions yet also
requires the maintenance of enzyme activity, i.e., where the enzyme
may be damaged by the freeze/thaw event. In one alternative
embodiment, AFP, in the presence or absence of a carrier protein,
is combined with AMV to protect the reverse transcriptase activity
of the enzyme over a period of several slow freeze/thaw cycles.
Nucleic Acid Amplification Using AFP-Based Reaction Mixtures
[0058] In one aspect, the invention provides methods for amplifying
a nucleic acid template, comprising subjecting the nucleic acid
template to an amplification reaction in an amplification reaction
mixture. The amplification reaction mixture preferably comprises
AFP1, and still more preferably comprises AFP1 and a carrier
protein such as, e.g., BSA.
[0059] Nucleic acid molecules may be amplified according to any of
the literature-described manual or automated amplification methods.
Nucleic acid amplification results in the incorporation of
nucleotides into a DNA molecule or primer, thereby forming a new
DNA molecule complementary to a nucleic acid template. The formed
DNA molecule and its template can be used as templates to
synthesize additional DNA molecules. As used herein, one
amplification reaction may consist of many rounds or cycles of DNA
replication. DNA amplification reactions include, for example,
polymerase chain reactions ("PCR"). One PCR reaction may consist of
10 to 100 "cycles" of denaturation and synthesis of a DNA molecule.
Such methods include, but are not limited to PCR (as described in
U.S. Pat. Nos. 4,683,195 and 4,683,202, which are hereby
incorporated by reference), Strand Displacement Amplification
("SDA") (as described in U.S. Pat. No. 5,455,166, which is hereby
incorporated by reference), and Nucleic Acid Sequence-Based
Amplification ("NASBA" (as described in U.S. Pat. No. 5,409,818),
which is hereby incorporated by reference). For example,
amplification may be achieved by a rolling circle replication
system which may even use a helicase for enhanced efficiency in DNA
melting without heat (See Yuzhakou et al., "Replisome Assembly
Reveals the Basis for Asymmetric Function in Leading and Lagging
Strand Replication," Cell 86:877-886 (1996) and Mok et al., "The
Escherichia coli Preprimosome and DnaB Helicase Can Form
Replication Forks That Move at the Same Rate," J. Biol. Chem.
262:16558-16565 (1987), which are hereby incorporated by
reference). Most preferably, nucleic acid molecules are amplified
by the methods of the present invention using PCR-based
amplification techniques.
[0060] In a preferred embodiment, the amplification reaction
involves a high temperature denaturation step. Preferred
temperatures for the high temperature denaturation step range from
about 90.degree. C. to about 98.degree. C., with temperatures from
93.degree. C. to 94.degree. C. being especially preferred. Such
preferred amplification reactions include thermocycling
amplification reactions, such as polymerase chain reactions
involving from about 10 to 100 cycles, more preferably from about
25 to 50 cycles, and peak temperatures of from about 93.degree. C.
to about 94.degree. C.
[0061] In a preferred embodiment, a PCR reaction is done using a
desired polymerase to produce, in exponential quantities relative
to the number of reaction steps involved, at lease one specific
nucleic acid sequence, given (a) that the ends of the requisite
sequence are known in sufficient detail that oligonucleotides can
be synthesized which will hybridize to them and (b) that a small
amount of the sequence is available to initiate the chain reaction.
The product of the chain reaction will be discrete nucleic acid
duplex with termini corresponding to the ends of the specific
primers employed.
[0062] Any source of nucleic acid, in purified or nonpurified form,
can be utilized as the starting nucleic acid, provided it contains
the specific nucleic acid sequence desired. Thus, the process may
employ, for example, DNA or RNA, including messenger RNA, which DNA
or RNA may be single stranded or double stranded. In addition, a
DNA-RNA hybrid which contains one strand of each may be utilized. A
mixture of any of these nucleic acids may also be employed, or the
nucleic acids produced from a previous amplification reaction
herein using the same or different primers may be so utilized. The
nucleic acid amplified is preferably DNA. The specific nucleic acid
sequence to be amplified may be only a fraction of a larger
molecule or can be present initially as a discrete molecule, so
that the specific sequence constitutes the entire nucleic acid. It
is not necessary that the sequence to be amplified be present
initially in a pure form; it may be a minor fraction of a complex
mixture, such as a portion of the .beta.-globin gene contained in
whole human DNA or a portion of nucleic acid sequence due to a
particular microorganism which organism might constitute only a
very minor fraction of a particular biological sample. The starting
nucleic acid may contain more than one desired specific nucleic
acid sequence which may be the same or different. Therefore, the
method is useful not only for producing large amounts of one
specific nucleic acid sequence, but also for amplifying
simultaneously more than one different specific nucleic acid
sequence located on the same or different nucleic acid
molecules.
[0063] The nucleic acid or acids may be obtained from any source
and include plasmids and cloned DNA or RNA, as well as DNA or RNA
from any source, including bacteria, yeast, viruses, and higher
organisms such as plants or animals. DNA or RNA may be extracted
from blood, tissue material such as corionic villi or amniotic
cells by a variety of techniques such as that described by Maniatis
et al., Molecular Cloning: A Laboratory Manual, (New York: Cold
Spring Harbor Laboratory) pp 280-281 (1982).
[0064] Any specific nucleic acid sequence can be produced by the
present methods. It is only necessary that a sufficient number of
bases at both ends of the sequence be know in sufficient detail so
that two oligonucleotide primers can be prepared which hybridize to
different strands of the desired sequence and at relative positions
along the sequence such that an extension product synthesized from
one primer, when it is separated from its template (complement),
can serve as a template for extension of the other primer into a
nucleic acid of defined length. The greater the knowledge about the
bases at both ends of the sequence, the greater the specificity of
the primers for the target nucleic acid sequence, and, thus, the
greater the efficiency of the process. It will be understood that
the word primer as used hereinafter may refer to more than one
primer, particularly in the case where there is some ambiguity in
the formation regarding the terminal sequence(s) of the fragment to
be amplified. For instance, in the case where a nucleic acid
sequence is inferred from protein sequence information a collection
of primers containing sequences representing all possible codon
variations based on degeneracy of the genetic code can be used for
each strand. One primer from this collection will be homologous
with the end of the desired sequence to be amplified.
[0065] In some alternative embodiments, random primers, preferably
hexamers, are used to amplify a template nucleic acid molecule. In
such embodiments, the exact sequence amplified is not
predetermined.
[0066] Oligonucleotide primers may be prepared using any suitable
method, such as, for example, the phosphotriester and
phosphodiester methods or automated embodiments thereof. In one
such automated embodiment diethylophosphoramidites are used as
starting materials and may be synthesized as described by Beaucage
et al., Tetrahedron Letters, 22:1859-1862 (1981), which is hereby
incorporated by reference. One method for synthesizing
oligonucleotides on a modified solid support is described in U.S.
Pat. No. 4,458,006, which is hereby incorporated by reference. It
is also possible to use a primer that has been isolated from a
biological source (such as a restriction endonuclease digest).
[0067] The specific nucleic acid sequence is produced by using the
nucleic acid containing that sequence as a template. If the nucleic
acid contains two strands, it is necessary to separate the strands
of the nucleic acid before it can be used as the template, either
as a separate step or simultaneously with the synthesis of the
primer extension products. This strand separation can be
accomplished by any suitable denaturing method including physical,
chemical, or enzymatic means. One physical method of separating the
strands of the nucleic acid involves heating the nucleic acid until
it is completely (>99%) denatured. Typical heat denaturation may
involve temperatures ranging from about 80.degree. C. to
105.degree. C. for times ranging from 1 to 10 minutes. Strand
separation may also be induced by an enzyme from the class of
enzymes known as helicases or the enzyme RecA, which has helicase
activity and is known to denature DNA. The reaction conditions
suitable for separating the strands of nucleic acids with helicases
are described by Cold Spring Harbor Symposia on Quantitative
Biology, Vol. XLIII "DNA: Replication and Recombination" (New York:
Cold Spring Harbor Laboratory, 1978), and techniques for using RecA
are reviewed in C. Radding, Ann, Rev. Genetics, 16:405-37 (1982),
which is hereby incorporated by reference.
[0068] If the original nucleic acid containing the sequence to be
amplified is single stranded, its complement is synthesized by
adding one or two oligonucleotide primers thereto. If an
appropriate single primer is added, a primer extension product is
synthesized in the presence of the primer, an agent for
polymerization, and the four nucleotides described below. The
product will be partially complementary to the single-stranded
nucleic acid and will hybridize with the nucleic acid strand to
form a duplex of unequal length strands that may then be separated
into single strands, as described above, to produce two single
separated complementary strands. Alternatively, two appropriate
primers may be added to the single-stranded nucleic acid and the
reaction carried out.
[0069] If the original nucleic acid constitutes the sequence to be
amplified, the primer extension product(s) produced will be
completely complementary to the strands of the original nucleic
acid and will hybridize therewith to form a duplex of equal length
strands to be separated into single-stranded molecules.
[0070] When the complementary strands of the nucleic acid or acids
are separated, whether the nucleic acid was originally double or
single stranded, the strands are ready to be used as a template for
the synthesis of additional nucleic acid strands. This synthesis
can be performed using any suitable method. Preferably, a molar
excess (for cloned nucleic acid, usually about 1000:1
primer:template, for the genomic nucleic acid, usually about
10.sup.6:1 primer:template) of the two oligonucleotide primers is
added to the buffer containing the separated template strands. It
is understood, however, that the amount of complementary strand may
not be known if the process herein is used for diagnostic
applications, so that the amount of primer relative to the amount
of complementary strand cannot be determined with certainty. As a
practical matter, however, the amount of primer added will
generally be in molar excess over the amount of complementary
strand (template) when the sequence to be amplified is contained in
a mixture of complicated long-chain nucleic acid strands. A large
molar excess is preferred to improve the efficiency of the
process.
[0071] Nucleoside triphosphates, preferably dATP, dCTP, dGTP, and
dTTP are also added to the synthesis mixture in adequate amounts,
and the resulting solution is preferably heated to a temperature
from about 90.degree. C.-95.degree. C. for about 1 to 10 minutes,
preferably from 15 sec to 2 minutes. After this heating period, the
solution is allowed to cool to a temperature from about 60.degree.
C., which is preferable for the primer hybridization. The
polymerase then performs nucleic acid synthesis at a temperature
well above room temperature, preferably at a temperature from about
60 to 75.degree. C.
[0072] The newly synthesized strand and its complementary nucleic
acid strand form a double-stranded molecule which is used in the
succeeding steps of the process. In the next step, the strands of
the double-stranded molecule are separated using any of the
procedures described above to provide single-stranded
molecules.
[0073] New nucleic acid is synthesized on the single-stranded
molecules. Additional polymerase, nucleotides, and primers may be
added if necessary for the reaction to proceed under the conditions
prescribed above. Again, the synthesis will be initiated at one end
of the oligonucleotide primers and will proceed along the single
strands of the template to produce additional nucleic acids.
[0074] The steps of strand separation and extension product
synthesis can be repeated as often as needed to produce the desired
quantity of the specific nucleic acid sequence. The amount of the
specific nucleic acid sequence produced will increase in an
exponential fashion.
[0075] When it is desired to produce more than one specific nucleic
acid sequence from the first nucleic acid or mixture of nucleic
acids, the appropriate number of different oligonucleotide primers
are utilized. For example, if two different specific nucleic acid
sequences are to be produced, four primers are utilized. Two of the
primers are specific for one of the specific nucleic acid sequences
and the other two primers are specific for the second specific
nucleic acid sequence. In this matter, each of the two different
specific sequences can be produced exponentially by the present
process. Of course in instances where nucleic acid sequences are
the same, primer sequences will be the same.
[0076] Additionally, as mentioned above, in an alternative
embodiment, random primers, are used to amplify a template nucleic
acid molecule.
[0077] The present invention can be performed in a step-wise
fashion where after each step new reagents are added, or
simultaneously, wherein all reagents are added at the initial step,
or partially step-wise and partially simultaneously, wherein fresh
reagent is added after a given number of steps, for example adding
new enzyme solution comprising AFP and optionally carrier protein.
After the appropriate length of time has passed to produce the
desired amount of the specific nucleic acid sequence, the reaction
may be halted by inactivating the enzymes in any known manner or
separating the components of the reaction.
[0078] Thus, in amplifying a nucleic acid molecule according to the
present invention, the nucleic acid molecule is contacted with a
composition comprising a polymerase in an appropriate buffer,
preferably having AFP, and preferably AFP1, and most preferably
having AFP and a carrier protein.
Method for Enhancing Amplification Reactions after One or More
Freeze/Thaw Events
[0079] The present invention provides methods for maintaining the
functional stability of an enzyme in a solution, preferably a
polymerase in a PCR or real time-PCR buffer, although other enzymes
like reverse transcriptase polymerase III, etc. are within the
scope of the present invention. Initially, a polyol, AFP or AFP
with carrier protein is combined with a desired amplification
enzyme. The combination can occur in a standard amplification
buffer or buffers consistent with more specific embodiments of the
present invention. The combined enzyme-polyol, enzyme-AFP, or
enzyme-AFP-carrier protein is then utilized in the reaction mixture
for an amplification reaction. Remaining material, when frozen, is
protected from some or all of the freeze/thaw effects. The enzyme
solution thus obtained maintains the requisite enzymatic activity
and can be used over the course of numerous freeze/thaw events,
preferably as many as five or ten freeze/thaw events, more
preferably as many as fifteen freeze/thaw events. In a preferred
embodiment, the amplification reaction is a real time-PCR reaction.
In another embodiment, the method incorporates an enzyme mixed with
at least two of: a polyol, an AFP, and a carrier protein, and most
preferably all three components are included. In an additional
embodiment, an unconventional nucleotide is included in the
amplification reaction mix to enhance the efficiency of the
reactions by lowering the formation of primer-dimers, as described
in co-pending Patent Cooperation Treaty Application No. ______,
filed Feb. 4, 2005, entitled dUTP BASED COMPOSITIONS FOR REDUCING
PRIMER-DIMER FORMATION DURING NUCLEIC ACID AMPLIFICATION,
incorporated by reference herein.
[0080] In an alternative embodiment of the present invention,
inclusion of one or more polyols in an amplification enzyme
solution or buffer further increases stability under freeze/thaw
conditions. In a preferred embodiment the amplification buffer is a
standard PCR buffer. In one embodiment, sorbitol or other like
polyol is included in a PCR buffer at a concentration of from about
50 mM to about 500 mM, preferably from about 50 mM to about 400 mM,
and most preferably from about 100 mM to about 300 mM. As above,
inclusion of other disruptive agents, i.e., DMSO, SSBP, and the
like, with the polyol is anticipated to enhance the overall
positive affect.
Compositions of High Performance Real Time PCR Buffers
[0081] The present invention further provides high performance
amplification buffers for use in amplification reactions,
preferably in standard PCR reactions and real-time PCR reactions.
Buffers in accordance with the present invention can include
anti-freeze protein (preferably AFP1, AFGP, mixtures of AFP1 and
AFGP), carrier protein, dNTP mix, polyol, nucleic acid polymerase,
preferably a thermophilic or hyperthermophilic polymerase, and have
a modified pH, obtained through a buffer system that utilizes a
zwitterionic formulation. Illustrative zwitterionic formulations
include HEPES-KOH, TAPS-Tris, HEPES-Tris, HEPES-KOH, TAPS-KOH or
TAPS-Tris. Preferred embodiments of the present invention utilize a
buffer system that includes TAPS-KOH and/or TAPS-Tris, and most
preferably TAPS-Tris 0. Preferred pH ranges for these buffers are
dependent on the final use, for example, buffers for use in
real-time PCR are buffered to have a pH of from about 7.9 to about
8.7, and preferably from about 8.2 to about 8.7. Note that buffers
for use in standard PCR are modified to have a pH of from about 7.9
to 8.9. In preferred embodiments, the potassium salt concentration
his between 10 mM to about 80 mM. In preferred embodiments, the
dNTP mix of the buffer system includes from about 10% to about 50%
dUTP (in replacement of dTTPs in the dNTP mix), and more preferably
from about 10% to about 40% dUTP (in replacement of dTTPs in the
dNTP mix). Further, in some embodiments n-propyl sulfoxide, and/or
trehalose can be included in the high performance buffer.
[0082] Concentrations of ingredients useful in embodiments of the
high performance buffer are as shown in Table 2. Note that the
preferred concentration for TAPS-KOH is 150 mM KCl and for the
TAPs-Tris is 500 mM KCl, both with a final buffer pH of about
8.
TABLE-US-00002 TABLE 2 High Performance PCR Buffer/Real-Time PCR
Buffer Useful Concentration Range Preferred Concentration Buffer
Ingredient (Final) (Final) Sorbitol, Trehalose, , maltitol 10
mM-300 mM 100 mM and/or mixtures thereof dNTP Mix/% dUTP in dATP
100 .mu.M-500 .mu.M dATP 200 .mu.M Replacement of dTTP (or other
dCTP 100 .mu.M-500 .mu.M dCTP 200 .mu.M dNTP analogs) dGTP 100
.mu.M-500 .mu.M dGTP 200 .mu.M dTTP + dUTP 100 .mu.M-500 .mu.M dTTP
+ dUTP 200 .mu.M dTTP/dUTP 75%:25% - 25%-75% dTTP/dUTP 75%:25%
Nucleic Acid Polymerase 0.5 to 2 Units 2 Units Mg Ion Concentration
3 mM to 10 mM 5 mM and 8 mM Potassium Salt Concentration 10 mM to
80 mM 40 mM to 60 mM anti-freeze protein, e.g., AFP 10 .mu.g/ml to
200 .mu.g/ml//100 .mu.g/ml 50 .mu.g/ml//100 .mu.g/ml type I, AFGP
or mixtures of to 300 .mu.g/ml same//Carrier Protein Buffering
Ingredient, e.g., Taps 10 mM-40 mM Taps 25 mM TAPS-Tris Tris 5
mM-25 mM Tris 10.3 mM
[0083] The present invention further provides the AFP zwitterionic
buffer compositions discussed above having a monovalent potassium
salt ranging in concentration from about 30 mM to about 80 mM, and
preferably between about 40 mM and 60 mM. Additionally, magnesium
ions in buffers of the present invention can be from about 2.5 mM
to about 10 mM, and preferably from about 5 mM and about 8 mM.
[0084] It is also envisioned that embodiments using modified dNTP
mixtures to limit primer-dimer formation in accordance with
embodiments previously described can be included with embodiments
of the high performance PCR buffers of the instant invention. See
co-pending Patent Cooperation Treaty Application No. ______, filed
Feb. 4, 2005, and entitled dUTP BASED COMPOSITIONS FOR REDUCING
PRIMER-DIMER FORMATION DURING NUCLEIC ACID AMPLIFICATION,
incorporated herein by reference. As such, embodiments using the
zwitterionic buffer formulations can have one or more of AFP,
carrier protein, sorbitol, mannitol, DMSO, SSBP, and a dNTP mix
having a percentage of the dTTP or other dNTP replaced with an
unconventional nucleotide like dUTP. Note also that the embodiments
of the present invention can include a combination of AFP 1 with
DMSO (or other like compound) and a polyol, for example, sorbital
(See for example, U.S. Pat. No. 6,783,940, which is incorporated
herein by reference). Typically, the composition has a pH of
between about 7.9 and 8.2 for optimal effects. Other pH can be used
but with limited results.
PCR and Real-Time PCR Buffer Kits
[0085] The present invention further provides kits that include the
composition embodiments of the present invention. Kits can include
PCR buffers of the invention, for example embodiments of the high
performance PCR buffers of the invention, or alternatively,
pre-determined stand alone amounts of AFP, sorbitol, mannitol, or
other compositions of the invention, which are added to PCR buffers
or are combined with amplification enzymes appropriate for the
target use, combined with the invention. In addition, kit
compositions can include combinations of AFP with different
polymerase enzymes in the same or different tubes.
[0086] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLES
Example 1
Anti-Freeze Proteins (AFPs) Enhance Stability of Real Time-PCR
Master Mix During Extended Periods of Storage at -20.degree.
[0087] The effects of AFP1 on the freeze/thaw stability of
real-time PCR buffers were investigated. Initially, AFP1 was
titrated into real time-PCR reactions to determine what
concentration of AFP1 did not detrimentally affect amplicon yield.
Real-time PCR was performed as described in Eppendorf RealMasterMix
Probe +/- ROX (catalog #00320900.525), and products visualized on
ethidium bromide stained 1% agarose gels. As shown in FIG. 1,
product yields were not detrimentally affected until approximately
100 .mu.g/ml AFP1 was included in the reaction mix. Conversely, the
addition of 50 .mu.g/ml AFP1 to the real time-PCR had little or no
effect on the reaction yields (compare the lane having 0 AFP1 to
the lane having 50 .mu.g/ml AFP1).
[0088] Prior to determining the freeze/thaw capacity of Real-Time
PCR buffers in the presence and absence of AFP1, the ability of
AFP1 to enhance fresh Real-Time PCR samples was tested. Real-Time
PCR was performed as above and samples incubated with either 200
.mu.g/ml BSA, 50 .mu.g/ml AFP1, or 200 .mu.g/ml BSA mixed with 50
.mu.g/ml AFP1. FIGS. 2A and 2B graphically show that the addition
of AFP1 alone actually has a slightly negative effect on the
average RFU (A) and Threshold cycle (B) in this experiment. BSA
alone had little or no effect on real-time PCR. However, the
combination of BSA and AFP1 provided marked improvement on both
signal amplification (A) and threshold cycle (B). As such, the
combination of AFP1 and a carrier protein like BSA provides a
synergistic benefit during real-time PCR while maintaining maximal
yield of the amplicons (FIG. 1).
[0089] AFP1:BSA containing real-time PCR samples were next tested
for stability during freeze/thaw conditions over zero, one week,
two weeks and three weeks at -20.degree. C. (see FIGS. 3A and 3B).
Real-time PCR conditions were as described above except for the
inclusion of the different amounts of carrier protein and AFP1. As
shown in FIGS. 3A and 3B, the enhanced signal obtained during
Real-Time PCR was maintained when AFP1 and BSA were added to the
reaction mix (A) and the threshold cycle was also maintained as
compared to the changes noted in BSA alone, AFP1 alone or no
additives (B).
[0090] Similar Real Time PCR studies were performed using AFGP in
the presence and absence of a carrier protein to determine whether
the AFP1 effects were limited to AFP1 or were extendable to other
AFP family members, and particularly to AFP family members having
similar primary and secondary protein structure. AFGP includes a
primary and secondary protein structure similar to
AFP1--particularly, strong similarity exists between the
alanine-rich motif of AFP1 having an alpha helical amphiphilic
secondary structure (see Table 1) and the alanine-alanine-threonine
repeat disaccharide of AFGP. Like AFP1, AFGP in combination with
BSA, had a significant effect on maintaining or lowering threshold
cycle number as compared to control samples. Experimental
parameters are shown in Table 3 below.
TABLE-US-00003 TABLE 3 AFP1 and AFGP Reaction Set-up MasterMix
nuclease free Component Stock Final .mu.L/20 .mu.L rxn Volume water
Buffer Master Mix for PCR (The combined master mixes are 2.5X) 10X
PCR 10 2.5 5 800 -- buffer (1M sorbitol) dNTP with 10 1 2 320 --
UTP mix (mM) glycerol (%) 100 1 0.2 32 -- DNA poly 5 2 0.4 64 --
(U/.mu.L) (Eppendorf Hot Master) Total 7.6 1216 Aliquot Master Mix
into 4 tubes 167.2 .mu.L (22 rxns) each. Add 272.8 .mu.L
corresponding additive mix. Buffer Master Mix With BSA BSA (mg/ml)
50 0.5 0.2 4.4 268.4 Buffer Master Mix With AFGP AFGP (mg/ml) 1
0.125 2.5 55 217.8 Buffer Master Mix With BSA and AFP1 BSA (mg/ml)
50 0.5 0.2 4.4 213.4 AFP1 (mg/ml) 1 0.125 2.5 55 -- Buffer Master
Mix No Additives No Additive 0 0 0 0 272.8 Control Buffer Master
Mix With BSA and AFGP BSA (mg/ml) 50 0.5 0.2 4.4 213.4 AFGP (mg/ml)
1 0.125 2.5 55 -- Buffer Master Mix With BSA, AFP1, AFGP BSA
(mg/ml) 50 0.5 0.2 4.4 158.4 AFGP (mg/ml) 1 0.125 2.5 55 -- AFP1
(mg/ml) 1 0.125 2.5 55 -- Buffer Master Mix With AFP1, AFGP AFP1
(mg/ml) 1 0.125 2.5 55 -- AFGP (mg/ml) 1 0.125 2.5 55 --
[0091] As shown in FIG. 7, AFGP showed similar results in threshold
cycle for fresh and freeze/thawed samples (-20.degree. C.) for one,
two or three weeks. Note that the combination of AFP type I, AFGP
and BSA provided the best overall performance during real time-PCR
stability testing, as compared to the controls but also as compared
to AFP type I alone, AFGP alone, AFP type I with BSA and AFGP with
BSA.
[0092] The results in this Example show the utility of including
AFP1 and AFGP, in the presence of a carrier protein, in Real-Time
PCR reactions for facilitating the stability of the buffer during
multiple freeze/thaw events at -20.degree. C. This Example also
provides data that at least two anti-freeze protein family members
supported long term stability of a Real Time-PCR buffer, AFP type I
and AFGP. The data provides strong evidence that proteins that
exhibit similar anti-freeze protein like functional properties will
be effective in similar manners to these proteins, and that the
functional motif of AFP type I and AFGP (see Table 1 above) will be
effective in a similar manner.
Example 2
pH Modification and Buffer Type have Significant Effect on
Optimizing Sorbitol, AFP1 Containing Real-Time PCR Buffer
[0093] Several real time-PCR buffer combinations were tested for
signal amplification and for threshold cycle in the presence of a
standard amount of both sorbitol and AFP1/BSA. Both the buffering
component and salt were modified to provide different ionic
concentrations and pHs. In particular, 25 mM HEPES-KOH with 15 mM
KCL pH 8.0; 25 mM TAPS-Tris with 15 mM K GLUTpH 8.0; 25 mM
HEPES-Tris with 50 mM KCL pH 8.0; 25 mM Bicine-Tris with 15 mM K
GLUT pH 8.7; 25 mM HEPES-KOH with 15 mM K GLUTpH 8.0; 25 mM
Bicine-Tris with 50 mM K GLUT pH 8.0; 25 mM TAPS-KOH with 15 mM KCL
pH 8.0; 25 mM Bicine-Tris with 15 mM KCL pH 8.0; 25 mM TAPS-Tris
with 50 mM KCL pH 8.0; and 25 mM Bicine-Tris with 50 mM KCl pH 8.4
were compared for ability to support highly accurate real time-PCR
(each buffer also included 50 .mu.g/ml AFP1, 200 .mu.g/ml BSA and
100 mM sorbitol).
[0094] As shown graphically in FIGS. 4A and 4B (and Table 4),
buffers composed on TAPS-KOH 150 mM KCl pH 8.0 and TAPS-Tris 500 mM
KCl pH 8.0 provided the lowest threshold cycle and highest signal
amplification in Real-Time PCR.
TABLE-US-00004 TABLE 4 Real-Time PCR Buffer Test Data For
Alternative Buffer Constituents Standard Standard Buffer Type Ave
Ct Deviation of Ct Ave RFU deviation of RFU 25 mM HEPES-KOH
18.86667 0.057735 1751.46 90.14655 with 15 mM KCL pH 8.0 25 mM
TAPS-Tris 18.83333 0.11547 2037.563 64.23551 with 15 mM K GLUT pH
8.0 25 mM HEPES-Tris 18.63333 0.057735 1873.903 32.29925 with 50 mM
KCL pH 8.0 25 mM Bicine-Tris 19.7 0.173205 1308.253 99.58234 with
15 mM K GLUT, pH 8.7 25 mM HEPES-KOH 19.0333 0.11547 1691.167
78.98864 with 15 mM K GLUT pH 8 25 mM Bicine-Tris 19.2333 0.057735
1666.757 31.04646 with 15 mM K GLUT pH 8.0 25 mM TAPS-KOH 18.4333
0.057735 2243.12 29.99653 with 15 mM KCL pH 8.0 25 mM Bicine-Tris
18.8 0.1 1786.747 53.66013 with 15 mM KCL, pH 8.0 25 mM TAPS-Tris
18.36667 0.057735 2110.447 77.15043 with 50 mM KCL pH 8.0 25 mM
Bicine-Tris 19.1 0.1 1489.81 28.70814 with 50 mM KCl pH 8.4
[0095] Further characterization was made on the buffer compositions
by comparing the 25 mM TAPS-KOH with 15 mM KCLpH 8.0, 25 mM
TAPS-Tris with 50 mm KCLpH8.0 and 25 mM Bicine-Tris with 50 mM KCl
pH 8.4 for ability to support Real-Time PCR. Results shown in FIG.
5 graphically illustrate RFU and threshold cycle for each buffer
condition, showing that 25 mM TAPS-Tris with 50 mM KClpH 8.0
out-performed both the 25 mM TAPS-KOH with 15 mM pH 8.0 and 25 mM
Bicine-Tris with 50 mM KCl pH 8.4. Finally, results shown in FIG. 6
illustrate that 25 mM TAPS-Tris with 50 mM KCLpH 8.0 outperformed
both 25 mM Bicine-Tris with 50 mM KCl pH 8.4, and Invitrogen
Platinum qPCR Supermix-UDG (Invitrogen product number 11730-017).
Real-Time PCR conditions for the Invitrogen Platinum qPCR
Supermix-UDG were performed using manufacturer recommendation.
[0096] This data shows that a Real-Time PCR buffer composed of 25
mM TAPS-Tris with 50 mM KCl, pH of about 8, and including sorbitol,
AFP1 and BSA, provided significant improvement in both signal size
and threshold cycle over the Invitrogen based product. These same
buffers also help stabilize buffer storage at -20.degree. C. for
longer-term storage needs of the investigator.
Example 3
AFP Containing PCR Master Mix Functionally Outperforms Competitor
PCR Mix
[0097] An embodiment of a PCR Master Mix prepared in accordance
with the present invention was functionally compared to Invitrogen
Platinum qPCR Supermix-UDG for its ability to support long term
stability within real time-PCR. Long term stability test
experimental parameters are shown in Table 5. Cycling parameters
included: 95.degree. C. for one minute and forty cycles of
95.degree. C. for twenty seconds, 56.degree. C. for ten seconds and
68.degree. C. for thirty seconds.
TABLE-US-00005 TABLE 5 Long Term Stability Test Component Stock
Final .mu.l/50 .mu.l rxn 3.5 Aliquot 70 .mu.l of each of the
RealMaster, 2.5X master mix into a biopure tube 2.5X Real Master
2.5 1 20 70 Mix +/- ROX(x) Primer-Probe Template Mix For 2.5X MM
B2M fwd primer 10 0.2 1 22 (.mu.M) B2M rev primer 10 0.2 1 22
(.mu.M) B2M FAM probe 7.5 0.15 1 22 (.mu.M) gDNA male 50 1 1 22 (50
ng/.mu.l) MBGW 26 572 Total 30 660 Add 105 .mu.l (3.5 rxn) of
primer-probe-template mix to each aliquot of 2.5X master mix.
Aliquot 87.5 .mu.l of each of the competitors 2X master mixes into
a biopure tube. 2X Invitrogen PCR 2 1 25 87.5 probe Mix
Primer-Probe-Template Mix For 2X MM B2M fwd primer 10 0.2 1 33
(.mu.M) B2M rev primer 10 0.2 1 33 (.mu.M) B2M FAM probe 7.5 0.15 1
33 (.mu.M) gDNA male 50 1 1 33 (50 ng/.mu.l) MBGW 21 693 Total 25
825 Add 87.5 .mu.l (3.5 rxns) of primer-probe-template mix to each
aliquot of 2X master mix
[0098] As shown in FIGS. 8A and 8B, the AFP containing Real
Time-PCR buffer of the present invention supported stronger signal
amplification (RFU)(8A) and earlier Ct(8B) at -4.degree. C.,
-20.degree. C. and -80.degree. C. as compared to Invitrogen
Platinum qPCR Supermix-UDG over a six week course of experiments.
The data illustrates that the buffers of the present invention
provide comparable or better results for long-term stability,
comparable to buffers sold as state of the art. As such,
embodiments of the present invention have utility in providing long
term buffer stability useful in real time PCR. Note that Invitrogen
product was purchased and used per the manufacturer's
recommendations.
Example 4
Sorbitol Enhances Stability of Real Time-PCR Master Mix During
Extended Periods Of Storage at -20.degree.
[0099] Sorbitol was included in real time-PCR master mix to
determine its effects on storage stability of real time-PCR master
mix buffers. Master mix samples were prepared with and without 100
mM sorbitol, stored at 4, -20 or -80.degree. C. for one, two or
three weeks and tested for overall performance in real
time-PCR.
[0100] As shown in FIGS. 9A and 9B, 100 mM sorbitol provided for an
earlier Ct for reactions conducted with master mix buffers frozen
at -20.degree. C. for one, two or three weeks (10A). Corresponding
RFU values were also maintained in the sorbitol containing master
mixes at this temperature. Samples stored at either 4.degree. C. or
-80.degree. C. showed smaller or no change between the sorbitol
containing and non-containing samples. It is believed that sorbitol
is most effective at stabilizing master mix storage at -20.degree.
C. due to the slow freeze nature of this temperature range. In
comparison, storage of the master mix at either 4.degree. C. or
-80.degree. C. was not expected to provide the same protection, as
4.degree. C. does not freeze samples and -80.degree. C. is a very
fast snap freeze, less likely to involve formation of damaging
water crystals, although as shown below some benefit is
obtained.
[0101] FIGS. 10A and 10B provide corresponding yield comparisons
for one and three week storage, again indicating the usefulness of
sorbitol in stabilizing the master mix at -20.degree. C. in RT-PCR.
Note that the yield is significantly lowered when no sorbitol is
included in the master mix stored at -20.degree. C. (lane 8 in 10A
and lane 5 in 10B). However, a dramatic increase for corresponding
samples occurs when 100 mM sorbitol is included in the master mix
buffers.
Example 5
AFP1 Provides Significant Stability Protection Over the Course of
Numerous Freeze/Thaw Events
[0102] The effects of AFP1 on DNA poly based gDNA amplification was
tested over the course of 15 freeze thaw cycles. Reaction mixes
including a 2.5.times. RealMasterMix with or without ROX
(5-carboxy-x-rhodamine; triethyl ammonium salt). RealMasterMix is
composed of 10.times. RealMaster Probe Buffer (0.5 mg/ml AFP1, 250
mm Taps, 103 mm Tris, 500 mm KC1, 50 mm MgAc and 2 mg/ml BSA), 10
mm dNTP mix (Eppendorf product #W40460) and 100 U.mu.l HotMaster
Taq DWA polymerase. Each reaction, therefore, includes
approximately 200 .mu.g/ml BSA and 50 .mu.g/ml AFP1. Reactions were
set up as shown in tables 6-7 and 8, using either a AB1 7000
thermal cycler or a Bio-Rad iCycler. Note that each reaction mix
was gently vortexed.
TABLE-US-00006 TABLE 6 #RXNS [STOCK] [FINAL] ul/50 uL RXN 6.500
REACTION COCKTAIL: ABI 7000 SDS-EPPENDORF RealMasterMix Probe ROS
2.5X RealMasterMix + ROX (X) 2.50 1.000 20.000 130.000 B2M fwd
primer (uM) 10.00 0.200 1.000 6.500 B2M rev primer (uM) 10.00 0.200
1.000 6.500 B2M FAM probe (uM) 7.50 0.150 1.000 6.500 MBGW 26.000
169.000 TOTAL 49.000 318.500 4.500 gDNA (ng/ul) 50.00 1.000 1.000
4.500 TOTAL 225.000 REACTION COCKTAIL: BIO-RAD IQ iCYCLER-EPPENDORF
RealMasterMix Probe 2.5x RealMasterMix (X) 2.5 1.000 20.000 130.000
B2M fwd primer (uM) 10.00 0.200 1.000 6.500 B2M rev primer (uM)
10.00 0.200 1.000 6.500 B2MFAM probe (uM) 7.50 0.150 1.000 6.500
MBGW 26.000 169.000 TOTAL 49.000 318.500 4.500 gDNA (ng/ul) 50.00
1.000 1.000 4.500 TOTAL 225.000
TABLE-US-00007 TABLE 7 THERMAL PROTOCOL: ABI 7000 # CYCLES
TEMPERATURE TIME INITIAL 1 95.degree. C. 1:00* DENATURATION CYCLING
PARAMETERS 40 DENATURE 95.degree. C. 00:20 ANNEAL 56.degree. C.
00:10 EXTEND 68.degree. C. 00:30 HOLD 1 4.degree. C. Indefinite
*Incubate AB rxns 9:00 at 95o C. then transfer all reactions to the
7000
TABLE-US-00008 TABLE 8 THERMAL PROTOCOL: BIO-RAD iCYCLER # CYCLES
TEMPERATURE TIME INITIAL 1 95.degree. C. 01:30 DENATURATION CYCLING
PARAMETERS 40 DENATURE 95.degree. C. 00:20 ANNEAL 56.degree. C.
00:10 EXTEND 68.degree. C. 00.30 HOLD 1 4.degree. C. Indefinite
[0103] As shown in FIG. 11, AFP1, mixed with BSA, provided
protection to the DNA polymerase activity over the course of 15
freeze/thaw events. Each freeze/thaw cycle included a slow freeze
to -20.degree. C. and a thaw up to room temperature.
[0104] Each tested composition was evaluated for consistency of
performance after each of the 15 freeze/thaw cycles under two
alternative PCR methods. The data illustrates that AFP1, combined
with BSA, functions to maintain DNA polymerase activity over the
course of numerous freeze/thaw events. It is envisioned that this
protection can be extended to other enzyme systems as well as for
use in protecting enzymes over long periods of time at a freezing
temperature.
[0105] The invention has been described with reference to specific
examples. These examples are not meant to limit the invention in
any way. It is understood for purposes of this disclosure, that
various changes and modifications may be made to the invention that
are well within the scope of the invention. Numerous other changes
may be made which will readily suggest themselves to those skilled
in the art and which are encompassed in the spirit of the invention
disclosed herein and as defined in the appended claims.
[0106] This specification contains numerous citations to patents,
patent applications, and publications, each is hereby incorporated
by reference for all purposes.
* * * * *