U.S. patent application number 14/399491 was filed with the patent office on 2015-06-11 for electrochemical detection of polymerase reactions by specific metal-phosphate complex formation.
This patent application is currently assigned to ALACRIS THERANOSTICS GMBH. The applicant listed for this patent is ALACRIS THERANOSTICS GMBH. Invention is credited to Jorn Glokler, Fred Lisdat.
Application Number | 20150159208 14/399491 |
Document ID | / |
Family ID | 48485133 |
Filed Date | 2015-06-11 |
United States Patent
Application |
20150159208 |
Kind Code |
A1 |
Glokler; Jorn ; et
al. |
June 11, 2015 |
ELECTROCHEMICAL DETECTION OF POLYMERASE REACTIONS BY SPECIFIC
METAL-PHOSPHATE COMPLEX FORMATION
Abstract
The present invention relates to determining pyrophosphate by
electrochemical means via the depletion of metal ions as a result
of its binding to and/or precipitation with pyrophosphate. This
principle is of particular interest for polymerase catalysed
reactions such as nucleic acid sequencing.
Inventors: |
Glokler; Jorn; (Berlin,
DE) ; Lisdat; Fred; (Wildau, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALACRIS THERANOSTICS GMBH |
Berlin |
|
DE |
|
|
Assignee: |
ALACRIS THERANOSTICS GMBH
BERLIN
DE
|
Family ID: |
48485133 |
Appl. No.: |
14/399491 |
Filed: |
May 8, 2013 |
PCT Filed: |
May 8, 2013 |
PCT NO: |
PCT/EP2013/059615 |
371 Date: |
November 6, 2014 |
Current U.S.
Class: |
205/777.5 ;
204/403.14 |
Current CPC
Class: |
C12Q 1/6869 20130101;
G01N 33/84 20130101; C12Q 1/6869 20130101; C12Q 2531/113 20130101;
C12Q 2563/137 20130101; C12Q 2565/301 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
May 11, 2012 |
EP |
12167723.1 |
Claims
1. An electrochemical nucleic acid sequencing method comprising the
steps of: a. providing a sample comprising a template nucleic acid
to be sequenced; b. annealing a primer molecule to said template
nucleic acid, or creating a nick in the non-template strand for a
site-specific elongation; c. carrying out nucleic acid elongation
steps with a polymerase on said template in the presence of metal
ions, wherein each step comprises the addition of a single type of
nucleotide, wherein pyrophosphate is released in a stoichiometric
ratio to the number of nucleotides incorporated, and wherein the
metal ions are capable of preferentially binding to pyrophosphate
in solution; d. determining in said sample the potential, current
or charge at an electrode at least twice, wherein between the first
time and the second time one or more elongation steps take place
or, alternatively, determining the potential, current or charge
continuously; and e. correlating the change in potential, current
or charge over the number of elongation steps with the type of
nucleotides added.
2. The method according to claim 1, wherein the nucleic acid
sequencing method is based on pyrosequencing,
reversible-terminator-pyrosequencing, or closed complex formation
sequencing.
3. The method according to claim 1 or 2, wherein step d. and
optionally step e. is done after each elongation step c.
4. A method for detecting a nucleic acid polymerase reaction
comprising the steps of: a. providing a sample comprising a nucleic
acid; b. carrying out a nucleic acid polymerase reaction step on
said sample in the presence of metal ions, wherein pyrophosphate is
released or depleted depending on the type of polymerase reaction,
and wherein the metal ions are capable of specifically binding to
pyrophosphate in solution; c. determining in said sample the
potential, current or charge at an electrode at least twice,
wherein between the first time and the second time one or more
nucleic acid polymerase reaction steps take place or,
alternatively, determining the potential, current or charge
continuously; and d. correlating the difference in potential,
current or charge over the number of polymerase reaction steps
and/or time with the progress of the nucleic acid polymerase
reaction.
5. The method according to claim 4, wherein the nucleic acid
polymerase reaction is a synthesis reaction, an amplification
reaction and/or a transcription reaction.
6. The method according to claim 4 or 5, wherein step c. and
optionally step d. is done after each polymerase reaction step
b.
7. A method of detecting the formation or depletion of
pyrophosphate in a sample comprising the steps of: a. providing a
sample in which pyrophosphate is formed or depleted; b. adding to
said sample metal ions, wherein the metal ions are capable of
specifically binding pyrophosphate in solution; c. determining in
said sample the potential, current or charge at an electrode a
first and at least a second time or continuously; d. correlating
the difference in potential, current or charge over time with the
formation or depletion of pyrophosphate in the sample.
8. A method of electrochemically quantifying pyrophosphate in a
sample comprising the steps of: a. adding to a sample comprising
pyrophosphate a predetermined amount of metal ions or metal ions of
a known potential, current or charge, wherein the metal ions are
capable of specifically binding pyrophosphate in solution; b.
determining in said sample the potential, current or charge at an
electrode; and c. correlating the potential, current or charge with
the amount of pyrophosphate present in said sample.
9. The method according to any of the preceding claims, wherein the
metal ions are selected from the group of manganese ions, aluminium
ions, tin ions and iron ions.
10. The method according to any of the preceding claims, wherein
the metal ions are manganese ions.
11. The method according to any of the preceding claims, wherein
the electrode is based on manganese dioxide.
12. An electrochemical nucleic acid sequencing apparatus
comprising: a. means for carrying out the steps of annealing and
elongating a primer molecule to a template nucleic acid to be
sequenced; and b. an electrochemical cell comprising metal ions
being capable of preferentially binding to pyrophosphate and at
least one working electrode responsive to said metal ions.
13. The electrochemical nucleic acid sequencing apparatus according
to claim 12, wherein the metal ions are manganese ions and the
working electrode is based on manganese dioxide.
Description
FIELD OF THE INVENTION
[0001] The present invention is in the field of chemistry and
biology. Specifically, the invention relates to the electrochemical
detection of pyrophosphate and to electrochemical nucleic acid
sequencing.
INTRODUCTION
[0002] Nucleic acid polymerase reactions are central to a multitude
of sensitive detection methods. Especially many different solutions
have been developed for PCR product formation. More specifically, a
certain reaction used to identify a DNA sequence termed
pyrosequencing (Ahmadian et al. Clin. Chim. Acta. 2006.
363(1-2):83-94) is of great interest. In pyrosequencing the four
different nucleotides are added to a polymerase reaction in
alternating cycles and the release of pyrophosphate is monitored
indirectly.
[0003] Currently, two main approaches for the detection of product
formation from a polymerase reaction such as a nucleic acid
sequencing method exist:
[0004] Pyrophosphate is used to generate ATP via sulfurylase and
ADPS, which leads to light emission by luciferase (Ahmadian et al.
Clin. Chim. Acta. 2006. 363(1-2):83-94). The light is detected and
corresponds to a given reaction by polymerase. As a disadvantage,
this method utilizes expensive and unstable agents, such as enzymes
and ATP-derivates.
[0005] Another method is employed by Ion Torrent that measures the
polymerase reaction by a change in pH (Rothberg et al. Nature. 2011
Jul. 21; 475(7356):348-52). The reaction liberates one extra
H.sup.+-ion which is detected by a chemFET or ISFET (see
WO2010047804). The advantage of this method is a simple adaptation
for integrated circuits which allows considerable miniaturization
and parallelization. No extra enzymes other than the polymerase are
necessary. However, the reaction needs to occur largely unbuffered
and suffers from a low sensitivity.
[0006] In other methods pyrophosphate is detected by complexation
with certain metal ions. Calcein/Mn.sup.2+ is a colourless complex,
which becomes fluorescent when the manganese is depleted and
occupied with magnesium (Tomita et al. Nat Protoc. 2008;
3(5):877-82.). Alternatively, pyrophosphate has been used to
measure manganese ions in solution by optical means (Takashima et
al. J. American Chem. Soc. [Internet]. 2011 Dec. 28).
[0007] Aluminium ions also form a tight specific complex with
pyrophosphate. A fluorescent rhodamine/Al.sup.3+ complex is
depleted of aluminium by the synthesis of pyrophosphate and thus
becomes colorless for detection (Lohani et al. Analyst. 2010.
135(8):2079-84). Free, uncomplexed AI(III) can be detected with
ISFET (Abbaspour et al. Anal. Chim. Acta. 2010 Mar. 3;
662(1):76-81.). A chelator-modified silicon-on-insulator
field-effect transistor (SOI-FET) with bound Zn.sup.2+ ions has
been shown to detect pyrophosphate liberated by a polymerase
reaction directly by the immobilized complex (Credo et al. The
Analyst [Internet]. 2012 Jan. 19).
[0008] Several other metal ions are known to be specifically
displaced from complexes (e.g. transferrin) by pyrophosphate, e.g.
Fe.sup.3+, Ga.sup.3+, In.sup.3+ (Harris et al. Coordination
Chemistry Reviews. 2002. 228(2):237-62), Sn.sup.2+ and Zn.sup.2+
(Cigala et al. Journal of Molecular Liquids. 2012.
165:143-153).
[0009] Some lanthanide metal ions such as terbium, europium and
ytterbium are known to bind to phosphate species such as
triphosphates, DNA, pyrophosphate, and phosphate, albeit with
different affinities (Spangler et al. Ann. N. Y. Acad. Sci. 2008.
1130:138-48.). These metal ions can be used either free or in
complex with certain ligands that may increase their specificity.
So far, these measurements were conducted by using the unique
fluorescence properties of lanthanides.
[0010] The main approaches suffer from the drawback that they
either require expensive and unstable agents, or the reaction is
carried out in a largely unbuffered system and has a low
sensitivity. Methods based on the formation of metal
ion/pyrophosphate complexes require extra hardware such as optical
devices or special surface receptors (for the method of Credo et
al.) which is expensive and hard to implement into existing
sequencing units.
[0011] Recently, synthetic nucleotide analogues lacking terminal
pyrophosphate moieties were successfully tested in polymerase
reactions (Herdewijn and Marliere. FEBS Lett. 2012. Jul. 16;
586(15):2049-56.). The pyrophosphate leaving group was replaced by
synthetic compounds that were equally liberated upon base
incorporation by various polymerases. Since many of the effective
leaving groups are metal ion chelating groups such as aspartic
acid, iminodipropionic acid, or iminodiacetic acid, it is
conceivable that the detection principle of polymerase reactions by
metal ion binding leaving groups using such nucleotide analogues
remains the same.
[0012] The problem to be solved by the present invention may be
formulated as the provision of an inexpensive and simple method for
a fast and sensitive detection of nucleic acid polymerase activity
with a potential for miniaturization and parallelization. Ideally,
the method may be integrated into current systems such as
sequencing devices without or with few modifications.
DEFINITIONS
Sample
[0013] A sample refers to any kind of chemical or biological
substance or substance mixture comprising a nucleic acid, wherein
the sequence or part of the sequence of the nucleic acid is of
interest. A sample may stem from or comprise a prokaryote or an
eukaryote, such as an archaeon, a bacterium, a protist, a fungus, a
virus, a viroid, a plant, an animal or a synthetic
oligonucleotide.
Nucleic Acid
[0014] The term (template) nucleic acid is here used in its
broadest sense and comprises ribonucleic acids (RNA) and
deoxyribonucleic acids (DNA) from all possible sources, in all
lengths and configurations, such as double-stranded,
single-stranded, circular, linear or branched. All sub-units and
sub-types are also comprised, such as monomeric nucleotides,
oligomers, plasmids, viral and bacterial nucleic acids, as well as
genomic and non-genomic DNA and RNA from animal and plant cells or
other eukaryotes or prokaryotes, mRNA (messenger RNA) in processed
and unprocessed form, tRNA (transfer RNA), hn-RNA (heterogeneous
nuclear RNA), rRNA (ribosomal RNA), LNA (locked nucleic acid),
mtRNA (mitochondrial), nRNA (nuclear RNA), siRNA (short interfering
RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA),
scaRNA (Small Cajal Body specific RNA), microRNA, dsRNA
(double-stranded RNA), ribozyme, riboswitch, viral RNA, dsDNA
(double-stranded DNA), ssDNA (single-stranded DNA), plasmid DNA,
cosmid DNA, chromosomal DNA, viral DNA, mtDNA (mitochondrial DNA),
nDNA (nuclear DNA), snRNA (small nuclear DNA) or the like or as
well as all other conceivable nucleic acids.
[0015] Sometimes the concentration of the nucleic acid to be
sequenced (template nucleic acid) may be too low for sequencing. In
this case, the sample may be subjected to an amplification method
prior to sequencing. Nucleic-acid amplification can be accomplished
by any of the various nucleic-acid amplification methods known in
the art, including but not limited to the polymerase chain reaction
(PCR), ligase chain reaction (LCR), transcription-based
amplification system (TAS), nucleic acid sequence based
amplification (NASBA), rolling circle amplification (RCA),
transcription-mediated amplification (TMA), self-sustaining
sequence replication (3SR) and Q.beta. amplification. In a
preferred embodiment the amplification method is selected from the
group of polymerase chain reaction (PCR), real-time PCR (rtPCR),
helicase-dependent amplification (HDA) and recombinase-polymerase
amplification (RPA).
Primer
[0016] The sequence of the primer (molecule) may be a random
sequence. However, in cases where part of the sequence of the
template nucleic acid is already known the primer can be designed
to be complementary to such a sequence. In one embodiment, the
primer anneals to either the 3' or the 5' end of the template
nucleic acid. The primer 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. 1981. 22:1859-1862. 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 which has been isolated from a
biological source (such as a restriction endonuclease digest).
[0017] Preferred primers have a length of about 15-100, more
preferably about 20-50, most preferably about 20-40 bases.
Nucleotides
[0018] As used herein, the term nucleotides refer to
deoxyribonucleoside polyphosphates, such as deoxyribonucleoside
triphosphates (dNTPs). Non-limiting examples of such dNTPs are
dATP, dGTP, dCTP, dTTP, dUTP, which may also be present in the form
of labelled derivatives, for instance comprising a fluorescent
label, a radioactive label, a biotin label. dNTPs with modified
nucleotide bases are also encompassed, wherein the nucleotide bases
are for example hypoxanthine, xanthine, 7-methylguanine, inosine,
xanthinosine, 7-methylguanosine, 5,6-dihydrouracil,
5-methylcytosine, pseudouridine, dihydrouridine, 5-methylcytidine.
Deoxyribonucleoside polyphosphates, i.e. nucleotides with more than
3 phosphates, are also utilized by polymerases. In this case the
polymerase reaction does not generate pyrophosphate.
[0019] However, the principle of selecting a metal ion for binding
to other phosphates (such as triphosphate) and using it for
electrochemical detecting said phosphate can be applied
correspondingly.
Elongation
[0020] Nucleic acid elongation herein means the stepwise addition
of nucleotides to the growing nucleic acid chain. Elongation is
catalyzed by a polymerase. Herein, a polymerase include but are not
limited to T7 DNA polymerase, DNA Polymerase y, Escherichia coli
DNA pol I, Thermus aquaticus pol I, Bacillus stearothermophilus pol
I, Pol II (bacterial), Phi29 DNA polymerase, Pol B
(archaebacterial), Pol .alpha., .delta., .epsilon. and .zeta.,
eukaryotic polymerase pol .beta., pol .sigma., pol .lamda., pol
.mu., terminal deoxynucleotidyltransferase (TdT), Pol X polymerase
and Pol IV.
Sequencing
[0021] Nucleic acid sequencing can consist of determining whether a
particular nucleic acid differs in sequence from a reference
nucleic acid, confirming the presence of a particular nucleic acid
sequence in a sample, determining partial sequence information such
as the identity of one or more nucleotides within a nucleic acid,
determining the identity and order of nucleotides within a nucleic
acid, etc.
[0022] Most sequencing methods relate to the sequencing of DNA.
Current methods, thus, typically require RNA to be converted to
complementary DNA (cDNA) via reverse transcription prior to
sequencing. Thus, if RNA is to be sequenced, the RNA may be first
reverse transcribed into cDNA.
Annealing
[0023] Annealing refers to the pairing of the primer by hydrogen
bonds to a complementary sequence on the template nucleic acid,
forming a double-stranded polynucleotide. Annealing may be
facilitated by decreasing the temperature.
Electrode Measurements
[0024] Electrochemical measurements may be made in an
electrochemical cell consisting of two or more electrodes and the
electronic circuitry for controlling and measuring the current and
the potential.
[0025] The simplest electrochemical cell uses two electrodes. The
potential of one electrode is sensitive to the analyte's
concentration, and is called the working electrode or the indicator
electrode, herein only electrode. The second electrode, which is
called reference electrode, completes the electrical circuit and
provides a reference potential against which the working
electrode's potential is measured. Ideally the reference
electrode's potential remains constant so that one can assign to
the working electrode any change in the overall cell potential.
[0026] Potentiometry is the field of electroanalytical chemistry in
which potential is measured under the conditions of no current
flow. The measured potential may then be used to determine the
analytical quantity of interest, generally the concentration of
some component of the analyte solution. The potential that develops
in the electrochemical cell is the result of the free energy change
that would occur if the chemical phenomena were to proceed until
the equilibrium condition has been satisfied.
[0027] A reference electrode is an electrode which has a stable and
well-known electrode potential. Reference electrodes are well-known
to the skilled person.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The inventors have unexpectedly found that biological
reactions which release pyrophosphate may be detected by
electrochemical methods via the formation of pyrophosphate/metal
ion complexes and/or precipitates.
[0029] The present invention is based on the principle that
pyrophosphate (PPi; inorganic diphosphate) can be bound and/or
precipitated directly in a biological reaction (in a reaction
mixture). The binding and/or precipitation decreases the
concentration of unbound and/or dissolved (free) PPi which changes
the concentration of a metal ion, subsequently resulting in a
change of signal generation in an electrochemical sensor.
[0030] A typical PPi releasing reaction is a reaction that uses the
synthetic activity of a polymerase. For example, DNA polymerase
catalyses the polymerization of deoxyribonucleotides (dNTPs) into a
DNA strand. The addition of one of the four complementary dNTPs
onto the template releases PPi stoichiometrically. This principle
can be exploited in that the synthetic activity of the polymerase
is determined indirectly over the release of PPi. In contrast to
the present methods, the depletion of metal ions as a result of the
binding to and/or the precipitation with PPi is detected by
electrochemical means. Metal ions that bind to the pyrophosphate
anion specifically in the presence of triphosphates and nucleic
acids can thereby be used as an indicator for polymerase reactions.
Free metal ions can be detected by electrochemical methods and thus
indirectly indicate the amount of pyrophosphate complex formed by
the reaction despite the presence of triphosphates and
oligonucleotides. The decrease in free metal ion concentration
results in a corresponding signal drop at an electrode. This will
occur only if a nucleotide matching to the template is offered to
the polymerase. Thus, this technique can be applied to the
detection of nucleic acid sequencing reactions.
Sequencing
[0031] Accordingly, in a first aspect the present invention relates
to an electrochemical nucleic acid sequencing method comprising the
steps of (a) providing a sample comprising a template nucleic acid
to be sequenced; (b) annealing a primer molecule to said template
nucleic acid or introducing a nick at a predetermined site into a
double stranded template; (c) carrying out nucleic acid elongation
steps with a polymerase on said template in the presence of metal
ions, wherein each step comprises the addition of a single type of
nucleotide, wherein pyrophosphate is released in a stoichiometric
ratio to the number of nucleotides incorporated, and wherein the
metal ions are capable of specifically binding to pyrophosphate;
(d) determining in said sample the change of potential, current or
charge at an electrode and (e) correlating this change in
potential, current or charge over the number of elongation steps
with the type of nucleotides added.
[0032] To correlate the signal observed with the sequence of the
template nucleic acid to be sequenced one may proceed as
follows:
[0033] A change in the signal may be observed, once a nucleotide is
successfully incorporated. Since only one type of nucleotide
(adenine A, cytosine C, guanine G, or uracil U/thymine T) is added
at the same time, one can deduce the type of incorporated
(elongated) nucleotide. Repeating the steps of adding types of
nucleotides and observing the signal one may further deduce the
whole sequence of the template nucleic acid.
[0034] Preferably, a calibration curve is generated by measuring
the signal at various known concentrations of metal ions used for
binding and/or precipitating pyrophosphate in order to ascertain
confidence parameters. For example pyrophosphate may be added in
various known concentrations to a solution of constant metal ion
concentration in another experiment. The calibration curve is
preferably made in a solution (e.g. a buffer) which is comparable
to the test solution (e.g. sequencing buffer). In the test solution
the initial metal ion concentration may be known or calculated from
the initial signal.
[0035] Preferably, the nucleic acid sequencing method is based on
pyrosequencing, reversible-terminator-pyrosequencing, or closed
complex formation sequencing.
[0036] It is preferred that step d. and optionally step e. is done
after each elongation step c. Alternatively, step d. and optionally
step e. is done after every second, third, fourth, firth or even
more elongation steps c.
[0037] The sequencing method according to the invention is
preferably based on pyrosequencing. Pyrosequencing is based on the
detection of the pyrophosphate (PPi) that is released during DNA
polymerization (see, e.g., U.S. Pat. Nos. 6,210,891 and 6,258,568).
While avoiding the need for electrophoretic separation,
pyrosequencing suffers from a large number of drawbacks that have
as yet limited its widespread applicability (Franca et al.
Quarterly Reviews of Biophysics. 2002. 35(2):169-200).
[0038] In an alternative embodiment the step of sequencing involves
reversible-terminator-pyrosequencing. The method is extensively
explained in Wu et al. PNAS. 2007. 104(42):16462-16467, which is
hereby incorporated by reference.
[0039] In yet another embodiment sequencing involves closed complex
formation sequencing. The method is described in WO 2007/048033,
which is hereby incorporated by reference.
Polymerase Reaction
[0040] The inventive concept is particularly advantageous in the
case of sequencing a nucleic acid. There are, however, other cases
where it is desirable to detect and/or quantify the amount (e.g.
concentration) or a change in said amount of PPi with a fast and
cost-effective method. Consequently, the invention relates to a
method for detecting a nucleic acid polymerase reaction comprising
the steps of (a) providing a sample comprising a nucleic acid; (b)
carrying out a nucleic acid polymerase reaction step on said sample
in the presence of metal ions, wherein pyrophosphate is released or
depleted depending on the type of polymerase reaction, and wherein
the metal ions are capable of specifically binding to
pyrophosphate; (c) determining the potential, current or charge at
an electrode at least twice, wherein between the first time and the
second time one or more nucleic acid polymerase reaction steps take
place; and (d) correlating the difference in potential, current or
charge over the number of polymerase reaction steps and/or time
with the progress of the nucleic acid polymerase reaction.
[0041] It is preferred that the nucleic acid polymerase reaction is
a synthesis reaction, an amplification reaction and/or a
transcription reaction.
[0042] It is further preferred that the step of determining the
potential, current or charge (step c.) and optionally the step of
correlating the difference in potential, current or charge step
(step d.) is done after each polymerase reaction step (step b.).
Alternatively, step c. and optionally step d. is done after every
second, third, fourth, fifth or even more elongation polymerase
reaction steps.
[0043] As described above, the principle can generally be utilized
for assessing whether the concentration of pyrophosphate changes in
a sample. The method of detecting the formation or depletion of
pyrophosphate in a sample comprises the following steps: (a)
providing a sample in which pyrophosphate is formed or depleted;
(b) adding to said sample metal ions, wherein the metal ions are
capable of preferentially binding pyrophosphate; (c) determining
the potential, current or charge in the sample a first and at least
a second time; (d) correlating the difference in potential, current
or charge over time with the formation or depletion of
pyrophosphate in the sample.
[0044] Further, the invention can further be used for detecting the
presence and/or the quantity, e.g. concentration, of pyrophosphate
in a sample. This method comprises the steps of (a) adding to a
sample comprising pyrophosphate a predetermined amount of metal
ions or metal ions of a known potential, current or charge, wherein
the metal ions are capable of specifically binding pyrophosphate;
(b) determining in said sample the potential, current or charge at
an electrode; and (c) correlating the potential, current or charge
with the amount of pyrophosphate present in said sample.
Metal Ions
[0045] There are a number of different metal ions that can bind to
and/or precipitate pyrophosphate and, thus, be used in the present
method. Preferred metal ions are manganese ions, tin ions, zinc
ions, copper ions, molybdenum ions, aluminium ions, iron ions,
indium ions, gallium ions, zirconium ions, and lanthanides. More
preferred metal ions are manganese ions, aluminium ions, tin ions
and iron ions. Most preferred metal ions are manganese ions.
Electrochemical Measurements
[0046] The present invention is not restricted to a particular
electrochemical technique. For example, potentiometry,
controlled-current coulometry, amperometry, controlled-potential
coulometry, stripping voltammetry, hydrodynamic voltammetry,
polarography and stationary electrode voltammetry, pulse
polarography and voltammetry and cyclic voltammetry may be
used.
[0047] Herein, however, potentiometry is preferred, since it is
simple, inexpensive and may be further miniaturized. Furthermore,
potentiometry is a quantitative technique. The skilled person will
readily understand that and how the principle may be transferred to
other electrochemical techniques.
[0048] The working electrode must be selected such that it responds
to the metal ion used for binding and/or precipitation. This means
that e.g. for a potentiometric detection the potential of the
electrode depends on the concentration of said metal ion. In
certain cases it may be advantageous that the electrode is
selective for the metal ion. But this may not be necessary in cases
where the composition of the solution can be controlled such as in
the case of nucleic acid elongation. Preferred working electrodes
are electrodes based on manganese oxide. They show a signal which
correlates to the concentration of the potential determining ion,
i.e. manganese, in solution.
[0049] As reference electrode, which provides a stable potential,
for example a silver/silver chloride or a saturated calomel
electrode (SCE) may be used in the inventive method. Preferably,
the reference electrode is a silver/silver chloride electrode with
a salt bridge. But also miniaturized reference electrode
constructions are possible.
[0050] In a specific embodiment, the metal ions are manganese ions
and the electrode is a manganese oxide electrode and the reference
electrode is a silver/silver chloride electrode.
[0051] The present method has a number of advantages: The method is
simple, cost-effective and may be easily implemented into existing
devices. Moreover, since buffer conditions and components during
pyrosequencing can be highly controlled, even less specifically
binding and/or precipitating metal ions can be used to monitor the
polymerase reaction.
Identification Procedure for Suitable Combination of Metal
Ion/Working Electrode
[0052] In general, for identifying further working electrodes one
may first select suitable metal ions and then chose the
corresponding working electrode. The type of metal ion should be
selected such that they meet one or more of the following criteria.
Ideally, the metal ions selected meet all criteria: (1) The metal
ions bind and/or precipitate PPi, preferably specifically. In some
cases, metal ions binding to and/or precipitating Phosphate (Pi)
may be also used. In this case, a pyrophosphatase should be added
to the sample. (2) The metal ions selected do not or not
substantially bind to DNA, NTP, dNTP and proteins or are
efficiently blocked/displaced by other ions present in the buffer
such as magnesium. (3) The metal ions selected do not affect the
chemical composition and structure of DNA and/or RNA. (4) The metal
ions selected do not affect (e.g. inhibit) the polymerase at
typical concentrations used in the method. (5) The metal ions
interact with the electrode used in such a way that slight changes
in the concentration or complex formation of metal ions can be
measured.
[0053] By way of example, DNA is known to be damaged e.g. by
platinum. Hence, such metals do not appear to be suitable. In
contrast, tin can be present in at least two oxidation stated, i.e.
Sn.sup.2+ and Se. It binds more specific to PPi (and Pi) than to
ATP. It is assumed that tin does not bind strongly to DNA; it
might, however, be necessary to additionally add magnesium to the
sample. Thus, tin may be an alternative to manganese. Further, PPi
(and/or Pi) binding metal ions are molybdenum Mo, aluminium Al,
iron Fe, gallium Ga, indium In, zirconium Zr and chromium Cr.
[0054] For identification of a suitable metal ion the candidate
metal ion should be assessed with regard to the foregoing criteria
if not known. For example, Cigala et al. describe the interaction
of Sn.sup.2+ and Zn.sup.2+ with phosphate ligands (Cigala et al.
2012. J. Molecul. Liquids. 165:143-153). Further, others describe
the effect of different metal ions on HIV-1 reverse transcriptase
(Sabbioni et al. 1999. Biol. Trace Element Res. 68:107-119).
Electrochemical Nucleic Acid Sequencing Apparatus
[0055] The invention further relates to an electrochemical nucleic
acid sequencing apparatus comprising: (a) means for carrying out
the steps of annealing and elongating a primer molecule to a
template nucleic acid to be sequenced; and (b) an electrochemical
cell comprising metal ions being capable of specifically binding to
pyrophosphate and a working electrode responsive to said metal
ions. In a preferred embodiment the metal ions are manganese ions
and the electrode is based on manganese dioxide.
[0056] In a preferred arrangement these parts are combined in a
small reaction chamber (see FIG. 2).
[0057] The means for carrying out the steps of annealing and
elongating a primer may comprise a temperature control unit for
heating and cooling, such as one or more peltier elements. It may
also comprise means for supplying reagents and buffers to the
reaction chamber. Such means can comprise one or more channels
(e.g. tubes). Preferably the reagents and buffers can be supplied
through different channels, i.e. one channel for each nucleotide or
alternatively for all nucleotides, one channel for the sample, one
channel for the polymerase, etc. The channels are preferably
controllable, e.g. by valves.
Example 1
[0058] Manganese ions and their depletion by enzymatically
generated pyrophosphate were followed by potentiometry using a
manganese oxide/carbon paste electrode. For this purpose a template
DNA was amplified by PCR using the following conditions:
Volume=50 .mu.l
[0059] dNTP 10 mM 0.4 .mu.l
Forward Primer 10 mM 0.2 .mu.l
Reverse Primer 10 mM 0.2 .mu.l
Template 2 .mu.l
1 u Taq Polymerase 10.times. Puffer (10 mM Tris, 50 mM KCl, 1.5 mM
MgCl.sub.2, pH 8.3 (at 20.degree. C.)) 5 .mu.l
MnCl.sub.2 10 mM 0.4 .mu.l
[0060] Several samples from reaction mixtures after different
numbers of PCR cycles yield different potentials (FIG. 1). It was
shown that with a simple potential measurement the concentration of
unbound Mn.sup.2+ ions can be analyzed in a complex mixture and
thus the progress in the biochemical amplification reaction can be
followed.
FIGURE CAPTIONS
FIG. 1: MnO.sub.2/Carbon-Paste-Electrode Measurement of Different
PCR Cycles.
[0061] Manganese ions and their depletion by enzymatically
generated pyrophosphate were followed by potentiometry using a
manganese dioxide/carbon paste electrode. For this purpose a
template DNA was amplified by PCR using the conditions set out in
the example section.
FIG. 2: Examples of the Electrochemical Sequencing Apparatus.
[0062] The upper part illustrates a measuring set up with a two
electrode arrangement [metal ion sensitive working electrode (dark)
and reference electrode (white)] dipping into the small reaction
chambers and connected to a voltmeter. The lower part shows
schematically an integrated version with enclosed working and
reference electrodes within the reaction chambers.
* * * * *