GB2509481A - Ionic signal enhancement in nucleic acid synthesis reactions - Google Patents

Abstract

A method of detecting a change in a proton concentration in a nucleic acid synthesis reaction mixture comprising the steps of providing a nucleotide analogue to a reaction mixture comprising a nucleic acid template and a nucleotide hydrolysing enzyme such that the nucleotide hydrolysing enzyme hydrolyses the nucleotide analogue to produce a greater change in the proton concentration than would occur with a corresponding natural nucleotide, and detecting a change in proton concentration with an ion sensitive apparatus, such as an ion sensitive field effect transistor (ISFET). The nucleic acid synthesis reaction may include primer extension, nucleic acid sequencing, nucleic acid amplification, reverse transcription or combinations thereof. Preferred nucleotide hydrolysing enzymes include DNA polymerases, RNA polymerases, reverse transcriptases, ligases and helicases. Preferred nucleoside analogues include deoxyadenosine 5-(beta,gamma-difluromethylene) triphosphate (DMFB-dAMP) and iminodipropionic acid deoxyadenosine monophosphate (IDP-dAMP).

Description

Ionic Signal Enhancement

Field of the invention

The present invention relates to a method and apparatus for increasing the detectable ionic signal from an enzymatic reaction, which is particularly, though not exclusively, suitable for DNA sequencing, nucleic acid amplification and SNP genotyping. More particularly, disclosed herein are nucleotide analogues that can be used to increase ionic signal strength detected by an ion sensitive apparatus.

Background of the invention

Over the last two decades, there has been a rapid development in nucleic acid analysis, specifically in the field of nucleic acid amplification and DNA sequencing technology, with an increasing range of instrumentations now available The conventional methods of detecting and analysing a nucleic acid sequence primarily rely on fluorescent nucleic acid intercalating dyes, fluorescent-labelled oligonucleotide probes, fluorescent-or radioactive-labelled nucleotides.

Subsequently, a new method of analysing nucleic acid synthesis and sequence has been developed using a semiconductor-based detection system such as an Ion Sensitive Field Effect Transistor (ISFET) (Rothberg JM, Nature, 2001). An Ion Sensitive Field Effect Transistor -based platform, unlike conventional fluorescent-based nucleic acid analysis systems, does not require expensive optical instruments or dangerous radioactive isotopes for detection, thus making this platform a cost effective, safe and simple alternative for sequencing and nucleic acid amplification analysis.

Specifically, an ISFET, which measures ion concentrations in solution, has been employed to detect nucleotide incorporation into a nucleic acid strand by detecting the change in hydrogen ion (Hf, proton) concentration resulting from the reaction.

Hydrogen ions are released during the nucleic acid polymerization reaction. For example, Equation I below demonstrates the release of hydrogen ion facilitated by DNA polymerase mediated hydrolysis of a single deoxynucleotide: dNTP -3 dNMP + FPi + zH (Equation I) Wherein dNTP is a nucleoside triphosphate, dNMP is a nucleoside monophosphate, z is an integer or fraction describing the average number of protons generated per nucleotide turnover, H is a proton and PPi is a Pyrophosphate (leaving group or reaction product).

The change in electiical output signal strength of the ISFET depends on the amount of hydrogen ions released, which largely depends on the quantity of nucleic acid (for instance RNA or DNA) present in the sample to be measured. However, in some cases, the quantity of hydrogen ions released directly from the incorporation reaction may be too small to be detected accurately by the ISFET and signal processing. This is especially true for detection of single nucleotide extension reactions as well as detection of low quantities of nucleic acids. Additionally, there may be high levels of background noise, which may be a result of by-products of the reaction.

Therefore, given that the detection or quantification of the nucleic acids depends on the ability to discriminate the true signal against a background noise, the inventor has appreciated that it would be useful to amplify the proton signal generated without increasing the turnover of the enzymatic reaction. Such signal enhancement may increase efficiency and accuracy of nucleic acid analysis by, for example, decreasing the detection time for SNP genotyping and increasing the confidence of base calling for semiconductor sequencing platforms. To date, strategies to improve the ionic signal detection have focused on optimizing the ISFET, nucleotide hydrolysis reaction turnover and composition of the reaction mixture. In the present invention, the inventor offers an alternative approach by adding modified nucleotides as substrates in a nucleotide hydrolysis reaction. The present invention provides a method useful for ion sensing nucleic acid analysis applications, including polymerase reaction monitoring, SNP genotyping and non-gel-based sequence determination methods.

Summary Of The Invention

According to a first aspect of the invention there is provided a method of detecting a change in a proton concentration in a nucleic acid synthesis reaction mixture comprising: providing a nucleotide analogue to a reaction mixture comprising a nucleic acid template and a nucleotide hydrolysing enzyme, such that the nucleotide hydrolysing enzyme hydrolyses the nucleotide analogue to produce a greater change in the proton concentration than would occur with a corresponding natural nucleotide; and detecting a change in proton concentration with an ion sensitive apparatus.

Preferably, the nucleotide analogue is represented by the formula: 0 fo 0 0 II I ii II II R4-P-R1-j-P-O-P-R6-P-R5-S-X n wherein X is a nucleobase or nucleobase analogue; S is an acyclic or carbocyclic sugar moiety; n is an integer between 0 and 2; at least one of 51, 52, 53, P4 and RL is a moiety without a pKa or with a pKa outside a range of 6.0-8.5, more preferably 6.0- 9.5; R5, 56 and R7 may be the same or different and are independently selected from the group consisting of -0, difluoromethylene and carbonyl, with the proviso that at least one of R6 or R7 is -0; and with the proviso that if R5, 56, and R7 are -0, then at least one of 51, 52, 53, R4 and SL is not -OH.

It is also preferred that the ion sensitive apparatus is an Ion Sensitive Field Effect Transistor (ISFET).

Alternatively, the ion sensitive apparatus is a conductive, a capacitative or an inductive ion sensitive device.

The nucleic acid synthesis reaction mixture is preferably a reaction mixture for primer extension reaction, nucleic acid sequencing reaction, nucleic acid amplification, reverse transcription reaction, or combination thereof.

According to a second aspect of the invention there is provided a method of increasing the quantity of an ion signal in a nucleic acid synthesis reaction mixture comprising: mixing a nucleic acid template with a polymerase enzyme and a primer to form a complex of the template, the primer and the polymerase enzyme; exposing the complex to one or more nucleotides wherein at least one non-bridging oxygen group of at least one terminal phosphorous group of the nucleotide and its reaction byproduct is substituted with a moiety without a pKa or with a pKa outside a range of 6-8.5; and detecting the ion signal with an ion sensitive apparatus.

Brief Description Of The Drawings

Specific embodiments of the invention will now be described by way of example only with reference to the accompanying figures, in which: Figure lisa profile view of an ISFET exposed to a sample; Figure 2 is a profile view of a pH detection system; Figure 3A, 3B and 3C show results of a simulated pH-PCR reaction comparing DFMB-dAMP and dAIP as substrates; and Figure 4A, 4B and 4C show results of a simulated pH-PCR reaction comparing IDP-dAMP and dAIP as substrates.

Detailed Description

As used herein, the term "nucleotide" refers to a compound composed of a nucleobase, a sugar and a mono-or oligo-phosphate group, or analogues thereof. Nucleotides are monomeric units of a nucleic acid sequence (DNA or RNA). Examples include, but are not limited to, ribonucleotides such as ATP, GTP, CIP, UTP; deoxyribonucleotides such as dATP, dCTP, dTTP, dUIP, dGTP and dideoxyribonucleotides such as ddATP, ddCTP, ddTTP, ddUTP, ddGTP, or analogues thereof.

As used herein, the term "natural nucleotide" refers to a nucleotide that is naturally occurring and has not been modified. Examples include ATP, GIP, CTP, TTP, UTP, dATP, dGTF, dCTP and dTTP.

As used herein, the term "nucleotide analogue" refers to a compound that resembles a nucleotide but has been modified whereby some elements within the nucleobase, sugar and/or triphosphate group have been substituted with moieties at the equivalent position. For nucleic acid synthesis, the deoxyribonucleotide or ribonucleotide analogues described herein are capable of being hydrolysed and can be incorporated into a nucleic acid (DNA or RNA) chain, and are thereby capable of base-pairing with a nucleotide residue in a complementary chain or base stacking in the appropriate nucleic acid chain.

As used herein, the terms "modified nucleotide" and "nucleotide analogue" are used interchangeably and are intended to describe non-natural or modified molecular compounds.

As used herein, the term "leaving group" refers to a molecular group of the triphosphate or modified triphosphate moiety that departs upon hydrolysis of the nucleotide or nucleotide analogue. Examples include pyrophosphate, secondary amines or monophosphate and modified versions thereof. Monophosphate will also be referred to as Pi, and pyro-or diphosphate will also be referred to as PPi.

As used herein, the term phosphate is a group of the chemical structure OP(O)(O)0 (written in SMILES, Weininger D, J Chem lnj Comput Sci. 1988. 28: 31-36) and is applied broadly to include phosphate analogues wherein one or more of the oxygens in the phosphate are replaced by other atoms or moieties.

As used herein, the term "substrate" refers to nucleotide triphosphate, or deoxynucleotide triphosphates or their analogues.

As used herein, the term "reaction product" refers to pyrophosphate (PPi), phosphate (Pi), polymeric dNMP (DNA) or monomeric dNMP.

As used herein, the term "primer" refers to a single stranded oligonucleotide that is extended by covalent bonding of nucleotide monomers during amplification, single nucleotide extension or polymerization of a DNA or RNA molecule.

As used herein, the term "template" refers to double-stranded or single-stranded nucleic acid molecules, which are to be amplified, synthesized or sequenced.

As used herein, reference to protons, hydrogen ions, and H are intended to be synonymous, all associated with the pH of a reaction mixture.

As used herein, the terms -OH, -OH group, hydroxyl, hydroxyl group, and "non-bridging oxygen" or "non-bridging oxygen atom" where referring to moieties that are part of a phosphate group, are meant to be synonyms.

The present invention relates to a method for increasing the proton signal generated from a nucleotide hydrolysis reaction that can be detected and quantified by an ion sensitive apparatus such as an ISFET. This is achieved by replacing the natural enzyme substrates (i.e. natural nucleotides), partly or in total, with modified nucleotides.

In some embodiment, the present invention relates generally to a modified nucleotide comprising one or more modifications that increase proton release and/or reduce buffering capacity of the modified nucleotide relative to the corresponding natural nucleotide. Generally, a natural nucleotide is characterized by a triphosphate group, a sugar group and a nucleobase, and the modified nucleotide can contain one or more modifications to the triphosphate group, the 3-OH of the sugar group, the nucleobase, or combination thereof.

The preferred nucleotide hydrolysis reactions are nucleic acid synthesis reaction, in particular those catalysed by DNA-and RNA-polymerases. However, other biochemical reactions that hydrolyse nucleotides such as protein phosphorylation, nucleic acid unwinding and ligation can be envisaged.

In general, the nucleic acid synthesis reaction involves synthesis of RNA or DNA via nucleic acid polymerase activity. RNA and DNA polymerase synthesize oligonucleotides via transfer of a nucleoside monophosphate from nucleoside triphosphate (NTP) or deoxyribonucleoside triphosphate (dNTP) to the 3' hydroxyl of a growing oligonucleotide chain. The incorporation of nucleotide leads to the release of protons in the reaction mixture, and when in the presence of low buffering activity, the released proton leads to a decrease in the overall pH of the reaction mixture. In addition, the decrease in pH is also contributed by the combination of the hydrolysis of the nucleotide and the accumulation of reaction product(s) (i.e. PPi). Thus the inventor appreciates that replacing the natural nucleotides in a nucleic acid synthesis reaction mixture, partly or in total, with modified nucleotides that alters the proton yield of the reaction and/or buffering capacity of the reaction mixture can lead to an increase in the magnitude of pH change of the reaction mixture. Such signal enhancement may increase efficiency and accuracy of nucleic acid analysis by, for example, decreasing the detection time for SNP genotyping and increasing the confidence of base calling for semiconductor sequencing platforms.

Modifications to the triphosphate group of a nucleotide In general, the nucleotide analogues of the present invention have different pKa compared to the natural nucleotides. The present invention utilises the finding that altering the pKas can affect the proton change by one or both of the following mechanisms. The first mechanism involves the proton yield of the nucleotides and the second mechanism involves the buffering capacity of the nucleotides. The proton yield', are the H+ ions generated from the turnover of a single substrate molecule (i.e., dATP -) (dAMP)n + PPi). The amount is equal to the net difference between the average protonation states of a substrate (i.e., dATF) and its reaction product (i.e., (dAMP)n and PPi) at a given reaction pH. Generally the reaction is conducted in the pH range of 6-9.5, more preferably 6-8.5. Typically, moieties with low pKas compared to the reaction pH will release H+ ions, whereas moieties with higher pKas will bind protons. The second mechanism relates to the buffering of the reaction medium. This effect depends on the concentrations of the buffering compounds, in addition to their pKas. Buffering will be strongest when the reaction pH is closest to the pKa(s) of the buffering compound(s). Many chemical and biochemical compounds, including natural nucleotide have some buffering capacity, and thus contribute to the buffering of the reaction mixture. In particular, the terminal phosphate groups of the triphosphate group of a nucleotide (i.e. y-phosphate) and its reaction by-products [i.e. PPi and (dAMP)n] exhibit buffering capacity which may hinder the detection of pH change by an ion sensitive apparatus. This is because, in contrast to internal phosphates of a polyphosphate chain, terminal phosphates (e.g. gamma phosphate of a nucleotide triphosphate) carry not one but two -OH group. While the first pKa of the -OH group (ionization state -1) is low, which is similar to that of the single -OH group of the internal phosphates, the release of the proton from second -OH group is hindered by the negative charge whereby the resulting pKa is close to 7. In fact, terminal phosphates are the only moieties on natural nucleotides and their reaction products with a pKa in the 6-8.5 range.

Therefore, one of the most efficient way of increasing the magnitude of pH change in a nucleic acid synthesis reaction mixture is by using modified nucleotides wherein at least one of the non-bridging -OH or bridging -o of a natural nucleotide or nucleotide analogue has been substituted with a moiety that increase release or absorption of protons and/or decrease buffering capacity. Increased protonation is achieved by increasing the difference between pKas of the OH groups of substrate nucleotide and its reaction by-products! in particular those which fall into the pH range of the reaction mixture that falls typically between pH 6-9.5. Decreased buffering at reaction pH is achieved mainly through changes affecting the terminal phosphates of polyphosphate chains such as in nucleotide triphosphate, nucleotide diphosphate, and pyrophosphate.

In one aspect of embodiments, the second -OH group is replaced with an atom or a moiety which does not exchange protons with the reaction mixture or whose pKa is outside the pH range of the reaction mixture. In another aspect of embodiments, the pKas of both -OH groups at the terminal phosphate is lowered by introducing moieties that are highly electronegative or moieties that are capable of delocalizing the negative charge of the deprotonated -OH groups.

In one embodiment of the method of detecting a change in proton concentration in a nucleic acid synthesis reaction mixture provided herein comprising: a) providing a nucleotide analogue according to formula I to a reaction mixture comprising a nucleic acid template and a nucleotide hydrolysing enzyme, such that the enzyme hydrolyses the nucleotide analogue to produce a greater change in the proton concentration than would occur with a corresponding natural nucleotide; and b) detecting a change in proton concentration with an ion sensitive apparatus: Formula I: 0 fo o o

II I II II II

R4 -P -R1-j-1P-0 -R6 -F-R5-S -X

II RL n

wherein X represents a nucleobase, preferably guanine, adenine, thymidine, cytosine or uracil, but may also include xanthine, hypoxanthione or nucleobase analogues; wherein S is a sugar moiety, preferably a ribose, deoxyribose or dideoxyribose, but can also be a similar cyclic or acyclic tetrose, pentose or hexose moiety; wherein n is an integer between 0 and 2, and describes the number of "linker" phosphate groups inserted before the terminal phosphate of the analogue; wherein Ri, P2, P3, P4 and RL may be the same or different and is a moiety without a pKa or with a pKa outside a range of 6-8.5; wherein PS, P6 and P7 may be the same or different and are independently selected from the group consisting of -0, difluoromethylene and carbonyl, with the proviso that at least one of P6 or P7 is -0; and with the proviso that if PS, P6, and P7 are -0, then at least one of P1, P2, P3, P4 and RL is not -OH.

The nucleotide analogues according to Formula I have the potential to be recognized and processed by a variety of nucleotide hydrolysing enzymes depending on whether P6 or P7 contain a hydrolysable bond (i.e. -0). For example, nucleotide analogues with a hydrolysable bond at P6 are substrates of DNA polymerases, PNA polymerases and reverse transcriptases, or mutants thereof; and are capable of being incorporated into a growing nucleic acid chain. Similarly, ATP analogues with a hydrolysable bond at P6 are substrates of ligases, or mutants thereof, which use ATP, or analogues thereof as co-factors to ligate a broken nucleic acid strand. On the other hand, nucleotide analogues with a hydrolysable bond at P7 are substrates of helicases, or mutants thereof; and are utilized frequently as an energy source for nucleic acid unwinding.

According to Formula I of the present method, the choice of substitutions at the side positions of the phosphorus group (i.e. P1, P2, P3, P4 and PL) and the oxygen atoms linking the phosphorus groups (i.e. PS, P6 and P7) are aimed to alter the pKa of the nucleotides relative to the natural nucleotides, and will depend on the enzymatic reaction and the reaction by-products.

Both reaction substrates (e.g. dNTP or dNTP analogues) and the various reaction by-products will contribute to the pH change through their buffering capacity at the end of the reaction. The effect of substituting a phosphate -OH group depends on its position and the site at which the enzyme cleaves the polyphosphate chain. P3 and P4 of Formula I are of particular significance because they are carried by the terminal phosphorous group of the reaction substrate as well as most reaction products. P1, R2, and RL can become part of terminal phosphorus groups of reaction by-products depending on the site at which the nucleotide hydrolysing enzyme cleaves. To illustrate in more detail, the scheme below shows how terminal phosphorous groups in both reaction substrate and by-products may contribute to the buffering capacity of a nucleotide hydrolysis reaction.

0 [0 0 0

II I II II II

R4 -P Rrf-P0 PR6P R55 -x R4 --R7 Ei_ OH + Y -R5- -In the above scheme, X represents a nucleobase, preferably guanine, adenine, thymidine, cytosine or uracil, but may also include xanthine, hypoxanthione or nucleobase analogues. S is a sugar moiety, preferably a ribose, deoxyribose or dideoxyribose, but can also be a similar cyclic or acyclic tetrose, pentose or hexose moiety. n is an integer between 0 and 2 and describes the number of "linker" phosphate groups inserted before the terminal phosphate of the analogue. R5, R6 and R7 are -0, wherein R6 is hydrolysed by the polymerase. Y is -O of the reaction product and is covalently attached to the nascent strand of DNA or RNA. The hydrolysis at R6 by the polymerase results in a new -OH arisen at the same phosphate as R2, creating a new terminal phosphorous group. Therefore in nucleic acid polymerase reactions, substituting at least one of the -OH moiety of natural nucleotide at the equivalent R2, R3, and R4 positions of formula I with a moiety without a pKa or with a pKa outside a range of 6-8.5 can increase the magnitude of pH change in a nucleic acid synthesis reaction. On the other hand, substitution at Ri and/or RL (internal phosphorous group) position has no significant effect on altering the pH of the reaction.

Similarly, the scheme above can represent a ligase reaction where X is an adenosine, S is a sugar moiety, preferably a ribose and n is 0. R5, R6 and R7 are -0, wherein R6 is hydrolysed by the ligase. Y is -OH of the reaction product AMP. AMP contains a terminal phosphate carrying both Si and Y. Theiefoie, in addition to substituting at at least one of R2, R3 or R4, substituting Ri can be advantageous for the pH monitoring of ligase, but not polymeiase reactions.

It will be obvious from the above that there is a variety of chemical moieties that could replace phosphate -OH groups of the natural nucleotide to reduce buffering, as long as these moieties do not have a pKa close to 7 In addition, for the ease of synthesis as well as acceptance by nucleotide hydrolysing enzymes, the use of small polar groups that are similar in conformation and electronic structure to the -OH group will generally be preferable.

In certain preferred embodiments! the method uses nucleoticle analogues of formula I wherein Ri, R2, R3, R4 and RL of fomiula I are independently selected from the group consisting of -OH, -SCH3, -OCH3, -Cl, -F, -BH3 and -NH2, and derivatives thereof; and wherein 55, R6 and R7 may be the same or different and aie independently selected from the group consisting of-O, difluoromethylene and carbonyl, with the proviso that at least one of R6 or R7 is -0; and with the pioviso that if R5, R6, and R7 aie -0, then at least one of Ri, R2, 53, R4 and RL is not -OH.

In a pieferied embodiment, the nucleotide analogues differ from the natural nucleotides in that only one of the -OH groups of the terminal phosphate (i.e. gamma phosphate) group of a nucleotide is substituted. In other words, according to formula I, Ri, R2 and R3 are -OH, R8, R9 and RiO are -0, and R4 is selected from a moiety that has no pKa value or has a pKa outside the range of 6-8.5, and that is not -OH. More preferably, R4 is selected from a group consisting of -SCH3, -OCH3, -Cl, -F, -BH3 and -NH2, and derivatives thereof. The advantage of substituting one of the -OH group of the terminal phosphate of the natural nucleotide is that such substitution will have the largest impact in the buffering capacity of the nucleic acid synthesis reaction mixture, while lemain closely analogous to the natural nucleotides, thus minimizing any enzymatic related effects of processing modified nucleotides into a growing nucleic acid chain.

Modifications of the substrate terminal phosphates have been described before. These were large fluorescent moieties that are usually attached to the terminal phosphate via long linkers, and whose potential to affect the pH was neither suggested nor observed.

Nevertheless, it is possible that some of these nucleotide analogues can also be used to improve pH changes provided that these fluorescent moieties have low buffering capacity.

In another embodiment, the present method utilise nucleotide analogues of Formula II in the nucleic acid synthesis reaction to increase the magnitude of pH change in positive direction. The nucleotide analogues of formula II have an increased ability to absorb protons from the reaction mixture when compared with natural nucleotides: Formula II:

II

L-P-O-S-x

OH

wherein X represents a nucleobase, preferably guanine, adenine, thymidine, cytosine or uracil, but may also include xanthine, hypoxanthione or nucleobase analogues.

wherein S is a sugar moiety, preferably a ribose, deoxyribose or dideoxyribose, but can also be a similar cyclic or acyclic tetrose, pentose or hexose moiety.

wherein L is one of a secondary amine or a tertiary amine.

The nucleotide analogues according to Formula I and II are not exhaustive, other modifications to the triphosphate group can be incorporated into the nucleotide hydrolysis reaction mixture to increase the magnitude of the pH change of the reaction mixture by shifting the pKa of the nucleotide analogues to increase protonation or decrease the buffering capacity. Therefore, it should be understood that these embodiments are illustrative. Those skilled in the art will be able to make modifications and alternatives in view of the disclosure which are contemplated as falling within the scope of the appended claims.

The pKa values can be estimated from the chemical structure and can be used to calculate the protonation state of a molecule at a given pH. For a series of related compounds, variation of electronegativity at the proton acceptor/donor group results in a predictable trend, e.g. higher electronegativity results in a lower pKa and vice versa.

Quantitative values for pKa or protonation states can be calculated from chemical structure using programs such as ACD (www.acdlabs.com), Marvin (www.chemaxon.com), or SPARC (sparc.chem.uga.edutsparc/). The pH of the mixture can be calculated from pKa values and concentrations of compositions using programs such as CurTipot (http:IIwww2.ig.usp.br/docente/gutz/Curtiiot html).

Calculation of the pKas of the nucleotide analogues and the pH change (ApH) in a biochemical reaction can also be calculated using other software or programs available in the art.

The design and synthesis of various nucleotide analogues according to Formula I and II are available in the prior art. For novel nucleotide analogues, they may be synthesized using standard techniques of organic chemical synthesis known to those of skill in the art. By way of example, nucleotide analogues of Formula I wherein R7 is a difluoromethylene group can be synthesized by the methods of Burton et al. or Arabshahi et al. (Burton DJ et al., J Fluorine Chem 20: 611-626 (1982), Arabshahi L et al., Biochemistry 29: 6820-6826 (1990)). Briefly, the synthesis of difluoromethylene phosphates of guanine nucleosides can be achieved by reaction of the corresponding 5'-phosphates, activated by 1, 1 -carbonyldiimidazole, with difluoromethanediphosphonate.

Nucleoside Scz-(R-P-Borano)-triphosphates can be synthesized via phosphoramidite or phosphite approaches (reviewed in: Li P et al., Chem Rev 107: 4746-4796 (2007)).

The synthesis of IDP-dAMP and related compounds has been described by Song et al.. Briefly, dAMP is first coupled to an appropriate amino acid diester, with dicyclohexylcarbodUmide (DCC) as the coupling reagent, followed by deprotection with 0.4m sodium hydroxide in a methanol-water solution (Song XP et al., Chem Bio Chem 12:1 -14 (2011)). Synthesis of guanosine 5'-triphosphate derivatives with various terminal phosphates modifications was described by Eckstein F et al., Biochemistry 14: 5225-5232 (1975)).

Modifications to the 3-OH of the sugar group and the nucleobase of a nucleotide In some embodiments, the modifications include conjugating a reporter group to the 3'-OH of the sugar group and/or nucleobase of the nucleotide. As used herein, the reporter group that are conjugated to the 3-OH of the sugar is termed Reporter B, and the reporter group that are conjugated to the nucleobase is termed Reporter C. The reporter group can be conjugated to the 3' -OH group and/or nucleobase, directly or via a linker. The reporter group can be cleaved from the 3' -OH group and/or nucleobase by an enzymatic cleavable reaction, a photo-cleavable reaction, a chemical cleavable reaction or a thermal cleavable reaction; and the released reporter group causes an ion change in the reaction medium.

Enzymatic cleavable reactions will include, but are not limited to, polymerases that possess 3'->5' exnuclease activity such as T5 polymerase, Klenow polymerase or T7 polymerase. Although enzymatic cleavable reactions are common, the preferred reaction is using photocleavable compounds and linkers as no additional chemical reagents are required in the reaction that potentially could affect ion change.

While nucleotides carrying reporters B or C do not reduce buffering capacity, they are useful for improving ion yield and controlling the time and rate of ion release.

The choice of the reporter B or C to be conjugated will depend on the type of cleavable reaction as well as ion yield and, in the case of protons, buffering capacity of the reporter group. In addition, the reporter group can be a nucleotide reversible terminator that prevents further nucleotide polymerization until it is removed. For example, the allyl group conjugated to the nucleotide can be efficiently removed by Pd-catalyzed deallylation. In another example, a 2-nitrobenzyl moiety can be efficiently removed by laser irradiation at 355nm. The design and synthesis of both 3'-O-allyl-dNTP and 3'-O-(2-nitrobenzyl)-dNTP has been previously described (Ju, at a!., Pro Nat! Acad Sal USA., 103:52 (2006); Metzker, at at, Biotechniques, 25-5 (1998)). These nucleotide analogues are tolerated by polymerases such as 9°N polymerase and have been successfully used in pyrosequencing (Wu, at at, Pro Nat/Aced Sc! USA., 104:42 (2007)). Rather than converting the released PPi into a light signal, the present invention describes the use of a reporter group that when cleaved generate ion change that can be sensed by ion sensing apparatus in a sequencing reaction.

Examples of suitable reporter B or C groups can be found among the chemically or photo-cleavable blocking groups developed for kinetic studies (caged' substrates), as well as flourophores that are used in sequencing by synthesis. For example, the photo-FavorskU rearrangement generates an acidic product that lends itself to proton detection. Unlike enzymatic catalysis, the reaction is not inhibited by acidification-in fact the protonated species show higher quantum yield (Stensrud, et a!., Photochem Photobiol Sal., 7:6 14 (2008)). A wide variety of FavoiskU reagents has been synthesized with peak excitation peaks ranging from 271 -39mm and pKas from 3.9 - 8.2 (Stensrud, at at, Photochem Photobiol Sc!., 7:614 (2008); G!vens, et at, Can J C/Rem 89:364 (2011)). This avows proHng of the sequence composiUon by lahefling different bases with reporters that can be selectively released at different wavelength.

Possible appflcations are multiplexing and assays for rnethylation content which are at present not possible with ion-based sensing. The Favorskii rearrangement uses p-hydroxyphenacyl esters which are structurally similar to the nitrobenzoyloxymethyl group which has been employed as a reversible terminator in sequencing-by-synthesis.

Uridine triphosphates modified with this group have been shown to substitute for dTTP in PCR with Vent(exo-) polymerase without compromising spedlicity, yield or size (Litosh, eta!.. Nyc/Acids Res 39:e39 (2011)).

Applications The nucleotide analogues of the present invention are analogous to natural nucleotides meaning that, in a particular application, the nucleotide analogues function in a manner similar to or analogous to naturally occurring nucleotides. Therefore, the nucleotide analogues described herein may be added to a nucleic acid synthesis reaction mixture where natural nucleotides are normally used as substrates and protons are used as reaction indicators. The preferred nucleic acid synthesis reactions where the said nucleotide analogues may advantageously be used include, but are not limited to, nucleic acid amplification reactions, nucleic acid reverse transcription reactions, nucleic acid sequencing reactions, and SNF genotyping. For these reactions, the preferred method of detecting the proton signals is using an ISFET.

The compositions of the reaction mixture contain low buffering capacity such that a proton sensitive apparatus may be used to monitor nucleic acid polymerization in the reaction. Strong buffers such as Tris and Hepes are avoided or minimized. The pH of the reaction mixture at the start of the reaction is preferably between 5.0 and 9.0, preferably within 0.5 units of the optimum pH of the enzyme(s) used. A strong base (e.g., NaOH) or strong acid (e.g. HCI) may be added to the initial reaction mixture as required to achieve the above pH setting at the start of the reaction.

In addition, the reaction mixture preferably comprises one or more cofactors necessary for synthesis of a nucleic acid molecule. Cofactor salts such as those of potassium (preferably potassium chloride), ammonium (preferably ammonium chloride) and magnesium (preferably magnesium chloride and magnesium sulphate) are included in the compositions. The preferred reagents and concentration for optimal reaction conditions will depend on the type of nucleic acid synthesis reaction and will be readily apparent to one of ordinary skill in the art. The compositions and methods of detecting pH changes using ion sensitive apparatus for DNA sequencing, SNP genotyping, PCR and Loop mediated isothermal amplification are described in the art and incorporated herein by reference (see, e.g., Patent Application Nos. US811459; US7686929; US7888015; EP2129792; W006/005967; US2O1O/0255595; and GB1112140.7; Rothberg UM et. al., Nature 475:348-52 (2011)).

In a preferred embodiment, the nucleic acid synthesis reaction is a nucleic acid polymerisation reaction wherein the reaction mixture comprises of a nucleic acid template, one or more primers, one or more nucleic acid polymerase enzymes and one or more nucleotide analogues according to formula I or II. Nucleotide analogues are hydrolysed by nucleic acid polymerase enzymes, and protons are released during the incorporation of nucleotides into the growing nucleic acid chain. The nucleotide analogues described herein are aimed to have an impact on ion concentrations and in particular the pH of a fluid after or during a nucleic acid synthesis reaction! wherein the hydrolysis of the nucleotide analogue produces a greater change in the ion concentration than would occur with a natural nucleotide. The change in proton signal is measured using an ion sensitive apparatus.

The preferred nucleic acid amplification reactions include, but are not limited to, polymerase chain reactions (PCR), ligase chain reaction and isothermal amplification reactions.

The preferred Isothermal amplification reactions include, but are not limited to, Loop mediated isothermal Amplification (LAMP), Strand Displacement Amplifications (SDA), Helicase Dependent Amplification (HDA) and Rolling Circle Amplification (RCA).

In another embodiment, the present method may further comprise multiple nucleotide hydrolysing enzymes in the reaction mixture. The preferred nucleotide hydrolysing enzymes include, but are not limited to, DNA polymerase, RNA polymerase, reverse transcriptase and helicases. The nucleotide hydrolysing enzymes can be combined in a nucleic acid synthesis reaction and the corresponding said nucleotide analogues are added to the reaction. The advantage of combinatorial hydrolysis is to further enhance proton signals generated as the reaction proceeds, so as to further enhance the detection of the reaction and the accuracy of quantification. For example, reverse-transcription-PCR combines one or more reverse transcriptases with one or more Taq polymerases, and a plurality of said deoxynucleotide analogues are added to this mixture. Similarly, for reverse-transcription-LAMP reaction, the Taq polymerase in the reaction mixture is substituted with Bst DNA polymerase.

Another example of combinatorial hydrolysis reaction is Helicase Dependent Amplification, which combines one or more helicases with one or more isothermal DNA polymerases or derivatives thereof. Helicases for use in embodiments of the method are enzymes capable of unwinding double-stranded DNA under normal (20°C -37°C) or elevated temperatures (above about 60°C). The latter are preferable because they do not require a single strand binding protein, which is required by a thermolabile helicase. Helicases are abundant in nature and while the number of examples that could be provided is extensive, Tte-UVrD helicase is representative and provides a person of ordinary skill in the art how to perform the invention. The preferred DNA polymerases for HDA are those which are thermostable, lack 5' to 3' exonuclease activity and possess strand displacement activity. Examples include Klenow fragment of E. coli DNA polymerase I, T7 DNA polymerase (Sequenase, USB Corporation, Cleveland, Ohio), Vent® DNA polymerase, Vent® (exo') DNA polymerase (New England Biolabs, Inc., lpswich, MA), Bst DNA polymerase large fragment, and Phusion DNA polymerase (Finnzymes, Espoo, Finland). A pH-changing HDA (pH-HDA) reaction is set up like other HDA reactions known in the art (patents WO 2007/120808, WO 2006/074334), with the exception that any strongly buffering substances, in particular Tris buffer and ammonium salts, are omitted. Instead the pH is adjusted by addition of HCI or NaOH as described above and in the Patent Application No. GB1112140.7.

The natural substrate of helicases is ATP, thus for pH-HDA, a mixture of said deoxynucleotide analogues (A, C, G and T) and said ATP analogues are added to the HDA reaction mixture.

In some embodiments, the nucleotide analogues also has utility in procedures other than nucleic acid synthesis reactions. Various biochemical reactions utilize nucleotide or ribonucleotide triphosphates as substrates. Under suitable conditions, the hydrolysis of these nucleotides can lead to a net change in the amount of hydrogen ions which can then be detected by the ion sensing apparatus. The following are selective examples of such biochemical reactions: a) In vitro nucleic acid polymerization, including primer extension and DNA amplification, such as sequencing, polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP).

b) Ligation of two nucleic acid fragments (i.e. ligation chain reaction).

c) AMP transfer reactions (i.e., synthesis of desamido-NAD+, and FAD, ATP sulfurylase reaction).

d) Phosphorylations of nucleic acid or protein catalysed by kinases.

e) Separation of two annealed nucleic acid strands by helicases.

Detection system A preferred system using the present method comprises an ion sensitive apparatus, microfluidic devices, a reference electrode, a nucleic acid template, primers, polymerases and nucleotide analogues according to Formula I or II.

In the preferred embodiment shown in Figure 2, the ion sensitive apparatus(s) 3 may be one or more ISFET on a CMOS microchip 7, having thereupon microfluidic chambers 8 defined by manifold 2. The nucleic acid synthesis reaction mixture containing nucleic acid template, one or more nucleotide hydrolysing enzymes and one or more nucleotide analogues is added to the chamber(s) exposed to the ISFET(s).

Each ISFET outputs an electrical signal which is monitored by a signal processor. The passivation layer of the ISFET can be functionalized to be sensitive to protons, and the preferred passivation layer is silicon nitride. As the nucleotides are hydrolysed and incorporated into the growing nucleic acid chain, protons will be released and be detected by the signal processor as a change in the electrical output of the ISFET. The change in electrical signal of the ISFET is indicative of nucleotide incorporation during nucleic acid synthesis.

Preferably, each ISFET generates a normalised output signal from the difference between the ISFET signal and a reference signal. Preferably the reference signal is derived from an ISFET or FET located on the chip but not exposed to a fluctuating pH.

Thus any common drift or noise on the chip will be cancelled by taking the difference between these signals.

Figure 1 shows an ISFET with a floating gate and sensing layer made of Silicon Nitride, which is exposed to the reaction mixture. ISFETs are further described in patent US20041 34798 (Al), incorporated herein by reference.

The microfluidic devices may be a well, chamber, or channel to receive the sample proximate the ion sensitive sensor and may be comprise means for delivering the sample to the sensor. The microfluidic devices may also be isolated from each other to help reduce cross-diffusion of protons.

The methods of detecting pH changes with ion sensitive apparatus for DNA sequencing, SNP genotyping, PCR and Loop mediated isothermal amplification are described in the art and incorporated herein by reference (see, e.g., Patent Application Nos. US311459; US7686929; US7888015; EP2129792; W006/005967; US201010255595; and GB1 112140.7; Rothberg JM et. al., Nature 475:348-52 (2011)).

In an alternative embodiment, the ion sensitive apparatus to detect protons released during nucleic acid synthesis is a pH indicator. For example, the pH indicator may be a colorimetric or fluorescent dye, which changes optical properties, such as emitted wavelength from the dye, as the pH of the contacting reaction mixture changes.

In another embodiment, the ion sensitive apparatus is a conductive, a capacitative or an inductive sensor known in the art and incorporated herein by reference (Saremi-Yarahmadi S et al., 2011 IEEE International Conference on Dielectric Liquids, Richter A et al., Sensors 2008, 8: 561-581 and Korostynska 0 et al., Sensors 2007, 7: 3027-3042).

Although the invention has been described in terms of preferred embodiments as set forth above, it should be understood that these embodiments are illustrative only.

Those skilled in the art will be able to make modifications and alternatives in view of the disclosure which are contemplated as falling within the scope of the appended claims.

Each feature disclosed or illustrated in the present specification may be incorporated in the invention, whether alone or in any appropriate combination with any other feature disclosed or illustrated herein.

Theoretical calculations of pH changes in a PCR model using exemplary nucleotide analogues The following are example calculations of how the extent and direction of the pH change can be influenced by the use of triphosphate-modified nucleotide analogues in a PCR reaction. For comparison, all were calculated with dAMP as the polymerized nucleotide.

The simple PCR model was generated assuming a starting amount of 25pgIul human DNA, containing a single copy of target/haploid genome, and generating approximately 6 ng!ul PCR products (2lObp long) in 35 cycles. Initial amplification efficiencies were assumed to be 0.5 for copies generated from genomic DNA, and 1.0 for copies generated from PCR amplicons. Both amplification efficiencies were assumed to decrease in a linear fashion towards 0 at cycle 35. The results were sigmoidal curves describing the decrease in the concentrations of reaction substrates (primers, dNTPs) and corresponding increase in the concentrations of reaction products (pyrophosphate, polymerised dNMPs) with cycle number. While the shapes of these curves mimic experimentally observed kinetics, no attempt was made to validate their quantitative accuracy. It should be noted that a PCR reaction can be set up in many different ways, resulting in slower or faster kinetics, and different concentrations at beginning and end.

The examples are therefore for illustration, but within the range of common experimental results. Similar (Rutledge RG and Stewart D, BMC Molecular Biology 2008, 9:96 doi:10.1186/1471-2199-9-96) as well as more sophisticated models (i.e., Booth CS et al.,Chem Eng Sci 2010, 65: 4996 -5006) have been described in detail and could be used instead without affecting the conclusions of this work.

Calculation of pH changes was done with the program CurtiPot (Gutz, I. G. R., CurliPot -pH and Acid-Base Titration Curves: Analysis and Simulation software, version 3.6.1, http://wvvw2.iq.usp.br/docente/gutz/Curtipot html). Calculated pKa values of reaction substrates and reaction products were added into the program's database, as well as their maximum negative charges, defined as the number of -OH groups (including 3' OH). Next, starting concentrations of the reaction mix were entered consisting of 50 -500uM dNTP or dNTP analogue, luM primers (2Obp long), 25pg/ul template DNA, 50mM KCI, 3.5 mM MgCI2. For simplicity, all nucleotides and DNA molecules were assumed to have only one nucleobase, adenine, and be single stranded. Starting pH was adjusted by adding HCI or hydroxide ions to the mix until a pH of 7.500 was reached, then adding Na+ ions to balance the charge, recalculating the pH and repeating this procedure until the pH remained unchanged upon recalculation. New concentrations, reached after a certain number of amplification cycles were obtained from the PCR model and entered. Care was taken to choose the protonation states of substrates and products such that the net charge remains very close too (< 10"-lB in all examples). The pH of the new mixture was then calculated.

Actual changes in pH will depend on (1) the pKa of compounds, (2) concentrations of acids and bases, (3) concentration of salts, and (4) temperature. Table 1 illustrate the theoretical calculations of pH changes in a model PCR using dATP and exemplary dATP analogues. The value represents the amount of change in the initial and final pH of the nucleotide hydrolysis reaction. Negative value indicates a decrease in pH as the reaction proceeds whereas positive value indicates an increase in pH. Ri, R2, R3, R4 and Ri referto formula I. [refers to formula II.

Table 1: Theoretical calculations of pH changes in a model PCR using dATP and exemplary dATP analogues Hydrolysis product _____________________ PPi Pi Natural dATP -0.44 -0.56 Modification: PPi Pi R3or R3or non-bridging-OH Ri R2 R4 Ri R2 R4 amino -0.45 -2.54 -2.61 -0.57 -2.53 -2.60 dimethyl amino n.t. n.t. -2.09 nt n.t. -1.93 ethylenediamino n.t. n.t. -1.92 rLt. n.t. -1.79 methyl thio -0.44 -1.51 -1.54 -0.56 -1.70 -1.73 fluoro -0.44 -1.45 -1.48 -0.56 -2.07 -2.11 methoxy -0.44 -1.44 -1.45 -0.56 -1.82 -1.84 y methyl PP1 n.t. n.t. -1.58 itt. n.t. -1.96 borano -0.44 0.44 -1.36 -0.56 0.35 -1.59 thio -0.44 -0.42 -0.54 -0.56 -0.71 -1.01 Bridging 0 R7 carbonyl -1.75 difluoromethylene -0.63 amino -0.49 methylene -0.11 PPi leaving group [ IDP 1.35 Underlined text above indicates modifications that do not result in an improved pH change compared to natural dATP.

As additional information, graphical representation of pH change and proton yield of DFMB-dAMP and IDP-dAMP, two published compounds, are illustrated in a modelled PCR reaction and compared with the natural dATP (Figure 3 and Figure 4). .Eroton yield (average number of protons per hydrolysed nucleotide) was calculated as the net difference of the protonation states of dNTP and its reaction products. The average negative charge of each species was calculated in 0.5 pH unit intervals over a pH range from 2 -12. The average number of protons released at a given pH is the sum of the average charges of the reaction products minus the average charge of the substrate dNTP or dNTP analogue.

In summary, calculations for DEMB-dAMP display greater drops in pH than with the natural nucleotide dATP (Figure 3). The IDP-dAMP analogue increases the pH change; however, the pH rises as the reaction proceeds (Figure 4).

Example 1: DMFB-dAMP versus dATP Deoxyadenosine 5 (, y-difluoromethylene) triphosphate (DMFB-dAMP) is an analogue where the oxygen atom bridging the 3-y phosphates is substituted with a fluoromethylene group. The f3-y modification has been shown to accommodate a range of halomethylene groups having diverse steric and electronic properties. Replacing the f3-y-bridging triphosphate oxygen with (halo)methylene groups is a tolerated modification in terms of substrate binding in the DNA polymerase 13 active site (Sucato CA and 2007). For the purpose of this example, the predicted protonation ability of DMFB-dAMP was compared to that of dATP. As shown in Figure 3A, the predicted proton yield for the incorporation of DFMB-dAMF is greater than the corresponding reaction with dATP over a pH range of 6-8. The calculation suggests that generation of DFMB from DMFB-dAMP may lead to a greater change in pH in the PCR reaction compared to the production of pyrophosphate from dATP. As shown in figure 3B, a 35-cycle PCR reaction with starting concentration of 500uM of either DMFB-dAMP or dATP shows that the predicted pH change is greater with DMFB-dAMP, even if the natural nucleotide is further and completely hydrolysed to inorganic phosphate (Pi).

This is due to increased proton yield as well as the lower buffering effect of DFMB-dAMP versus dATP. The buffering effect was further explored by calculating the ApH (pHendPHinitialfor 35 cycles) in the PCR reaction mixture over a range of nucleotide concentrations. As shown in Figure 3C, DMFB-dAMP displays an even greater drop in pH at lower starting concentrations suggesting that DMFB-dAMP has lower buffering capacity than dATP. Even greater ion yield and lower buffering were found with y-fluoro ATP (not shown). Collectively, these data predict that substitutions, which increase proton yield and lower the pKa of the triphosphate group, can result in a greater pH change and thus improved signal detected by an ion sensing device.

Example 2: lOP-dAMP versus dATP During the polymerase-catalysed synthesis of DNA, the 3'-hydroxyl group of the primer nucleotide attacks the a-phosphate group of the incoming nucleoside triphosphate, and the resulting pyrophosphate acts as the leaving group. It has been shown that the pyrophosphate of triphosphate dATP can be replaced with an alternative leaving group, such as an iminodipropionic acid to form an iminodipropionic deoxyadenosine monophosphate (IDP-dAMP). IDP-dAMP was synthesized as an alternative substrate for polymerase, in order to investigate the role of the pyrophosphate moiety in the mechanism of the polymerization reaction itself. Interestingly, as shown in Figure 4A, the predicted proton yield for the incorporation of IDP-dAMP in a PCR reaction is negative, meaning that protons are consumed rather than generated as in the corresponding reaction with dATP. Calculating ApH (pHefld-pH of a PCR reaction with 500uM lOP-dAMP predicts that hydrolysis over 35 cycles of PCR reaction results in an increase in pH from pH 7.5 to over 7.9 (Figure 4B and 4C). Collectively, these data predict that the hydrolysis of a phosphoamidate-dAMP analogue will result in a pH change of opposite direction, but with greater absolute value than dATP, ultimately generating an improved signal by an ion sensing devise.

Note, it is understood that these examples and embodiments are for illustrative purposes and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of its claims.

Claims (15)

1. Claims: 1. A method of detecting a change in a proton concentration in a nucleic acid synthesis reaction mixture comprising: a) providing a nucleotide analogue to a reaction mixture comprising a nucleic acid template and a nucleotide hydrolysing enzyme, such that the nucleotide hydrolysing enzyme hydrolyses the nucleotide analogue to produce a greater change in the proton concentration than would occur with a corresponding natural nucleotide; and b) detecting a change in proton concentration with an ion sensitive apparatus.
2. 2. The method of claim 1, wherein the nucleotide analogue is represented by the formula: 0 fo 0 0II I II II IIL RL nwherein X is a nucleobase or nucleobase analogue; S is an acyclic or carbocyclic sugar moiety; n is an integer between 0 and 2; at least one of Ri, R2, R3, R4 and RL is a moiety without a pKa or with a pKa outside a range of 6.0-8.5, more preferably 6.0-9.5; PS, R6 and Ri may be the same or different and are independently selected from the group consisting of-O, difluoromethylene and carbonyl, with the proviso that at least one of R6 or Si is -C; and with the proviso that if R5, R6, and R7 are -0, then at least one of Ri, R2, R3, P4 and RL is not-OH.
3. 3. The method of claim 2, wherein R5, R6 and R7 are -0; Ri, R2, R3 and RL are -OH; and R4 is a moiety without a pKa or with a pKa outside the range of 6.0- 8.5, more preferably 6.0-9.5, and wherein P4 is not -OH.
4. 4. The method of claim 3, wherein R4 is selected from the group consisting of -SCH3, -SCH3 derivatives, -OCH3, -OCH3 derivatives, -Cl, -F, -BH3 and -NH2, and NH2 derivatives.
5. 5. The method of claim 2, wherein Ri, R2, R3, R4 and RL may be the same or different and are independently selected from the group consisting of-OH, -SCH3, -SCH3 derivatives, -OCH3, -OCH3 derivatives, -Cl, -F, -BH3 and -NH2, and NH2 derivatives.
6. 6. The method of claim 2, wherein R5 and R6 are -0; Ri, R2, R3, R4 and RL are -OH; and R7 is difluoromethylene.
7. 7. The method of claim 1, wherein the nucleotide analogue is represented by the formula:IIL-P-0--xOHwherein X is a nucleobase or nucleobase analogue; S is an acyclic or carbocyclic sugar moiety; and L is a secondary amine.
8. 8. The method of any preceding claim, wherein the ion sensitive apparatus is anIon Sensitive Field Effect Transistor (ISFET).
9. 9. The method of claim 1-7, wherein the ion sensitive apparatus is a conductive, a capacitative or an inductive ion sensitive device.
10. 10. The method of any preceding claim, wherein the nucleic acid synthesis reaction mixture is a reaction mixture for primer extension reaction, nucleic acid sequencing reaction, nucleic acid amplification, reverse transcription reaction, or combination thereof.
11. ii. The method in any preceding claim, wherein one or more nucleotide hydrolysing enzymes used in the reaction mixture is selected from a group consisting of a DNA polymerase, a RNA polymerase, a reverse transcriptase, a ligase and a helicase, and mutants and variants thereof.
12. 12. A method of increasing the quantity of an ion signal in a nucleic acid synthesis reaction mixture comprising: a) mixing a nucleic acid template with a polymerase enzyme and a primer to form a complex of the template, the primer and the polymerase enzyme; b) exposing the complex to one or more nucleotides wherein at least one non-bridging oxygen group of at least one terminal phosphorous group of the nucleotide and its reaction byproduct is substituted with a moiety without a pKa or with a pKa outside a range of 6-8.5; and c) detecting the ion signal with an ion sensitive apparatus.
13. 13. The method of claim 12, wherein the ion sensitive apparatus is an Ion SensitiveField Effect Transistor (ISFET).
14. 14. The method of claim 12-13, wherein the ion sensitive apparatus is a conductive! a capacitative or an inductive ion sensitive device.
15. 15. The method of claim 12-14, wherein the nucleic acid synthesis reaction mixture is a reaction mixture for primer extension reaction, nucleic acid sequencing reaction, nucleic acid amplification, reverse transcription reaction, or combination thereof.