Method for the quantification of nucleic acids, in particular bisulfite treated DNA.

The following invention relates to a method for the quantification of the amount (measurement of the total amount) of nucleic acids within a sample, said method is particularly suited to the quantification of bisulfite treated DNA. Further, the application relates to a method for the evaluation of the nucleic acid bisulfite treatment of a nucleic acid sample, as well as nucleic acid and kits for the quantification of nucleic acids.

The accuracy and precision of estimates of nucleic acid concentration are critical factors for efficient use of nucleic acid samples in high-throughput sequence analyses. Inaccuracy in quantification of nucleic acid can result in the unnecessary consumption of nucleic acids. Conservation of the original nucleic acid samples is important to validate previous findings and to allow for future studies. This is a particular problem with the bisulfite treatment procedure (commonly used for the analysis of DNA methylation) as DNA loss due to nonspecific degradation and purification procedures is a common problem. Under conditions for maximum bisulfite conversion it has been estimated that 84 - 96% of total DNA is degraded (Grunau, Clark & Rosenthal Nucleic Acids Research, 2001, Vol. 29, No. 13 e65). Accordingly, a bisulfite treated DNA sample will in fact consist of a mixture of converted fragments, unconverted fragments and DNA fragments which have been degraded such that they are no longer amplifiable. In this study comparison quantification of pre- and post- bisulfite treatment of DNA was measured using HPLC and competitive qPCR. Accordingly it is necessary to have accurate means for the quantification of total DNA within a sample, in particular for use with bisulfite-treated DNA.

In the state of the art a variety of methods for the quantification of nucleic acid are known. Traditionally nucleic acid concentration has been determined by UV absorbance spectroscopy (e.g. measuring the absorbance of the sample at 260/280nm). Disadvantages of this method include its poor sensitivity (nucleic acid contaminants such as proteins, RNA, and salts can increase the spectrophotometric estimation of nucleic acid concentration), and the inability to distinguish between single-stranded and double-stranded nucleic acids. As bisulfite-treated DNA is single stranded, UV spectroscopy is not a preferred means of quantitation. Furthermore, large amounts of nucleic acid are necessary for spectrophotometric analysis in current instrumentation for high-throughput environments.

Quantification of nucleic acids (in particular single stranded DNA) using intercalating fluorochromes and oligonucleotide hybridization methods have decreased nucleic acid consumption due to the increased sensitivity of these methods and has increased laboratory efficiency due to the high-throughput format of fluorometers. The use of oligonucleotide hybridization assays are popular, in particular PCR based assays such as Real-Time PCR assays. The amount of DNA is quantified by cross-reference of the CT (Threshold cycle) to a standard curve.

The most popular means of quantification of bisulfite treated DNA by means of sequence specific assay is carried out by means of amplification of a genomic locus that does not comprise any CpG positions (i.e. a genomic locus whose sequence can be accurately predicted after bisulfite treatment). The most commonly used of such assays is the MYODl gene assay initially described by Eads et al. (Nucleic Acids Research, 2000, Vol. 28, No. 8 E32-00). This document describes the use of an assay that amplifies a fragment of the MYODl gene that does not comprise any CpG position (but does comprise cytosine positions) thus ensuring that amplification of the target sequence is independent of the methylation status of genome. This is of particular importance as phenotype and disease conditions often result in changes in genomic methylation status, e.g. in cancer a general hypomethylation is observed, accompanied by increased methylation at specific loci. The amplification is carried out by real-time PCR, and the amount of input DNA is determined by comparing the CT of the amplification to a standard curve (i.e. a curve in which the CT of DNA of known amounts is plotted). However a heretofore unrecognized major disadvantage of this invention is that if the bisulfite reaction does not go to completion then the assay will only measure the amount of DNA that has been bisulfite converted, and not the total amount of DNA in the sample. Furthermore, as the amount of DNA is quantified by reference to a standard curve the same problem applies to the standard. The standard curve is determined by plotting the CT values of the assay of known quantities of a standard DNA. It is assumed that the standard DNA of known quantity is fully converted by bisulfite treatment. As it is not possible to make or buy guaranteed 100% bisulfite converted DNA there is no method of verification that the standard samples used to develop said standard curve are accurate. Furthermore, it is known that the bisulfite treatment procedure does not always go forward to completion (see Grunau, Clark & Rosenthal Nucleic Acids Research, 2001, Vol. 29, No. 13 e65). Accordingly the use of bisulfite treated DNA as a standard can lead to inaccuracies in creating a standard curve. This disadvantage has heretofore not been recognized.

In order to confirm the bisulfite conversion process Eads et al. describe the use of an assay specific for a non bisulfite converted region of the gene ACTB. This assay is specific to genomic (i.e. non-bisulfite converted DNA) and thereby provides an estimate of the sufficiency of the bisulfite treatment procedure. The bisulfite treated DNA is analyzed using a real-time assay specific for said gene, and if no signal is detected the reaction is assumed to have proceeded to completion. It is a further disadvantage of the method described by Eads et al. that two assays must be performed, a first assay to quantify the DNA and a second assay to ensure that bisulfite treatment was successful. The use of the quantification assay by itself, without verification of the success of the bisulfite treatment procedure is unreliable. However the major disadvantage of the assay described by Eads et al. is that the amount of DNA is quantified by reference to a standard curve that is derived from bisulfite treated DNA.

Taking into consideration the above mentioned state of the art there is a need for improved methods for the quantification of bisulfite treated nucleic acids. As it is not possible to accurately quantify a bisulfite treated DNA sample using bisulfite treated DNA as a standard there exists a need in the art for a method of quantification of bisulfite treated DNA by reference to a genomic standard. Such a method would have further utility for the assessment of bisulfite treatment procedures as it would allow the comparison of pre- and post- bisulfite treatment quantities of a DNA sample by means of a single assay that is calibrated against one standard thus allowing for the accurate comparison of the quantified values. The problem according to the invention can thus be stated as 'How to quantify bisulfite treated DNA with an improved accuracy'.

The invention solves this problem by providing an assay for the quantification of a nucleic acid sample which may be calibrated by reference to genomic (bisulfite untreated) DNA.

The assay achieves this by amplification of a target sequence of a nucleic acid by means of primer oligonucleotides, said target sequence characterized in that they do not comprise any cytosine bases. Due to the lack of cytosine bases within the target sequence, amplification of the target sequence genomic nucleic acid and bisulfite treated nucleic acid are amplified with equivalent efficiency (i.e. there is no bias in amplification). Furthermore, unlike the assay described by Eads et al. the accuracy of the quantification is not dependant on the complete bisulfite conversion of the DNA.

The assay may accurately be quantified by reference to a DNA standard that has been developed based on untreated DNA, irrespective of whether the DNA is bisulfite treated or not.

Therefore, the invention provides a single assay that is suitable for improved quantification of both genomic and bisulfite treated nucleic acids.

The advantage of increasing the accuracy of a bisulfite treated DNA quantification assay by reference of the assay to a genomic standard has heretofore not been recognized.

This provides a means for the quantification of nucleic acid within a sample that is ideally suited for the comparison of quantified amounts of bisulfite and genomic nucleic acids. In one embodiment this may be used to compare the quantity of genomic DNA inputted into a bisulfite treatment process and to compare the quantity of bisulfite treated DNA that is outputted from the said process. Furthermore, when used in combination with a further realtime PCR assay wherein said assay oligos cover conversion specific positions the method of the invention may be used to provide a measure of the degree of bisulfite conversion of the sample.

The technical characteristics of the invention (amplification assay of a bisulfite treated DNA sequence not comprising any cytosine positions and DNA quantification by reference to a genomic standard) have not been disclosed in any document, the invention is therefore novel. Furthermore considering the state of the art the invention is not obvious, as there is no recognized advantage in using genomic DNA as a standard reference for bisulfite treated DNA quantification.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the most preferred assays according to the present invention for the quantification of nucleic acids (bisulfite treated or genomic) by means of enzymatic amplification.

Figure 2 shows an alternative preferred assay according to the present invention for the quantification of nucleic acids (bisulfite treated or genomic) by means of enzymatic amplification.

Figure 3 shows an alternative preferred assay according to the present invention for the quantification of nucleic acids (bisulfite treated or genomic) by means of enzymatic amplification.

Figure 4 shows an alternative preferred assay according to the present invention for the quantification of nucleic acids (bisulfite treated or genomic) by means of enzymatic amplification.

Figure 5 shows an alternative preferred assay according to the present invention for the quantification of nucleic acids (bisulfite treated or genomic) by means of enzymatic amplification.

Figure 6 shows an alternative preferred assay according to the present invention for the quantification of nucleic acids (bisulfite treated or genomic) by means of enzymatic amplification.

Figure 7 shows an alternative preferred assay according to the present invention for the quantification of nucleic acids (bisulfite treated or genomic) by means of enzymatic amplification.

Figure 8 Shows the quantitation of total DNA within 12 test samples according to Example 1 by means of UV spectroscopy and the assay of the invention (quantitated by reference to both genomic and bisulfite treated standards).

Figure 9 Shows the quantitation of total DNA within 12 test samples according to Example 1 by means of two bisulfite treated DNA specific real-time PCR assays (HB 14 and C3 respectively) and assay of the invention (quantitated by reference to a bisulfite treated standard).

Figure 10 Shows the quantitation of unconverted (by bisulfite treatment) DNA within 12 test samples according to Example 1 by means of an assay specific to bisulfite untreated DNA.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the term 'target sequence' shall be taken to mean a sequence to be amplified by means of primer oligonucleotides.

As used herein the term 'methylation specific sequence conversion' shall be taken to the conversion of all cytosines within a nucleic acid to a base pair with dissimilar hybridization with the exception of 5-methylcytosine which is not converted to any other base. Methylation specific sequence conversion may be achieved by any means known in the art including but not limited to bisulfite treatment or use of the AID enzyme (as described in DE 10331107).

As used herein the term 'standard curve' shall be taken to mean a plot of DNA quantification against a measured parameter of an assay. The standard curve may be prepared by determining the measured parameter of DNA standards of known amount, plotting the measured value against on a two-dimensional axis of measured value against amount of DNA. The curve may then be drawn by interpolating between the points or by drawing a line of best fit. It is preferred that said standard curve is linear. Wherein the assay of the invention is implemented by Real-time PCR it is particularly preferred that said measure value is a threshold curve (CT).

As used herein the term 'quantification' shall be taken to mean a measurement of a quantity, for example but not limited to measurement of the amount of DNA within a sample, which may be expressed e.g. as a concentration or absolute value.

The invention according to the present application provides a method for the quantification of a nucleic acid sample by amplification of a target sequence of said nucleic acid by means of primer oligonucleotides, wherein said target sequence is characterized in that it does not comprise any cytosine bases. The invention is further characterized in that the quantification of the amount of amplified target sequence is achieved by reference to a standard curve based on genomic nucleic acids.

It is particularly preferred that the source of the nucleic acid sample is selected from the group consisting of cell lines, histological slides, biopsies, tissue samples, tissue embedded in paraffin, bodily fluids, ejaculate, urine and blood. It is particularly preferred that said nucleic acids are DNA and it is further preferred that said DNA consists of the entire genome.

In one embodiment of the method said nucleic acids are treated such that all cytosines within said nucleic acids are converted to uracil with the exception of 5-methylcytosine which is not converted to any other base by said treatment. It is preferred that said treatment is carried out using a bisulfite reagent. The term "bisulfite reagent" refers to a reagent comprising bisulfite, disulfite, hydrogen sulfite or combinations thereof, useful to distinguish between methylated and unmethylated CpG dinucleotide sequences. Methods of said treatment are known in the art (e.g. PCT/EP2004/011715, which is incorporated by reference in its entirety). It is preferred that the bisulfite treatment is conducted in the presence of denaturing solvents such

as but not limited to n-alkylenglycol, particulary diethylene glycol dimethyl ether (DME), or in the presence of dioxane or dioxane derivatives. In a preferred embodiment the denaturing solvents are used in concentrations between 1% and 35% (v/v). It is also preferred that the bisulfite reaction is carried out in the presence of scavengers such as but not limited to chromane derivatives, e.g., 6-hydroxy-2,5,7,8,-tetramethylchromane 2-carboxylic acid (see: PCT/EP2004/011715 which is incorporated by reference in its entirety). The bisulfite conversion is preferably carried out at a reaction temperature between 30°C and 70°C, whereby the temperature is increased to over 85°C for short periods of times during the reaction (see: PCT/EP2004/011715 which is incorporated by reference in its entirety). The bisulfite treated DNA is preferably purified priori to the quantification. This may be conducted by any means known in the art, such as but not limited to ultrafiltration, preferably carried out by means of Microcon™ columns (manufactured by Millipore™). The purification is carried out according to a modified manufacturer's protocol (see: PCT/EP2004/011715 which is incorporated by reference in its entirety).It is a further essential component of the method according to the invention that the target sequence (i.e. the fragment to be amplified) does not comprise any cytosine bases.

Particularly preferred is a target sequence selected from SEQ ID NO: 16 to SEQ ID NO: 37. Particularly preferred are primers selected from SEQ ID NO: 1 to SEQ ID NO: 15, according to Figures 1 to 7. The amplification of the target sequence may be carried out by any means known in the art, however it is preferred that it is carried out by enzymatic means. It is further preferred that it is carried out by means of a polymerase chain reaction. The amplification is monitored such that the quantity of amplified nucleic acid may be determined. Accordingly in the most preferred embodiment the amplification is carried out by means of real-time PCR, and all variants thereof including but not limited to SYBR Green, TaqMan®, Molecular Beacons, Sunrise and Scorpion.

Dependant upon the detection method used it may be necessary to quantify the amount of nucleic acid in a sample by comparison to a known standard. Wherein the detection method is real-time PCR a standard curve is required to reference the CT (Threshold curve) of the assayed sample with that of known standards in order convert the detected signal to a quantified value. Suitable standards include commercially available genomic DNA including but not limited to that supplied by Promega. This may be methylation specific sequence converted e.g. bisulfite treated or genomic. However wherein the aim of the invention is only

the quantification of nucleic acids it is particularly preferred that said nucleic acids are genomic DNA that has not been bisulfite treated. It is particularly preferred that standards of known quantity are used to develop a calibration curve against which tested samples may be compared to ascertain quantities of DNA present in the sample.

When quantifying methylation specific sequence converted nucleic acids (e.g. bisulfite treated) by means of the assay as herein disclosed compared to genomic non-treated nucleic acids twice as much genomic DNA will be detected as methylation specific sequence converted treated DNA. This is because subsequent to methylation specific sequence conversion treatment the change in DNA sequence results in two non-hybridizing strands, the DNA is thus single stranded and only one strand serves as the template for amplification. This must be taken into account when quantifying the amount of DNA either by comparison to a calibration curve or by reference to the quantification value of the sample prior to treatment.

Suitable reagents for carrying out the amplification of nucleic acids are known to the person skilled in the art. A variety of enzymes for use in the enzymatic amplification of nucleic acids, including for use in PCR, are known in the art and the person skilled in the art may select a suitable enzyme with undue burden of experimentation. Wherein said amplification is carried out by means of polymerase chain reaction said reagents may comprise one or more selected from the group consisting of primer oligonucleotides; Nucleotides; Dimethyl sulphoxide (DMSO); Taq polymerase; buffer. It is also preferred that the concentration of said primer oligonucleotides is 0.2 - 1.0 uM. It is also preferred that the concentration of said nucleotides is 50 - 200 uM of each dNTP, in a further embodiment of the method said dNTPs may consist of the bases thymine, adenine and guanine only. It is also preferred that the concentration of DMSO is 0 - 10% (v/v). Wherein the amplification enzyme is Taq polymerase it is also preferred that 0.5 - 1.0 Units/50ul reaction solution are used. It is also preferred that said buffer comprises a minimum of 1.5mM Mg2+, it may further contain detergent, gelatin or BSA.

Suitable reaction conditions for carrying out the amplification of the nucleic acids are known to the person skilled in the art. Said conditions are dependant on multiple variables including but not limited to length of primer oligonucleotides and nucleotide content of primer oligonucleotides. All temperature cycling variants of the polymerase chain reaction are

suitable for use with the present invention including but not limited to hot-start PCR and two- temperature PCR protocol.

The design of primer oligonucleotides is known to one skilled in the art, and is dependant on factors including A/T content, dimmer formation, specificity of annealing and Tm of the reaction. Suitable software is available to assist in the design and e-PCR of primer oligonucleotides. However it is essential to the invention that said primers amplify a target sequence which do not contain any cytosines bases. It is particularly preferred that said primers are between 15 and 30 bases in length.

Subsequent to or in parallel with the PCR amplification of the target sequence the presence of the amplificate is detected and measured. This may be by any means standard in the art including mass spectrometric analysis (wherein preferably the primer are labeled with mass labels), UV absorbance spectroscopy and intercalating dyes. However in the most preferred embodiment of the method the presence of the amplificate is observed and quantified by means of real-time PCR. All real-time PCR systems rely upon the detection and quantitation of a fluorescent reporter, the signal of which increases in direct proportion to the amount of PCR product in a reaction. All variants of Real-time PCR are suitable for use in the present invention including but not limited to SYBR Green, Taqman, Molecular Beacons, Sunrise and Scorpion. It is particularly preferred that quantity of nucleic acid in the sample is determined by comparison of the threshold cycle (CT) to a standard curve of CT against quantity that has been prepared by means of real-time analysis of known standard quantities of nucleic acids (e.g. Promega genomic DNA).

In one embodiment of the method according to the invention is used for the quantification of input and output nucleic acid of a methylation specific sequence conversion treatment procedure. It is particularly preferred that this is bisulfite treatment. In this embodiment the method comprises the following steps: i) quantification of the amount of nucleic acid within a genomic nucleic acid sample according to the above-described assay of the invention, ii) treatment of said genomic nucleic acid sample, and ii) quantification of the amount of nucleic acid within said treated nucleic acid sample according to the above-described assay of the invention.

In the first step of the method the amount of nucleic acid within a genomic DNA sample of interest is quantified. It is particularly preferred that said genomic DNA sample consists of the entire genome of a biological sample. The genomic DNA sample may be isolated from any source including but not limited to cell lines, histological slides, biopsies, tissue samples, tissue embedded in paraffin, bodily fluids, ejaculate, urine and blood.

The nucleic acids within said sample are quantified by means of an assay which amplifies a target sequence by means of primer oligonucleotides, wherein said target sequence and primer oligonucleotides are characterized in that they do not comprise any cytosine bases.

Subsequent to or in parallel with the PCR amplification of the target sequence the presence of the amplificate is detected and observed. This may be carried out by any means known in the art, however, it is preferred that this is carried out by means of Real-time PCR. The amount of DNA within the sample may then be determined by reference of the CT to a standard curve.

In the second step of the method the nucleic acids are treated such that all cytosines within said nucleic acids are converted to bases with different hybridization properties (e.g. uracil or thymine) with the exception of 5-methylcytosine which is not converted to any other base by said treatment. It is particularly preferred that said treatment is carried out by means of a bisulfite reagent. The term "bisulfite reagent" refers to a reagent comprising bisulfite, disulfϊte, hydrogen sulfite or combinations thereof, useful as disclosed herein to distinguish between methylated and unmethylated CpG dinucleotide sequences. Methods of said treatment are known in the art (e.g. PCT/EP2004/011715, which is incorporated by reference in its entirety). It is preferred that the bisulfite treatment is conducted in the presence of denaturing solvents such as but not limited to n-alkylenglycol, particularly diethylene glycol dimethyl ether (DME), or in the presence of dioxane or dioxane derivatives. In a preferred embodiment the denaturing solvents are used in concentrations between 1% and 35% (v/v). It is also preferred that the bisulfite reaction is carried out in the presence of scavengers such as but not limited to chromane derivatives, e.g., 6-hydroxy-2,5,7,8,-tetramethylchromane 2- carboxylic acid (see: PCT/EP2004/011715 which is incorporated by reference in its entirety). The bisulfite conversion is preferably carried out at a reaction temperature between 30°C and 70 ⁰ C, whereby the temperature is increased to over 85°C for short periods of times during the reaction (see: PCT/EP2004/01 1715 which is incorporated by reference in its entirety). The bisulfite treated DNA is preferably purified prior to the next (third) step of the method. This

may be conducted by any means known in the art, such as but not limited to ultrafiltration, preferably carried out by means of Microcon™ columns (manufactured by Millipore™). The purification is carried out according to a modified manufacturer's protocol (see: PCTVEP2004/011715 which is incorporated by reference in its entirety). In a further embodiment of the method the DNA may be purified subsequent to the first or second step in order to remove the amplification products of step i). This may be done by any means standard in the art.

In the third step of the method the nucleic acids within the methylation specific sequence converted sample are quantified by means of an assay which amplifies a target sequence by means of primer oligonucleotides, wherein said target sequence and primer oligonucleotides are characterized in that they do not comprise any cytosine bases.

Subsequent to or in parallel with the PCR amplification of the target sequence the presence of the amplificate is detected and observed. This may be carried out by any means known in the art, however, it is preferred that this is carried out by means of Real-time PCR. The amount of DNA within the sample may then be determined by reference of the CT to a standard curve.

In a further embodiment of the invention the method of the invention as described above comprising the following steps: i) quantification of the amount of nucleic acid within a genomic nucleic acid sample according to the above-described assay of the invention, ii) treatment of said genomic nucleic acid sample, iii) quantification of the amount of nucleic acid within said treated nucleic acid sample according to the above-described assay of the invention, comprises an additional step: iv) determination of any difference in quantity of nucleic acid post- and pre- step ii).

As described above, when quantifying methylation specific sequence converted nucleic acids (e.g. bisulfite treated) by means of the assay as herein disclosed compared to genomic non-treated nucleic acids twice as much genomic DNA will be detected as methylation specific sequence converted nucleic acids (e.g. bisulfite treated) DNA. This must be taken into account when comparing the amount of DNA as detected in step iii) to that detected in step i). Accordingly wherein 0.5 times the amount of amplificate is

detected step iii) as compared to step i) this indicates that the amount of nucleic acid in both samples is equal, wherein 0.25 times the amount of amplificate is detected step iii) as compared to step i) it is an indicator that post- step ii) only 50% of the quantity of nucleic acid is present in the sample. This method has many uses including but not limited to determining the loss of amplifiable nucleic acids under different conversion conditions and determining the optimum ratio between conversion efficacy and yield of amplifiable nucleic acids under varying conversion conditions.

A further aspect of the invention provides a method for the evaluation of the completeness of a methylation specific sequence conversion treatment procedure (preferably bisulfite treatment) comprising the following steps: i) quantification of the amount of nucleic acid within said treated nucleic acid sample according to the above-described assay of the invention wherein said assay is quantified by reference to a genomic standard ii) quantification of the amount of nucleic acid within said treated nucleic acid sample by means of an assay specific to fully methylation specific sequence converted DNA iii) determination of any difference in quantity of nucleic acid between that quantified in i) and that quantified in ii).

In the first step of the method the amount of nucleic acid within a bisulfite converted DNA sample of interest are quantified. It is particularly preferred that said genomic DNA sample consists of the entire genome of a biological sample. The bisulfite converted DNA sample may have been previously isolated from any source including but not limited to cell lines, histological slides, biopsies, tissue samples, tissue embedded in paraffin, bodily fluids, ejaculate, urine and blood.

The nucleic acids within said sample are quantified by means of an assay which amplifies a target sequence by means of primer oligonucleotides, wherein said target sequence and primer oligonucleotides are characterized in that they do not comprise any cytosine bases.

Subsequent to or in parallel with the PCR amplification of the target sequence the presence of the amplificate is detected and observed. This may be carried out by any means known in the art, however, it is preferred that this is carried out by means of Real-time PCR. The amount of DNA within the sample may then be determined by reference of the CT to a standard curve.

Said standard curve may be based on genomic DNA or methylation specific sequence converted DNA. However the methylation specific sequence conversion technique must be the same between the tested samples and that used to plot the standard curve.

In the second step of the method the DNA sample is quantified by analysis with an assay specific for the detection of methylation specific sequence converted sequences only. Said assays comprise a pair of primer oligonucleotides and may further comprise one or more detection probes. Wherein the conversion is achieved by means of bisulfite treatment the target sequence to be amplified preferably does not comprise any CpG positions, however it comprises at least one (preferably multiple) cytosine position(s) which is converted to uracil by means of a bisulfite treatment procedure. Amplification and/or detection only takes places if all cytosine positions covered by said primers and/or probe are converted to uracil. Quantification of the methylation specific sequence converted sequences is determined by reference to a standard curve plotted on the basis of said methylation specific sequence conversion technique treated DNA standards. A variety of such assays are known in the art including that as previously described by Eads et al., and the C3 and HB 14 assays described below in Example 1.

In the final step of the method the difference between the two quantified by reference of the CT to a standard curve which has been prepared by assay of known quantities of bisulfite treated DNA.

In the third step of the method any difference in quantity of nucleic acid between that quantified in the first step of the method and that quantified in the second step of the method is determined. Wherein less nucleic acids are quantified in the second step than the first, this provides a measure of the quantity of DNA which has not been fully converted by means of the bisulfite treatment procedure.

This method has many uses including but not limited to

- determining the loss of amplifiable nucleic acids after differing length of exposure time to said conversion conditions and

- determining the loss of amplifiable nucleic acids at different conversion temperatures and

- assessing the effectiveness of a treatment that converts un-methylated cytosines, but not methylated cytosines into uracil.

A further advantage of the method of the application is when verifying the presence of residual unconverted DNA in a bisulfite treatment reaction. In the state of the art an assay specific to unconverted DNA is used (see for example, Example 1 CGI assay figure 10).

Such an assay only provides a signal if totally unconverted DNA is present. There is no means in the art for quantifying the presence of partially converted DNA. Accordingly a further subject matter of the present invention is a means for quantifying the presence of partially converted bisulfite treated DNA in a sample comprising the following steps: i) quantification of the amount of nucleic acid within said treated nucleic acid sample according to the above-described assay of the invention wherein said assay is quantified by reference to a genomic standard ii) quantification of the amount of nucleic acid within said treated nucleic acid sample by means of an assay specific to fully bisulfite converted DNA iii) quantification of the amount of nucleic acid within said treated nucleic acid sample by means of an assay specific to bisulfite unconverted DNA iv) determination of any difference in quantity of nucleic acid between that quantified in i) and the sum of that quantified in ii) and that quantified in iii).

In the first step of the method the amount of nucleic acids within a bisulfite converted DNA sample of interest are quantified. It is particularly preferred that said genomic DNA sample consists of the entire genome of a biological sample. The bisulfite converted DNA sample may have been previously isolated from any source including but not limited to cell lines, histological slides, biopsies, tissue samples, tissue embedded in paraffin, bodily fluids, ejaculate, urine and blood.

The nucleic acids within said sample are quantified by means of an assay which amplifies a target sequence by means of primer oligonucleotides, wherein said target sequence and primer oligonucleotides are characterized in that they do not comprise any cytosine bases.

Subsequent to or in parallel with the PCR amplification of the target sequence the presence of the amplificate is detected and observed. This may be carried out by any means known in the art, however, it is preferred that this is carried out by means of Real-time PCR. The amount of

DNA within the sample may then be determined by reference of the CT to a standard curve. Said standard curve may be based on genomic DNA or methylation specific sequence converted DNA. However the methylation specific sequence conversion technique must be the same between the tested samples and that used to plot the standard curve.

In the second step of the method the DNA sample is quantified by analysis with an assay specific for the detection of methylation specific sequence converted sequences only. Said assays comprise a pair of primer oligonucleotides and may further comprise one or more detection probes. Wherein the conversion is achieved by means of bisulfite treatment the target sequence to be amplified preferably does not comprise any CpG positions, however it comprises at least one (preferably multiple) cytosine position(s) which is converted to uracil by means of a bisulfite treatment procedure. Amplification and/or detection only takes places if all cytosine positions covered by said primers and/or probe are converted to uracil. Quantification of the methylation specific sequence converted sequences is determined by reference to a standard curve plotted on the basis of said methylation specific sequence conversion technique treated DNA standards. A variety of such assays are known in the art including that as previously described by Eads et al, and the C3 and HB 14 assays described below in Example 1.

In the third step of the method the DNA sample is quantified by analysis with an assay specific for the detection of bisulfite unconverted (i.e. genomic) sequences only. Said assays comprise a pair of primer oligonucleotides and may further comprise one or more detection probes. It is preferred that the target sequence to be amplified preferably does not comprise any CpG positions, however it comprises at least one (preferably multiple) cytosine position(s). Amplification and/or detection only takes places if none of the cytosine positions covered by said primers and/or probe are converted to uracil. Quantification of the nucleic acids is determined by reference to a standard curve. A variety of such assays are known in the art including that as previously described by Eads et al, and the CGI assay described below in Example 1.

In the final step of the method the amount of partially converted nucleic acids is determined. In one embodiment this is determined as the difference between the amount quantified in step i) and the sum of the two amounts quantified in ii) and iii). Alternatively this may be

calculated by individually subtracting each of the values calculated in steps ii) and iii) from the amount quantified in step i).

Comparison of the quantified amount of DNA in a sample pre- and post- treatment as described in said three methods (to measure DNA loss and to provide a measure of DNA conversion) enables the evaluation and comparison of alternative treatment methods. When applied in a high-throughput nucleic acid analysis system the method may be utilized to alert the system operator to any dysfunction in a DNA treatment step that results in unacceptable loss of DNA or unacceptably low conversion rate. Accordingly a further aspect of the invention is a component of an automated nucleic acid analysis system, wherein said component provides feedback to the system operator if acceptable levels of DNA loss are breached.

A further aspect of the invention provides a kit for use in the quantification of nucleic acids by means of the assay of the invention. Said kit comprises a pair of primer oligonucleotides which do not comprise any cytosine wherein said primer oligonucleotides are suitable for the amplification of a fragment of a genome that does not comprise any cytosine positions. It is particularly preferred that said genome is the homo sapiens genome.

It is further preferred that the sequence of said primer oligonucleotides are selected from the group consisting SEQ ID NO: 1 to SEQ ID NO: 15.

The kit may further comprise a standard curve for the reference of amplificate amount to DNA quantity (e.g. by means of real-time PCT threshold cycles). In an alternative embodiment the kit may further comprise further components useful for performing the method of the invention such as but not limited to one or more components selected from the group consisting primer oligonucleotides; Nucleotides; Dimethyl sulphoxide Nucleotides; Dimethyl sulphoxide (DMSO); Taq polymerase; buffer.

While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following EXAMPLES and FIGURES serve only to illustrate the invention and are not intended to limit the invention within the principles and scope of the broadest interpretations and equivalent configurations thereof.

Example 1

In the following analysis the amount of bisulfite treated DNA quantified using the assay of the present invention was compared to that quantified by UV spectroscopy. Assay results were quantified by comparison to two different standards first by reference to a bisulfite treated standard and second by reference to a genomic DNA standard. The amounts quantified using the assay of the present invention (quantified by reference to a bisulfite treated standard curve) were then compared to two alternative assays (C3 and HB 14) as described below, which are specific for bisulfite converted DNA. Residual genomic DNA in each sample was then quantified using an assay specific for non-bisulfite converted DNA (CGI assay).

Twelve bisulfite treated DNA samples were quantified by means of UV spectroscopy. Each sample was then diluted until all contained 5ng/μl DNA. 10 μl samples of the DNA containing solution were then assayed using three alternative quantification assays: the C3 assay, the HB 14 assay and the 'cytosine free fragment' assay.

C3 Assay

The C3 assay is a DNA quantification assay specific for bisulfite converted DNA. The assay amplifies a fragment of DNA_(see below) that comprises multiple cytosine (but not CpG) positions in the genomic form which are initially converted to uracil and during amplification replaced by thymine in the bisulfite converted variant. Accordingly the assay does not quantify for unconverted or partially converted bisulfite treated DNA (i.e. wherein the target sequence comprises one or more cytosine positions which have not been converted to thymine). The quantity of DNA in the sample is deduced by comparison of the measured CT (Threshold cycle) to a standard curve relating such CT values to DNA amounts. The standard curve is based on measurements of known quantities of bisulfite converted DNA with the according assay.

The oligomer components of the assay are as follows:

Forward Primer: GGAGTGGAGGAAAtTGAGAt (SEQ ID NO: 38)

Reverse Primer: CCACACAaCAaaTaCTCAaAaC (SEQ ID NO: 39)

Amplified Fragment:

GGAGTGGAGGAAAtTGAGAtttAtTGAGGTTACGTAGTTTGtttAAGGTtAAGttTG GGTG ttTGtAATttTTGtttTGTGttAGGtTGttTtttAGGTGTtAGGTGAGtTtTGAGtAttT GtTGTGTGG (SEQ ID NO: 40)

Genomic equivalent of amplified fragment:

GGAGTGGAGGAAACTGAGACCCACTGAGGTTACGTAGTTTGCCCAAGGTCAAGC CTGGGTGCCTGCAATCCTTGCCCTGTGCCAGGCTGCCTCCCAGGTGTCAGGTGAG CTCTGAGCACCTGCTGTGTGG (SEQ ID NO: 41)

Detection Probe: TGGGTGTTTGTAATTTTTGTTTTGTGTTAGGTT (Reporter FAM; Quencher EclipseDQ) (SEQ ID NO: 42)

Reaction Solution

Cycling conditions:

Ninety six cycles were repeated under the following conditions:

HB 14 Assay

The HB 14 assay is a DNA quantification assay specific for bisulfite converted DNA, similar to the C3 assay. The assay amplifies a fragment of DNA (see below) that comprises multiple cytosine (but not CpG) positions in the genomic form which are converted to uracil initially and during amplification replaced by thymine in the bisulfite converted variant. Accordingly the assay does not quantify unconverted or partially converted bisulfite treated DNA (i.e.

wherein the target sequence comprises one or more cytosine positions which have not been converted to thymine). The quantity of DNA in the sample is deduced by comparison of the CT (Threshold cycle) to a standard curve of known quantities of bisulfite converted DNA (as described above).

The oligomer components of the assay are as follows:

Forward Primer: TGGTGATGGAGGAGGTTTAGTAAGT (SEQ ID NO: 43)

Reverse Primer: AACCAATAAAACCTACTCCTCCCTTAA (SEQ ID NO: 44)

Amplified Fragment:

TGGTGATGGAGGAGGtTtAGtAAGTtTTtTGGAtTGTGAAttTGTGTtTGttAtTGT GTGtTG GGTGGTGGTtATtTTTtttAttAGGtTGTGGttTtTGtAAttTTtAAGGGAGGAGtAGGT tttATT GGtT (SEQ ID NO: 45)

Genomic equivalent of amplified fragment:

TGGTGATGGAGGAGGCTCAGCAAGTCTTCTGGACTGTGAACCTGTGTCTGCCACT GTGTGCTGGGTGGTGGTCATCTTTCCCACCAGGCTGTGGCCTCTGCAACCTTCAAG GGAGGAGCAGGTCCCATTGGCT (SEQ ID NO: 46)

Detection Probe: ACC ACC ACCC AAC AC AC AAT AAC AAAC AC A (Reporter FAM; Quencher EclipseDQ although other non-fluorescent quenchers would also be suitable) (SEQ ID NO: 47)

Reaction Solution

Cycling conditions:

Ninety six cycles were repeated under the following conditions:

Temp °C|Time |

Cytosine free fragmentCCFF or CFFl) assay

This is the assay that is one subject of the invention. The assay comprises the amplification of a DNA fragment that does not comprise any cytosine positions (i.e. said target sequence which is going to be amplified is present in the genomic, as well as in the converted nucleic acids). The assay is therefore not specific to genomic or bisulfite treated DNA, both amplify with equivalent efficiency. This enables the quantification of the assay results by reference to a genomic standard.

The oligomer components of the assay are as follows:

Forward Primer: TAAGAGTAATAATGGATGGATGATG (SEQ ID NO: 1)

Reverse Primer: CCTCCCATCTCCCTTCC (SEQ ID NO: 2)

Amplified Fragment (Genomic and bisulfite treated):

TAAGAGTAATAATGGATGGATGATGGATAGATGAATGGATGAAGAAAGAAAGGA TGAGTGAGAGAAAGGAAGGGAGATGGGAGG (SEQ ID NO: 48)

Detection Probe: ATGGATG A AG AAAGAAAGGATGAGT (Reporter FAM; Quencher EclipseDQ) (SEQ ID NO: 49)

Reaction Solution

Cycling conditions:

Ninety six cycles were repeated under the following conditions: lTemp °C|Time

CGI assay

The CGI assay is a DNA quantification assay specific for non-bisulfϊte converted DNA. The assay amplifies a fragment of DNA (see below) that comprises multiple cytosine positions in the genomic form which are converted to uracil initially and during amplification replaced by thymine in the bisulfite converted variant. The oligomer components of the assay comprise multiple guanines that detect the presence of unconverted cytosine within the target sequence. Accordingly the assay does not quantify converted or partially converted bisulfite treated DNA (i.e. wherein the target sequence comprises one or more cytosine positions which have been converted to uracil). The quantity of DNA in the sample is deduced by comparison of the CT (Threshold cycle) to a standard curve of known quantities of genomic (non-bisulfite converted) DNA, as described above.

Forward Primer: ACTGTTCCTGGCAAATAAGA (SEQ ID NO:50) Reverse Primer: CTGGTCCCTAAGAACTCACA (SEQ ID NO:51)

Amplificate products were detected by means of SYBR green intercalating dye.

Reaction Solution

Cycling conditions:

Thirty four cycles were repeated under the following conditions:

Cycling conditions:

Thirty four cycles were repeated under the following conditions:

Results

The quantified amounts of DNA are shown in Figures 8 and 9. Figure 8 shows a comparison of the amount of DNA quantified by means of UV and a comparison of the amount of DNA quantified by comparison to a bisulfite treated standard and to a genomic standard. It can be seen that quantitation by comparison to a genomic standard provides a more accurate quantitation of the amount of DNA in the sample (as measured by means of UV spectroscopy). This is expected since the CFF is independent of the conversion rate. The conversion specific PCRs HB 14 and C3 measure less Bis-DNA than CFFl with one exception (sample 10). Figure 9 shows a comparison of the amount of DNA quantified by means of the C3 assay, the HB 14 assay and the CFF assay (quantified by reference to a bisulfite treated standard). Compared to the CFFl data there is a large degree of variation from sample to sample using the bisulfite treated DNA specific assays. By reference to the UV measurements the most accurate measurement appears to be the CFF assay, quantified by reference to a genomic DNA standard.

Figure 10 shows the quantification of residual genomic (non-bisulfite converted) DNA within each samples. With one exception (sample 6) only very low levels of residual unconverted DNA are detected. As can be seen sample 11 appears to have a large amount of partially converted DNA, a measure of the amount may be determined by subtracting the sum of the amount measured using a bisulfite specific assay (i.e. HB14, C3 or an average of both) and that of the genomic specific assay (i.e. the CGI assay) from the amount quantified by means of the CFFl assay.