WO1997012058A1 - Method for quantifying nucleic acid using multiple competitor nucleic acids - Google Patents

Method for quantifying nucleic acid using multiple competitor nucleic acids Download PDF

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Publication number
WO1997012058A1
WO1997012058A1 PCT/GB1996/002376 GB9602376W WO9712058A1 WO 1997012058 A1 WO1997012058 A1 WO 1997012058A1 GB 9602376 W GB9602376 W GB 9602376W WO 9712058 A1 WO9712058 A1 WO 9712058A1
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Prior art keywords
method
competitor
nucleic acid
target
amplification
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PCT/GB1996/002376
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French (fr)
Inventor
Joakim Lundeberg
Mathias Uhlén
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Dynal A/S
Dzieglewska, Hanna, Eva
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Priority to GB9519638.2 priority Critical
Priority to GBGB9519638.2A priority patent/GB9519638D0/en
Application filed by Dynal A/S, Dzieglewska, Hanna, Eva filed Critical Dynal A/S
Publication of WO1997012058A1 publication Critical patent/WO1997012058A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701Specific hybridization probes
    • C12Q1/702Specific hybridization probes for retroviruses
    • C12Q1/703Viruses associated with AIDS
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/6851Quantitative amplification

Abstract

A method of determining the amount of target nucleic acid in a sample which comprises the steps of: a) adding to said sample a known amount of at least two different competitor nucleic acids at different concentrations, which have at least a portion of the sequence in common with the target nucleic acid, said common sequence comprising a binding site for a complementary primer sequence, b) co-amplifying the target nucleic acid and competitor nucleic acids in the sample by an in vitro amplification reaction using at least one primer, wherein at least one of said primers comprises a region complementary to said common sequence and the amplification products carry a label or means for attaching a label, c) separation of the amplification products, d) assessing the amount of label associated with the amplification products and e) comparison of the amount of label associated with each of the amplified target nucleic acid and amplified competitor nucleic acids to assess the amount of target nucleic acid in said sample, a method of diagnosis using the assay and kits for performing the same.

Description

METHOD FOR QUANTIFYING NUCLEIC ACID USING MULTIPLE COMPETITOR NUCLEIC ACIDS

This invention relates to a method of quantifying nucleic acid and in particular to a competitive assay for nucleic acid which has particular utility in the diagnosis of medical conditions by the identification of specific target nucleic acid, e.g. viral DNA or RNA. Target DNA molecules are often present in cell lysates or other source materials in extremely small quantities and in order to amplify such DNA selectively, the polymerase chain reaction (PCR) method has been developed. In this technique a pair of polymerisation primers, specific to known sequences of the target DNA to be amplified, are selected, one primer hybridising at or towards the 5 ' end of one of the strands of the target DNA and the other primer at or towards the 5 ' end of the second strand, such that in the presence of a polymerase, each primer produces a DNA sequence corresponding to the length of the target DNA template from the terminal of the primer sequence to the other end of the DNA molecule. If the DNA so produced is then subjected to strand separation, typically by melting at a temperature of about 90°C, the newly formed single stranded DNA sequences will hybridise to excess primer present in the mixture, usually after reducing the temperature to the range suitable for annealing, whereupon in the presence of the polymerase, further DNA strands are synthesised, this time extending only between the termini of the two primers. The polymerase is preferably capable of surviving the high temperature used in the strand separation step, a suitable thermophilic polymerase, namely Taq DNA polymerase, 5 having recently become available. If an excess of the two primers and of nucleotides needed for DNA synthesis is maintained in the medium, it is possible to operate a repeated cyclic process in which the separate strands are synthesised, separated, annealed to primer and new strands synthesised, merely by raising and lowering the temperature between the optimal temperatures for each of the above stages. In this way, it is found that amplification of the original target DNA can be exponential and million-fold increases of concentration can be effected in a relatively short time.

However, this procedure is not always sufficiently selective due to a percentage of non-specific binding of the primers to other DNA sequences, thereby amplifying the latter in addition to the target DNA. Moreover, there is a need to establish how much target DNA was present in a sample before PCR amplification. In the detection of bacteria, virus and parasites for example, PCR has several advantages compared to conventional diagnostic methods, i.e. the generality and speed of the assay. However, the fact that conventional PCR assays are only qualitative limits their use to diagnostic applications where only the presence or absence of the pathogen is to be determined. For many diseases, a quantitative measurement is needed to make a proper diagnosis and advantageously it would be useful to be able to measure the amount of pathogen during treatment to make a relevant prognosis. As mentioned above, there is major concern regarding the usefulness of PCR assay because their extreme sensitivity makes it possible to obtain false positives as a result of single molecules contaminating the sample. There is therefore a need for a quantitative assay, e.g. one suitable for clinical assays, which overcomes the drawbacks associated with conventional PCR assays.

A number of systems to quantify the initial template DNA/RNA have been described (see for example A. C. Syvanen, M. Bengtstrόm, J. Tenhunen and H. Sδderlund Nucl. Acids Res., 16, 11327 (1988) ; G. Gilliland, S. Perrin and H. F. Bunn PCR protocols, pp. 60-69, Academic Press, San Diego (1990) ; M. Becker-Andre and K. Hahlbrock, Nucl. Acids Res. 17,9437 (1989) ; and N. Kato, O. Yokosuka, K. Hosoda, Y. Ito, iM. Okto and M. Omata, Hepatology 18, 16-20 (1993)) . Such methods involve quantization techniques e.g. based on isotope labelling or restriction analysis, which are difficult or cumbersome to operate and time consuming to perform. Such methods are not therefore readily amenable to automation or to the analysis of large numbers of different samples.

The competitive PCR-based quantitation technique described by Cemu Bioteknik AB in WO 92/01812 represents an improvement over such techniques, but nonetheless has drawbacks when it comes to the analysis of large number of samples, for example in a diagnostic situation. This method is based on the technique described in W0 90/11369 for the detection of immobilized amplified nucleic acids (designated DIANA) and involves competitive titration wherein amounts of target DNA are co-amplified with differing, known amounts of competitor DNA to produce different ratios of target: competitor DNA, the competitor DNA being substantially the same as the target DNA except that is comprises a recognition site which may be detected directly or indirectly by a labelled species. Different known amounts of the competitor DNA are added to aliquots of the sample generally as a series of equally stepped dilutions. A set of readings corresponding to the label values in each aliquot is obtained which, when plotted against the known amounts of added competitor DNA give a characteristic sigmoid curve; the point of inflection on the curve is defined by the sharp change in the amount of labelled DNA between those aliquots in which added competitor DNA predominated and those in which target DNA predominated and is approximately proportional to the amount of target DNA in the initial sample.

Whilst the so-called "quantitative DIANA" technique of WO 92/01812 avoids the use of disadvantageous quantification methods inherent in the other prior art methods mentioned above, it requires that a large number of determinations (and hence PCR reactions) be performed, in order to determine one unknown. Thus for example, for one sample at least 8 different measurement points are in practice required, in the form of a dilution series of the competitor DNA. The technique is therefore not only costly, in terms of the large number of reactions required, but requires large initial sample volumes . This renders the method unsuited to the analysis of large numbers of samples, such as occurs in a clinical diagnostic laboratory.

It will be seen therefore, that despite advances in quantitative PCR techniques, there remains a need for a quantitative assay which is simple, quick and cost- effective to perform, and in particular for an assay which avoids the need for large number PCR reactions. One method of achieving would be to modify the "Quantitative DIANA" assay to introduce a standard curve. In such a method a competitor DNA is amplified simultaneously with target DNA, and the copies of competitor DNA become labelled during or after amplification. As a result of the competition for primer between target and competitor DNA, the presence of target DNA is reflected by the amount of amplified competitor DNA, which is assessed by quantitation of amplified DNA carrying a label. The amount of target DNA may thus be assessed by comparison with a standard curve in which different known amounts of target DNA are amplified in the presence of a constant known amount of competitor DNA.

Although this method is simple and quick to perform, it has the shortcoming that it relies on the preparation of a standard curve prior to and/or separate from the actual quantitation assay. It is well known that despite the utility of PCR as an amplification procedure, the results are not always reproducible and do not give rise to the theoretically calculable number of copies which should be produced in principle according to the number of cycles of amplification. Thus efficacy of ampli ication may vary from experiment to experiment despite apparently constant conditions. There thus remains the need for an assay which quantifies the amount of target DNA which is independent of the variations associated with the PCR reaction. It has now been found that this can conveniently be performed by calibration from internal standards which are amplified under the same experimental conditions as the target nucleic acid.

Accordingly the present invention provides a method of determining the amount of target nucleic acid in a sample which comprises the steps of : a) adding to said sample a known amount of at least two different competitor nucleic acids at different concentrations, which have at least a portion of the sequence in common with the target nucleic acid, said common sequence comprising a binding site for a complementary primer sequence, b) co-amplifying the target nucleic acid and competitor nucleic acids in the sample by an in vitro amplification reaction using at least one primer, wherein at least one of said primers comprises a region complementary to said common sequence and the amplification products carry a label or means for attaching a label, c) separation of the amplification products, d) assessing the amount of label associated with the amplification products and e) comparison of the amount of label associated with each of the amplified target nucleic acid and amplified competitor nucleic acids to assess the amount of target nucleic acid in said sample.

Conveniently, the target nucleic acid may be DNA, although quantitation of target RNA is also within the scope of the invention. Optionally, the method may additionally include the step of generating cDNA from RNA when the target nucleic acid is RNA. Such preliminary synthesis can be carried out by a preliminary treatment with a reverse transcriptase, conveniently in the same system of buffers and bases to be used in the subsequent amplification. Since the amplification procedures require heating to effect strand separation, the reverse transcriptase may be inactivated in the first amplification cycle. The enzymatic activity of both a reverse transcriptase and a polymerase which is stable may thus be used which conveniently may take the form of an enzyme with both activities, for example rTth polymerase. When mRNA is the target nucleic acid, it may be advantageous to submit the initial sample, e.g. a serum sample, to treatment with an immobilized poly dT oligonucleotide in order to retrieve all mRNA via the terminal poly A sequences thereof. Alternatively, a specific oliognucleotide sequence may be used to retrieve the RNA via a specific RNA sequence. The oligonucleotide can then serve as a primer for cDNA synthesis, as described in International Patent Application PCT/EP89/00304. It will be appreciated that whilst the target RNA may first be reverse transcribed into cDNA prior to amplification in the presence of competitor DNAs, more conveniently, competitor RNAs may be introduced at the stage of reverse transcription such that cDNAs of the target and competitor RNAs are produced which may then be amplified. This latter method has the advantage that calibration from the standard curve will indicate the number of target RNA molecules rather than number of cDNA molecules reverse transcribed from the target RNA molecule. RNA competitors may also be used to prepare DNA competitors by reverse transcription or DNA competitors may be used to prepare RNA competitors by transcription (see for example, Example 2) for use in methods of the invention.

Thus, viewed from a further aspect, the present invention provides a method of determining the amount of target RNA in a sample which comprises the steps of : a) adding to said sample a known amount of at least two different competitor RNAs at different concentrations which have at least a portion of the sequence in common with the target RNA, said common sequence comprising a binding site for a complementary primer sequence, b) reverse transcribing the target and competitor RNAs into target cDNA and competitor cDNAs, c) co-amplifying the target cDNA and competitor cDNAs in the sample by an in vitro amplification reaction using at least one primer, wherein at least one of said primers has a region complementary to the said common sequence and the amplification products carry a label or means for attaching a label, d) separation of the amplification products, e) assessing the amount of label associated with the amplification products and f) comparison of the amount of label associated with each of the amplified target cDNA and amplified competitor cDNAs to assess the amount of target RNA in said sample.

The term "target nucleic acid" is intended to encompass inter alia RNA, mRNA, DNA, cDNA e.g. from retroviral RNA, genomic DNA, mitochondrial DNA etc. and PNA. The DNA may be single or double stranded.

The term "complementary" as used herein is intended to encompass any nucleic acid molecule which is complementary to the nucleic acid in question, its complementary sequence or its RNA or DNA equivalent or complementary sequence thereof.

The term "competitor nucleic acid" as used herein is intended to encompass any piece of DNA (or RNA after reverse transcription or PNA) which would compete with the target DNA (or RNA after reverse transcription or PNA) for binding to at least one the primers used in the amplification reaction. Competitor nucleic acid extends also to the use of chimers of RNA, DNA and/or PNA. It will be appreciated however that it is an essential requirement of the method that all the nucleic acid sequences which are amplified in the amplification reaction must be amplified at a comparable rate and must therefore not be restricted with regard to the availability of essential reagents, ie. primers. Thus although referred to as competitor nucleic acid, this nucleic acid would only compete with the target nucleic acid when limiting concentrations of primers were available. For performance of the invention, this would not be the case. The competitor may be single or double stranded.

The term "assessing" as used herein includes both quantitation in the sense of obtaining an absolute value for the amount of target nucleic acid in a sample, and also obtaining a semi-quantitative assessment or other indication, eg an index or ratio, of the amount of target nucleic acid in the sample.

As mentioned above, the method requires that the competitor and target nucleic acids are amplified to the same extent, i.e. substantially at the same rate or with the same or substantially the same amplification efficiency, albeit to different levels as a result of the presence of different concentrations in the starting reaction. It will thus be appreciated that it is advantageous to ensure that the size and GC-content of the nucleic acids are kept substantially the same and that the sequence/s where the primer/s binds is identical . For this reason it is preferable to use competitor nucleic acids which have substantially the same sequence as the target nucleic acid albeit non¬ contiguous portions of common sequence. Similarly, for the purpose of quantitation it will be appreciated that the amplification products of each of the competitor and target nucleic acids should be sufficiently different to allow their separate identification. By "separation" as used herein is meant that the amplification products of each of the competitor and target nucleic acids may be discriminated as a result of their differences, although this is not limited to physical separation. By "different" is meant that each of the competitors, and their amplification products, are distinguishable, both from each other and from the target. This may be performed by using target and competitor nucleic acids having different properties, for example by affinity binding through sequences unique to the different nucleic acid fragments (i.e. by each competitor and the target comprising a unique and different sequence) or, through the use of uniquely identifiable labels attached to the different amplification products. Competitors may be constructed which are of the same or different lengths prior to amplification, but by virtue of different sequences, for example placing of primer binding sites, have different lengths after amplification. Preferably however, separate identification of the target and competitors is conveniently achieved by using competitor nucleic acids which after amplification differ in size to each other and the size of the amplified target nucleic acid. In the case in which separate uniquely identifiable labels are to be used, it will be appreciated that the signals produced by the labels must be quantitatively comparable to allow preparation of the internal standard curve. As mentioned above, the minimum number of competitor molecules required to perform the invention is two. However, to improve accuracy and reliability the number of competitors may be increased e.g. up to 8. The use of three or four different competitors has been found to give reliable results. When separation on the basis of size is to be performed, to achieve minimal amplification differences, competitors and appropriate primers are selected which give rise to amplified products which are within a narrow length range which are also of comparable length to the amplified product of the target nucleic acid. The range is selected such that the amplification products are still separable on the basis of size. Preferably the amplification products are in the range of 50 to 800 bases, most preferably 50 to 200 bases in length. An amplicon of this size is considered preferable to reduce amplification differences and for convenience in the described method. The target nucleic acid and primers are conveniently selected such that the amplified target nucleic acid is similarly between 50 and 800 bases, preferably between 50 and 200 bases and separable from the other amplification products on the basis of size.

It will be clear to the person skilled in the art that the difference in length between products which still allows for separation on the basis of size will depend on the resolution of the method of separation. Thus, for example, using electrophoretic techniques, a difference of 10 to 20 bases is appropriate to achieve separation. It will however be appreciated that improved resolution may be achieved using different methods, for example, chromatography (e.g. HPLC) or capillary electrophoresis such that differences of less than 10 bases may be appropriate. Any method suitable for the separation of nucleic acids may be used, electrophoresis, preferably on a polyacrylamide gel, is convenient and generally preferred.

As mentioned previously, the competitor nucleic acid should have at least a portion of the sequence in common with the target nucleic acid for binding a primer. Preferably, the competitor nucleic acid includes at least 2 portions of sequence in common with - li ¬ the target nucleic acid such that sequences for binding primers occur at, or towards the 3' or 5' ends of the nucleic acid. It will however be appreciated that the target and competitor nucleic acids may be considerably longer than their amplification products. Selection of the appropriately sized amplification products for performance of the invention is achieved by appropriate placing (in the case of competitor nucleic acids) and selection of primer binding sites (in the case of the target nucleic acid) . Competitors of similar or the same lengths may be constructed, which by appropriate placement of the primer binding sites produce fragments of different sizes.

Optionally, the competitor nucleic acid may be provided with a means for immobilization, which may be inherently part of the nucleic acid sequence, or introduced during amplification, either through the nucleotide bases or the primer/s which is used to produce the amplified nucleic acid. The target nucleic acid may similarly be provided with a means for immobilization as a result of the amplification procedure. Preferably, therefore, one of the amplification primers will carry means for immobilization, or may be provided already immobilised on a solid support.

When immobilization means is present inherently or introduced during amplification, the nucleic acids may be attached to a solid support prior (when present inherently) or subsequent to the amplification procedure. Where a subsequent immobilization step is used, conveniently this may be effected prior to the amplification fragment analysis step, permitting an intervening washing step if required. In the case of the assay of target mRNA, the target may be chosen such that it is close to the 3' poly A tail, when this is present, such that this region can be used for immobilization and/or for binding primers. The competitor RNAs may similarly be provided with a corresponding poly A tail. The target and competitors may thus be "captured" from the sample by binding to a solid support carrying an oligo dT sequence complementary to the poly A tail. Such immobilisation may conveniently facilitate the reverse transcription step.

Likewise, in the case of a DNA target and competitors, or for subsequent processing of cDNA reverse transcripts, they may be immobilised on a solid support during or after the amplification steps. Immobilisation in this manner provides a convenient way of collecting the amplification products.

To facilitate immoblization, the primers used according to the invention may carry a means for immobilization either directly or indirectly. Thus, for example the primers may carry sequences which are complementary to sequences which can be attached directly or indirectly to an immobilizing support or may carry a moiety suitable for direct or indirect attachment to an immobilizing support through a binding partner.

Numerous suitable supports for immobilization of RNA or DNA before or after amplification, and methods of attaching nucleotides to them, are well known in the art and widely described in the literature. Thus for example, supports in the form of microtitre wells, tubes, dipsticks, particles, fibres or capillaries may be used, made for example of agarose, cellulose, alginate, teflon, latex or polystyrene. Advantageously, the support may comprise magnetic particles eg. the superparamagnetic beads produced by Dynal AS (Oslo, Norway) and sold under the trademark DYNABEADS. Chips may be used as solid supports to provide miniature experimental systems as described for example in Nilsson et al. (Anal. Biochem. (1995) , 224:400-408) .

The solid support may carry functional groups such as hydroxyl, carboxyl, aldehyde or amino groups for the attachment of the primer or capture oligonucleotide. These may in general be provided by treating the support to provide a surface coating of a polymer carrying one of such functional groups, eg. polyurethane together with a polyglycol to provide hydroxyl groups, or a cellulose derivative to provide hydroxyl groups, a polymer or copolymer of acrylic acid or methacrylic acid to provide carboxyl groups or an amino alkylated polymer to provide amino groups. US patent No. 4,654,267 describes the introduction of many such surface coatings .

Alternatively, the support may carry other moieties for attachment, such as avidin or streptavidin (binding to biotin on the nucleotide sequence) , DNA binding proteins (eg. the lac I repressor protein binding to a lac operator sequence which may be present in the primer or oligonucleotide) , or antibodies or antibody fragments (binding to haptens eg. digoxigenin on the nucleotide sequence) . The streptavidin/biotin binding system is very commonly used in molecular biology, due to the relative ease with which biotin can be incorporated within nucleotide sequences, and indeed the commercial availability of biotin-labelled nucleotides, and thus this represents one preferred method for attachment of the capture oligonucleotide or primer to the support. Streptavidin-coated DYNABEADS are commercially available from Dynal AS.

As mentioned above, immobilization may take place after amplification. To permit this, one or both of the amplification primers are provided with means for immobilization. Such means may comprise as discussed above, one of a pair of binding partners, which binds to the corresponding binding partner carried on the support. Suitable means for immobilization thus include biotin, haptens, or DNA sequences (such as the lac operator) binding to DNA binding proteins . When immobilization of the PCR products is not performed, the products of the PCR reaction may simply be separated by for example, taking them up in a formamide solution (denaturing solution) and separating the products, for example by electrophoresis or by analysis using chip technology (mentioned hereinafter) .

The concentration of competitor nucleic acid should be selected to provide a standard curve in which the concentration of target nucleic acid falls within the range of the lowest and highest concentration of the competitor nucleic acid. Furthermore, the range over which the determination of the standard curve is appropriate is reliant on assessment in the region of the curve in which the amount of signal is linearly related to the label, taking into account the starting concentration and the number of amplification cycles to be performed. Thus, for example, competitors at a concentration (or copy number) of lOx, lOOx and lOOOx would allow the determination of target nucleic acid at a concentration (or copy number) of lOx to lOOOx. This range of concentrations can be used in low-copy applications such as HIV-l using nested PCR or when the target is present at a high copy number/concentration such as expression analysis in which a single PCR run is sufficient to find relative variations in expression levels.

For the preparation of the internal calibration curve, at least two, preferably three or four different nucleic acid competitors at different concentrations should be used in the assay.

It will be appreciated that the sequence and length of the oligonucleotides to be used as primers according to the invention will depend on the sequence of the target nucleic acid, the desired length of amplification product, the further functions of the primer (eg. means for immobilization, label attachment) as well as the amplification procedure. The in vitro amplification reaction may be any process which amplifies the nucleic acid present in the reaction under the direction of appropriate primers. The method may thus preferably be performed by PCR, and the various modifications thereof e.g. the use of nested primers although it is not limited to this method. PCR will however generally be the method of choice. In the case of nested primers, if the label/means for labelling is introduced by the primers, only the inner set of primers will be provided with the label/means for labelling, or optionally with the means for immobilisation. Those skilled in the art will appreciate that the invention would also be appropriate with amplification procedures such as Self-sustained Sequence Replication (3SR) , NASBA, the Q-beta replicase amplification system and Ligase chain reaction (LCR) (see for example Abramson and Myers (1993) Current Opinion in Biotech., 4: 41-47) .

Advantageously, to minimise any possible differences due to amplification efficiency, sufficient cycles of amplification are performed, to reach a saturation level (the so-called "plateau" phase of amplification) . However, this is not essential and amplification may be performed in the exponential phase. If necessary, appropriate controls may be used to compensate for any differences in amplification, efficiency etc. The use of such controls is routine and widely known in the field of in vitro amplification.

The term "label" as used herein refers to any label which can be assessed quantitatively. It will be appreciated that the amplified products themselves inherently provide the label if their presence is assessed quantitatively, e.g. by absorbance or binding on a solid support such as in chip technology as described herein. Alternatively, labels may be attached to the nucleic acid sequences. Such labels or means for labelling include for example, enzymes, fluorescent compounds, radio-labels and chemiluminescent compounds. A label which uses enzyme activity to generate a colour for spectrophotometric assessment may also be used, for example a fusion protein containing an enzymatic portion fused to a DNA binding protein which recognises a DNA sequence introduced into both the amplified and target nucleic acids may be associated with the amplified DNA, which on the addition of a suitable substrate may generate a signal suitable for detection. Conveniently, the enzymatic portion of the fusion protein may be β- galactosidase, alkaline phosphatase or peroxidase.

Labels are conveniently introduced during amplification by using primers which are directly labelled or provide means for labelling. The latter may include the use of a primer with one partner of a binding pair, in which the second binding partner is provided with a label and may be attached to the first binding partner to introduce a label to the amplified product. The labelling means may not necessarily require the addition of another component for incorporation of the label. For example, a dual- labelled probe may be used in which the labels are of sufficient proximity and suitable type that they quench the possible fluorescence of the other label. If at least a portion of the probe is complementary to a sequence downstream of the amplification primer and thus binds to the template, then during amplification, on extension, the exonuclease activity of the Taq DNA polymerase will degrade the detector molecule such that a physical separation of the labels occurs with a concomitant disappearance of quenching and hence appearance of signal (see for example Holland, P.M., Abramson, R.D., Watson, R. and Gelfand, D.H. (1991) , Proc. Natl. Acad. Sci., 88:7276-7280) . This thus provides labelled amplification products.

The assessment of amount of different amplification products may be performed by any technique which allows the discrimination of the different products. This may be performed on the basis of, for example, the sequence characteristics, immobilization means, label or preferably the size of the amplified DNA fragments by techniques known in the art .

If the PCR products have different sequence characteristics these may be used to uniquely identify the different products and thus allow their discrimination and/or separation. For example, the specific sequences may be recognized by and thereby immobilized to a surface bearing a complementary sequence. For example, chip technology may be employed in which a target specific probe is attached to the surface of the chip (see for example Nilsson et al. , 1995, supra) . When the probe binds to the particular target, the extent of hybridization may be ascertained which indicates how much of the competitor or target nucleic acid, with the particular sequence, has been bound. Similarly an immobilization means provided on the products may be used for discrimination. Clearly a proviso of such a system is that the immobilization means on the PCR products of the different competitor and target nucleic acids must be sufficiently different to allow a means for discrimination. Thus, different binding partners may be used, for example biotin- streptavidin, an ibody-antigen, for the different competitor and target nucleic acids. Immobilization means which are discrete DNA, RNA or PNA sequences may also serve to bind to appropriate complementary partners for separation by differential affinity binding.

Unique labels may be employed which identify the different products by introducing unique labels during amplification. No physical separation of the products is necessary if the levels of the different labels can be determined using methods which are unaffected by the presence of the other labels.

For separation on the basis of size, the amplification products may be separated electrophoretically, for example in agarose or preferably in polyacrylamide or by capillary electrophoresis, or separated chromatographically, for example, by HPLC. Where the amplified products are immobilized, these are necessarily released prior to electrophoresis. The amount of label may then be determined by analysis of label associated with the separated products, for example by analysis of fluorescence associated with bands on a gel or eluting from a column after chromatography.

For the determination of the number of copies (or concentration) of target nucleic acid in the sample, the amount of label associated with the amplified competitor nucleic acids is used to generate a standard curve in which the level of signal is plotted against copy number (or concentration) prior to amplification. Different amounts of the competitor nucleic acids are used in the starting reaction to assess the label associated with the amplified DNA of different starting amounts of competitor nucleic acid. Once the standard curve has been generated this can be used to read off the amount of starting copies (or concentration) of target nucleic acid from the sample as reflected by the amount of label associated with amplified target nucleic acid.

This method thus not only qualitatively positively identifies the presence of target sequence, unlike a number of the assays based on competition between target and competitor nucleic acid sequences for primers, but also allows the quantitation of the amount of target nucleic acid present in the sample. This has previously not been possible using a one-tube amplification reaction and therefore offers considerable advantages in accuracy and reproducibility. Furthermore, by relying on a standard curve the method of the invention thus avoids the need for determination of the actual amount of amplified target DNA although this may in some cases be useful information.

Two-stage PCR (using nested primers) , as described in our co-pending application WO90/11369, may be used to enhance the signal to noise ratio and thereby increase the sensitivity of the method according to the invention.

Regardless of whether one-stage or two stage PCR is performed, the efficiency of the PCR is not critical since the invention relies on amplification of competitor and target nucleic acid in the same reaction and thus all nucleic acid is amplified to the same extent. The quantitative method according to the invention may be used for general quantitation of RNA and DNA both for research and clinical applications, including diagnosis of viral, bacterial and protozoan pathogens. It may also find applications in forensic medicine.

Any suitable polymerase may be used, although it is preferred to use a thermophilic enzyme such as Taq DNA polymerase to permit the repeated temperature cycling without having to add further polymerase, e.g. Klenow fragment, in each cycle.

The method of the present invention is particularly advantageous in diagnosis of pathological conditions characterised by the presence of specific DNA, particularly latent infectious diseases such as viral infection by herpes, hepatitis or HIV. Also, the method can be used with advantage to characterise or serotype and quantify bacterial, protozoal and fungal infections where samples of the infecting organism maybe difficult to obtain or where an isolated organism is difficult to grow in vitro for subsequent characterisation as in the case of P. falciparum or chlamydia species. Due to the simplicity and speed of the method it may also be used to detect other pathological agents which cause diseases such as gonorrhoea and syphilis. Even in cases where samples of the infecting organism may be easily obtained, the speed of the PCR technique compared with overnight incubation of a culture may make the method according to the invention preferable over conventional microbiological techniques.

The method of the present invention may advantageously be used in the detection of specific target RNA sequences. Thus, for example, the levels of RNA from retroviruses may be quantified. Alternatively, when present as a provirus, levels of target genomic viral DNA may be quantified. Subsequent references to viral RNA therefore include the possibility of assessing the levels of viral DNA. The method allows not only the positive identification of samples in which the target RNA is present, but also allow quantitation of the levels of the target RNA. This has considerable clinical utility, for example in assessing the levels of virally infected patients over time, possibly during the course of treatment to establish the efficacy of a particular treatment or to establish the extent of infection.

It may also be possible to use quantitation data acquired from the assay to determine the onset of viral infection by extrapolation with reference to the increasing levels of viral RNA in the same subject or analogous subjects. This may have significant implications in contagious diseases in which the identification of infection onset may allow the identification of other subjects which may be infected. One example of the use of the assay for quantifying viral RNA, is with regard to the quantitation of HIV as a means of monitoring HIV infection.

Thus viewed from a yet still further aspect the present invention provides a method of determining the amount of target HIV RNA in a sample from an infected patient using the aforementioned method. In this case, a portion or all (approximately 9000 base pairs in length) of the sequence of HIV RNA which is present in patients is selected and suitable primers are devised to generate a suitable amplification product, for example 50 to 800 bases, preferably 50 to 200 bases in length. Appropriate competitor nucleic acids, preferably competitor RΝAs, may be constructed in which the primer sites of the target fragments are retained at or towards the 3 ' and 5 ' ends.

Preferably the sequence between the primer sites also bears sequence homology to the portion of target RΝA chosen for analysis. Conveniently, for the quantitation of HIV-l the target RΝA may be the 3 'LTR region of HIV-l. Primers and competitor nucleic acids for this purpose as described in the Examples form a further aspect of the invention. Conveniently, the mRΝA has a poly A tail which may be used for immobilization and/or as the sequence for binding primers for reverse transcription. Competitor RΝAs may similarly be provided with a poly A tail for immobilization and/or for binding primers. Provision of the poly A tail conveniently allows the use of a common purification step of target and competitor RΝAs by immobilization on a solid support carrying oligo dT and thus minimizes differences in RΝA degradation. The invention also comprises kits for carrying out the method of the invention. These will normally include at least the following components: a) at least two different competitor nucleic acids which have at least a portion of their sequence in common with the target nucleic acid, said common sequence comprising a binding site for a complementary primer sequence; b) at least one primer, wherein at least one of said primers comprises a region complementary to said common sequence; c) a polymerase which is preferably heat stable, for example Taq DΝA polymerase; and d) buffers for the amplification reaction. Optionally, where the primer has a means for attaching a label, the necessary components for labelling the primer are also included. Where an enzyme label is used, the kit will advantageously contain a substrate for the enzyme and other components of a detection system. Preferably the competitor nucleic acids will be provided with known concentrations (copy numbers) . Conveniently, the target nucleic acid may be DNA, in which case the competitor nucleic acids will be DNA. However, kits for quantitation of target RNA is also within the scope of the invention. In the latter case, the kit may optionally also include a reverse trancriptase. This may conveniently take the form of an enzyme with both reverse transcriptase and polymerase activity, for example rTth polymerase. Competitor nucleic acids may be either competitor DNA or RNA depending on whether both the transcription and amplification steps or only the amplification steps are to be performed in the presence of competitor nucleic acid. As mentioned previously, it is however more convenient to introduce competitor RNAs at the stage of reverse transcription such that cDNAs of the target and competitor RNAs are produced which may then be amplified.

Thus, viewed from a yet further aspect, the present invention provides kits for determining the amount of target RNA in a sample. These will normally include at least the following components: a) at least two different competitor RNAs which have at least a portion of their sequence in common with the target RNA, said common sequence comprising a binding site for a complementary primer sequence; b) at least one primer, wherein at least one of said primers has a region complementary to said common sequence; c) a reverse transcriptase and a polymerase which are preferably heat stable; and d) buffers for the transcription and amplification reactions . The invention will now be described by way of non¬ limiting examples with reference to the drawings in which: -

Figure 1 shows a schematic diagram indicating how the amount of target DNA may be assessed using 4 different competitor DNAs;

Figure 2 shows a schematic diagram indicating how the amount of target RNA (HIV-l RNA) may be assessed using 4 different competitor RNAs;

Figure 3 shows the vector map, sequence of the multilinker region and scheme of insertions of the pGEM4zpA cloning vector;

Figure 4 shows the sequences of PCR primers and their location in the HIV-l 3'LTR region;

Figure 5 shows the sequence of the linkers used for cloning;

Figure 6 shows the sequence of the 89bp fragment (pGEM4zpA + UPLINK + DOWNLINK) as assessed by sequencing and fragment analysis;

Figure 7 shows the sequence of the 125bp fragment (pGEM4zpA + UPLINK + DOWNLINK + LACLINK) as assessed by sequencing and fragment analysis;

Figure 8 shows the sequence of the lOObp fragment (pGEM4zpA + UPLINK + DOWNLINK + ECOLINK) as assessed by sequencing and fragment analysis; Figure 9 shows the dynamic range of fluorescent fragment analysis;

Figure 10 shows the fragment analysis of multiplex quantitative PCR using 3 competitor DNAs and variable amounts of sample; Figure 11 shows the results obtained in Figure 9 in graphical form; Figure 12 shows the calibration curve generated from the competitor DNAs for quantitation of sample; Figure 13 shows the dependence of quantitative results on the number of PCR inner cycles; Figure 14 shows alternatives in competitor configurations. Panel a: 5-fold configuration of 4 DNA competitors (10:40:200:1000) copies and 500 copies HIV-l target (MN) ; panel b: the same PCR product, but diluted 5 times; panel c: 2-fold configuration of DNA competitors (10:20:40:80) copies and 30 copies HIV-l target (MN) ; panel d: the PCR product diluted 5 times; panels e and f: as panels c and d, respectively, but without the addition of target (MN) ;

Figure 15 shows the fragment analysis results of RT-PCR after solid-phase purification using 5-fold diluted RNA competitor no. 1. Panels 1 a-e correspond to 57, 12, 2.3, 0.5 and 0.1 RNA copies (size 89 bp) bound onto the Dynabeads Oligo(dT)25 and then subjected to RT-PCR. Panels 2 a-e correspond to 57, 12, 2.3, 0.5 and 0.1 RNA copies (size 89 bp) bound onto the Dynabeads Oligo (dT) 25, then eluted from solid-phase and subjected to RT-PCR in solution. Panels 3 a-e correspond to the same dilutions of RNA no.l amplified directly by RT-PCR in solution. Panel M in the middle shows the dye marked ladder: 50 nt, 100 nt, 150 nt and 200 nt and the corresponding length estimates are given below; and

Figure 16 shows the raw data from fragment analysis of RT-PCR with four RNA competitors. Panels a-b correspond to direct solid-phase RT-PCR with approximately 50 RNA copies for each competitor (in duplicate) . Panels c-d correspond to RT-PCR of four eluted RNAs from the beads with 50 RNA copies for each competitor (in duplicate) . Panels e-f correspond to control RT-PCR in solution with 50 RNA copies for each competitor (in duplicate) . In the bottom panel, dye labelled ladder: 50 nt, 100 nt, 150 nt and 200 nt. EXAMPLE 1; Ouantitation of HIV-l using a multiplex PCR reaction

This Example illustrates the use of a single reaction to quantify the amount of target HIV-l in a sample. The general protocol for performing this using DNA or RNA competitors is illustrated in Figures 1 or 2 , respectively in which multiple competitor DNAs or RNAs of different lengths and amounts are introduced into samples which contain HIV particles. In the case of competitor RNAs, the HIV-l mRNA and competitor RNA molecules are captured onto a solid support using the poly A tail of the HIV mRNA and similar extensions which are part of the competitor RNAs by virtue of their construction. RT-PCR is then performed and the resulting DNA fragments are separated by affinity chromatography using a biotin label incorporated during amplification. The fragments are then separated on the basis of size and assessed for the amount of associated fluorescence introduced during amplification by the use of a FITC-labelled primer. A calibration curve using the competitor RNAs amplification products' fluorescence is used to establish the amount of target RNA in the sample.

This may also be performed using DNA competitors in which target mRNA is first reverse transcribed into DNA. Alternatively, when present as a provirus, genomic DNA of HIV-l may be quantified.

The example below describes:

1) the construction and use of plasmid DNA competitors to ascertain the amount of target DNA, in which target DNA is genomic DNA of HIV-l which is a proviral form of HIV named MN strain, and

2) the use of RNA competitors to ascertain the amount of target HIV-l RNA. 1. Construction and use of DNA Competitors for Determination of Levels of Target HIV-l DNA

METHODS

General cloning vector.

A poly A tail was isolated from the cloning vector pGEM4pA (containing a 30bp poly A tail inserted between Sacl and EcoRI restriction sites) (Stalbom B.-M., Torven A. and Lundberg. L.G. (1994) Anal. Biochem. 217: 91-97) and was inserted in pGEM4z vector containing the LacZ' region (Promega) . This vector enabled standard PCR primers and USP/RSP sequencing primers suitable for PCR screening and subsequent solid-phase sequence (Hultman T., Stal S., Homes E., and Uhlen M. (1989) Nucl. Acids Res. 17: 4937-4946) . The structure was confirmed by PCR sequencing. Vector map, sequence of multilinker region and scheme of insertions are presented in Fig. 3.

PCR

Outer and inner PCR primers to the 3'LTR region of HIV-l were synthesised according to the manufacturer's recommendations (Pharmacia, Uppsala Sweden) . One of the inner primers (sense) was biotinylated to enable binding of PCR products onto streptavidin-coated magnetic beads (Dynal AS, Oslo, Norway) and another (antisense)- was FITC-labelled for using fluorescence-based detection. Sequences of PCR primers and their location in the HIV-l 3'LTR region are presented in Fig. 4. The outer polymerase chain reaction was carried out in 50μl PCR buffer (25mM TAPS-HCl, pH 9.3 at 20°C, 50mM KCl, 2.OmM MgCl2, 0.1% Tween 20 and 200μl of each deoxynucleotide triphosphate (dNTP) with 5pmol of each primer and 1 unit Tag DNA polymerase. The temperature profile consisted of a denaturation program 94°C, 5 min linked with the cycle program: denaturation, 96°C, 30 sec; primer annealing, 50°C, 30 sec and extension, 72°C, 30 sec. The reaction was carried through 32 cycles. The inner polymerase chain reaction was carried out in 50μl PCR buffer (as described for outer PCR) . 5μl of outer PCR product was used as DNA template in inner PCR. The temperature profile consisted of a denaturation program 96°C, 5 min linked with the cycle program: denaturation, 96°C, 30 sec; primer annealing, 60°C, 30 sec and extension, 72°C, 30 sec. The reaction was carried through 32 cycles (GeneAmp 9600, Perkin Elmer, Cetus, USA) .

Linkers

Sequences of the linkers used for cloning are presented in Fig. 5 and characteristics of competitor DNAs are listed in Table 1. Phosphorylation of oligonucleotides (100 pmol each) was performed with T4 PNK (0.5μl, lOu/μl) in 1 μl PNK buffer in the presence of 0. IM ATP (lμl) . Reaction volume was adjusted with distilled water to 10 μl . Reaction was carried out for 30 minutes at 37°C. Phosphorylated oligos were annealed for 5 min. at 70°C and then immediately chilled on ice.

Restriction cleavage.- isolation and ligation

5μg of pGEM4zpA vector (or its derivatives) in 5μl of appropriate restriction enzyme lOx buffer, containing 5μl of acetylated BSA (lmg/ml) was digested with appropriate restriction enzymes (5-25U) . Reaction volume was adjusted to 50μl with distilled water. The reaction mixture was incubated at 37°C for one hour. Enzyme activity was heat-activated at 70°C for 15 min. The total digest was loaded on a 1% agarose gel and electrophoresed in 1 X TAE. The fragment was excised from the gel and the DNA fragment was isolated by electroelution in 1XTE (Sambrook J. , Fritsch E.F., and Maniatis T. Molecular cloning; A laboratory Manual, 2nd Edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbor, NY 1989, Book 1) . Ligation was performed according to Promega Technical Bulletin (Promega Technical Bulletin 036:3/88; REV 12/93) . E.coli cells strain RRIΔM15 (150μl per sample) were transformed by 10 μl of ligation mixtures and incubated overnight at 37°C according to standard procedures (Sambrook et al, 1989, supra) . PCR screening with RIT28/29 was performed followed by solid-phase sequencing with standard primers.

Immobilisation of PCR products and elution of FITC labelled strand

The resulting PCR products were immobilized onto 40 μl (lOmg/ml) of streptavidin-coated paramagnetic beads (Dynabeads-M280, Dynal, Oslo, Norway) suspended in binding/washing buffer (10 mM Tris-HCl,pH 7.5, 1 mM EDTA, 2 M NaCl) and incubated for 15 minutes at room temperature. A neodymium-boron permanent magnet (MPC-E, Dynal, Oslo, Norway) was used to collect the beads during supernatant removal, washing procedures and final collection of the FITC-labelled strands after elution with alkali (Dynal Technical Handbook. (1995) Biomagnetic Techniques in Molecular Biology, 2nd Edition, Chapter 1: 9-27) .

Neutralisation and fragment analysis

Ten μl of the eluted FITC-labelled strands were neutralized with 6μl of 0. IM HCl. One μl of the eluted and neutralized strand was mixed with 9 μl of deionized formamide (100%) , containing Dextran Blue 2000 (Pharmacia sequencing kit) , heat-denatured for 5 minutes at 95°C, immediately chilled on ice and loaded on a 6% polyacrylamide gel (Ready Mix, Pharmacia Biotech, Uppsala, Sweden) and electrophoresed on an automated laser fluorescent sequence [ALF] (Pharmacia, Sweden) . A 50 bp FITC-marked ladder (50-500) was used as a size standard. Quantitation and interpretations of the raw data output from ALF Manager (Pharmacia, Sweden) was facilitated by using Fragment Manager software (Pharmacia, Sweden) .

Construction of competitor No.l (89 b.p. fragment) Upstream linker -28-mer duplex, harboring outer and inner HIV-l PCR primers, was inserted between Hind Ill/SphI restriction sites in the multilinker region of pGEM4zpA. The insert sequence was confirmed by sequencing. The corresponding plasmid was denoted pGEM4zpA+UPLINK.

Downstream linker - 25-mer duplex, harboring outer and inner HIV-l PCR primers, was inserted between KpnI/SacI restriction sites of multilinker region of pGEM4zpA+UPLINK. Fragment Analysis of pGEM4zpA+UPLINK+DOWNLINK confirmed the sequence of length of insert (89 bp) , Fig. 6.

Construction of Competitor No. 2 (125 b.p. fragment) Insertion of Laclinker - 36-mer duplex - between Xbal/BamHI restriction sites of construct No. 1 - pGEM4zpA+UPLINK+DOWNLINK was performed and confirmed by PCR sequencing and Fragment Analysis (Fig. 7) .

Construction of competitor No. 3 (100 b.p. fragment) Insertion of EcoLINK 17-mer duplex - between

Sphl/Sall restriction sites of construct No. 1 - pGEM4zpA+UPLINK+D0WNLINK. Correct structure of obtained clones was confirmed by PCR sequencing and Fragment

Analysis (Fig. 8) .

Determination of concentration/copy number of competitors

Prior to competitive PCR, quantitation of each competitor was performed by measuring absorbance on Gene Quant (Pharmacia Biotech, Uppsala, Sweden) and by PCR using limiting dilutions according to (Brinchmann J.E.,

Albert J. and Vartdal F. (1991) J. Virol. 65:2019-23) . To reach the plateau phase in PCR, the nested PCR approach was used. Using PCR of limiting dilutions (Brinchmann et al . , 1991, supra) we found that competitor 1 (the upper curve) was 2 times more concentrated than competitor 2 and 8 times more concentrated than competitor 3 for one and the same value of dilution coefficient 1. Comparative results of quantitation by measuring absorbance on Gene Quant (theoretical) and by PCR of limiting dilutions (actual) are presented in Table 2. Actual concentrations were taken into account while running competitive PCR with 3 competitors.

Linearity of fluorescence for electrophoresis in 6% PAAG on ALF is shown in Fig. 9. Ratios of peak areas corresponding to adjacent 2-fold dilutions of PCR product are equal to the constant value approx 1.9, that confirms the possibility of linear quantitation in this range of fluorescence.

Equal efficiency of PCR amplification for all three competitors with different size of PCR fragment in various starting concentrations: Competitor 1 -3000 copies; Competitor 2 - 400 copies and Competitor 3 - 50 copies in the presence of different dilutions of MN (proviral HIV-l) samples was confirmed using Fragment analysis (Fig. 10) . The concentration of the competitor DNAs was constant in Lanes, 4, 6, 8, 10 and 12 as mentioned above with the only difference in MN dilutions (from 1 to 5 μl) , respectively. The results of fragment analysis confirmed the sizes of individual PCR fragments: 89 bp for competitor 1; 125b bp - 2; 100 bp - 3 and 114 bp for the MN sample. Graphical interpretation of these results on the basis of Fragment Manager software is presented in Fig. 11. Constant slopes of the curves corresponding to each competitor proves similar efficiency in PCR amplification inspite of the increase of MN concentration. Thus a calibration curve for quantitation of the amount of target DNA (HIV- 1) in samples can be plotted on the basis of data from Fragment Manager as a function of the fluorescence competitors 1, 2 and 3 which depends on the number of amplified DNA copies of the respective competitors (Fig. 12) .

RESULTS

The amount of HIV-l (MN) in a sample was ascertained by finding the location of the MN peak area value (from the same set of PCR) on the calibration curve.

Efficiency of amplification for MN strain (114 bp PCR fragment) seems to be comparable with that for competitors. Thus, the theoretical number of apriori calculated MN copies matches the value obtained using the calibration curve of three standards in Fig. 12, by reading off the copy number from the x-axis which relates to the fluorescence of the sample and dividing by the volume of the sample. This gives one and the same value of 130 copies for both the theoretical and detected number of MN copies.

Calibration curves for 5 different dilutions of MN strain were found to have constant slopes. This confirms that similar efficiency of amplification of competitors occurred regardless of the amount of HIV-l nucleic acid, thus allowing confident use of this method to quantify HIV-l levels in samples.

In order to ascertain levels of HIV-l DNA in a sample prepared by reverse transcription of mRNA, appropriate competitor DNAs are prepared as above or may be excised from the vector before use. 2. CONSTRUCTION AND USE OF RNA COMPETITORS FOR THE DETERMINATION OF LEVELS OF TARGET HIV-l RNA

METHODS

Preparation of RNA competitors

RNA competitors are prepared by linearisation of competitor DNA using a restriction site adjacent to the poly (A) tail. In vitro transcription employing T7 RNA polymerase (Pharmacia Biotech) results in RNA competitors of a defined length and with a poly (A) tail. The RNA competitors are purified by standard procedures (Sambrook et al . , 1989, supra) and stored in water at -80°C.

RT-PCR

Reverse transcription is performed according to the methods known in the art (eg. Sambrook et al . , 1989, supra) using poly A tails of the target and competitor RNAs for immobilization and the site for binding appropriate primers. PCR is performed as mentioned previously with the exception that rTth polymerase is used for both reverse transcription and amplification. The primers allow for a one-step procedure for both reverse transcription and for PCR in which the PCR primer is directly adjacent to the poly A tail.

Determination of concentration/copy number of competitors Quantitation is performed as described previously for the DNA competitors using the method of limiting dilutions.

Determination of amount of target RNA in sample Blood of infected patients containing HIV particles containing two copies of a 9kb RNA genome are lysed and mixed with three competitor RNAs of different lengths and amount. The added competitor concentrations differ 10-fold from each other. The competitor and HIV-l RNAs are purified by attachment to oligo dT beads. RT-PCR is then performed. After nested competitive PCR, in which the PCR products are labelled by FITC and biotin, the products are bound to streptavidin coated beads, the FITC labelled strand is eluted with 0. IM NaOH and the fragments are separated as described above. The HIV-l target, amplified by outer primers JA159/JA162 and inner primers JA160-FITC/JA161-Bio, results in a PCR product of 114 b.p. The same primers used for the competitors results in inner amplified fragments of 89, 100 and 125 b.p. The number of mRNA copies of HIV-l in the blood are quantified by comparison to the internal calibration curve prepared as described for the assessment of target DNA above.

TABLE 1. Characteristics of internal standards constructions cloned in pGEM4zpA

Construction Cloned linker Restriction sites Size of PCR product

Construct 1 UPLINK, 28 bp and Hindlll/Sphl and

DOWNLINK, 25 bp Kpnl/Sacl 89 bp

Construct 2 UPLINK, 28 bp; Hindlll/Sphl;

DOWNLINK, 25 bp and Kpnl/Sacl and

LacLINK, 36 bp Xbal/BamHI 125 bp

Construct 3 UPLINK, 28 bp; Hindlll/Sphl;

DOWNLINK 25 bp and Kpnl/Sacl and

EcoLINK, 17 bp Sphl/Sall 100 bp

HIV-l (MN strain) 114 bp Table 2. Comparison of methods for quantitation of three competitors

Sample Theoretical calculation Practical calculation by based on absorbance on PCR of limiting dilutions,

Gene Quant, Pharmacia* Fluorescent detection on

ALF sequencer stndl, 89 bp 84 84

stnd2, 125 bp 64 22

stnd3, 100 bp 22 11

* Number of DNA copies for the same dilution coefficient

EXAMPLE 2; Ouantitation of HIV-l DNA or RNA using a multiplex PCR reaction

This example illustrates the use of RNA competitors for quantitating the amount of target RNA in a sample. Furthermore, improvements and optimizations of quantitation of DNA as described in Example 1 are shown. Samples containing target and known amounts of multiple competitors were co-analysed by RT-PCR. Four competitors were constructed to contain the same primer binding sequences as genomic HIV-l target, but each competitor was of different length. In addition, the competitors used for competitive RT-PCR harboured a poly A stretch enabling a common HIV-l RNA purification strategy to be developed based on oligo dT magnetic beads. The PCR primers were designed to anneal to the 3 'LTR region directly adjacent to the poly A region of the HIV-l RNA genome. One of the inner primers was fluorescent labelled to allow discrimination between the wild type DNA and the four competitors by fragment analysis using a standard automated sequencer. A calibration curve using the peak area of the competitors enabled accurate determination of the amount of target with minimal tube-to-tube variations.

Sample material

The model system described here for HIV-l DNA quantification was from HIV-l MN infected peripheral blood mononuclear cells (PBMC) . These were diluted in crude cell lysates of uninfected PBMC to contain various numbers of viral HIV-l copies. PBMC were isolated by Ficoll-Paque density centrifugation and lysed without prior cultivation in PCR lysis buffer (10 mM Tris-HCl pH 8.3, lmM EDTA, 0.5% NP40, 0.5% Tween 20 and 300 mg/ml Proteinase K) at a concentration of IO6 cells/100 μl as previously described (Wahlberg et al . , AIDS RES. and

Hum. Retrovir., 7, p983-990, 1991) . Crude cell lysates were used directly for PCR amplification.

Construction of competitor DNA HIV-l competitor DNAs were constructed by linker assembly into plasmid DNA. The restriction enzymes and DNA ligase were used according to suppliers' recommendations (Pharmacia Biotech, Uppsala, Sweden) and oligonucleotides were synthesized on a Gene Assembler Plus (Pharmacia Biotech, Uppsala, Sweden) by the phosphoamidite method and purified by a desalting and prepRPC 5/5 reverse phase column (Pharmacia Biotech, Uppsala, Sweden) . The PCR primer sequences were JA159 (outer, 432-452) ; JA160F (inner, 435-460) ; JA160Cy5 (inner, 435-460); JA161B (inner, 524-499) ; JA162 (outer, 528-508) as shown in Figure 4. Positions are given relative to MN (Myers et al. , in "Human Retroviruses and AIDS 1991", Los Alamos National Laboratory, Los Alamos, New Mexico, 1991) strain of HIV-l. The assembly of three of the four competitors has been described previously in Example 1, but is briefly summarised below. A 30 bp polyA stretch was inserted into the EcoRI site of pGEM4z vector (Promega, Madison, WI , USA) as described in Example 1. The first competitor (no. 1, 89 bp fragment, competitor 1 in Example 1) was constructed by insertion of an upstream linker harbouring annealing sites for outer and inner HIV-l PCR primers, between Hindlll/Sphl restriction sites in the multilinker region of the created pGEM4zpA. A downstream linker harbouring annealing sites for outer and inner HIV-l PCR primers, was inserted between Kpnl/Sacl restriction sites of the multilinker region. The construction of competitor no. 2 (100 bp fragment, competitor 3 in Example 1) was performed by insertion of EcoRV linker between Sphl/Sall restriction sites of construct no. 1. The construction of competitor no. 3 (125 bp fragment, competitor 2 in Example 1) was generated by insertion of a Lac operator linker between Xbal/BamHI restriction sites of construct no. 1. Competitor no. 4 (136 bp fragment) was constructed by insertion and blunt-ended ligation of HaJo linker (36- mer, 5 ' -GGGAACACCATGAACACCACCATGACCCG-3 ' and 3 -

TCGACCCTTGTGGTGGTACTTGTGGTGGTACTGGGCCTAG-5') at the EcoRV site of construct no. 2. The concentration and purity of competitor plasmid DNAs and in vitro transcribed RNA were determined by absorbance according to Sambrook et al (1989, supra) , and ultimately by limiting dilution experiments using PCR with HIV-l specific primers (see below) . The competitors were subsequently serially diluted in 10 mM Tris-HCl pH 8.3 and 10 ng/ml yeast tRNA (Boehringer Mannheim, Mannheim, Germany) .

Limiting dilution

Determination of competitor and HIV-l MN cell lysates (both of stock and diluted lysates) concentrations were performed using limiting dilution and nested PCR as previously described in Example 1. In short, the materials were diluted in five-fold steps and at least ten PCR determinations were performed on each dilution. The copy numbers were calculated by the Poisson distribution formula (Brinchmann, 1991, supra) (i.e. one starting copy corresponds to a dilution step in which 63% of the samples are positive) . The basis for the analysis is the ability of the nested PCR to reproducibly detect single HIV-l molecules.

Preparation of RNA competitors Plasmid preparations of competitors 1 to 4 were linearised by EcoRI (Pharmacia, Biotech, Sweden) restriction cleavage in a total volume of 50 μl reaction mixture. After heat-inactivation of the restriction enzyme, the restriction mixture was mixed with 100 μl of phenol: chloroform:isoamyl alcohol (25:24:1) , vortexed for 30 seconds and centrifuged for 1 minute at 12000 g at room temperature. The aqueous phase was removed and transferred into a RNase-free eppendorf tube and extraction was repeated. DNA was precipitated by addition of 30 μl 3.0 M sodium acetate and 750 μl 96% ethanol and after incubation at -70°C for one hour was centrifuged for 30 minutes at 12000 g at room temperature. The pellet was rinsed with 70% ethanol, dried and dissolved in DEPC-treated water. A half microgram of each enzyme-restricted DNA competitor 1 to 4 was incubated in a transcription buffer, containing 40 mM Tris-HCl (pH 8.0) , 30 mM MgCl2, 10 mM β-mercaptoethanol, 400 μm of each RNase-free dNTP, 50 μg/ml RNase-free BSA, with 70 units/ml of T7 RNA polymerase in a final volume of 50 μl reaction volume at 37°C for 30 minutes. Remaining DNA templates were then digested by addition of 10 units of RNase- free DΝase 1 and incubation at 37°C for 60 minutes. RΝA was immediately isolated according to the above described phenol extraction protocol with the exceptions that all procedures with RΝA samples were performed on ice and centrifugations were performed at 4°C. RΝA transcripts were dissolved in 50 μl DEPC-treated water and stored at -80°C. All precautions preventing RNAs degradation were taken (Innis et al . in "PCR Protocols", Academic Press Inc., San Diego, CA, 1990) .

Solid-phase purification of polyadenylated RNA

Twenty-five microliter (0.125 mg) Dynabeads Oligo (dT)25 (Dynal, Oslo, Norway) were transferred to an RNase- free Eppendorf tube placed in a magnet for magnetic sedimentation. After removal of supernatant the beads were washed once in 50 μl Lysis/binding buffer. Twenty five μl of Lysis/binding buffer was added to the sedimented beads and resuspended with 10 μl of RΝA competitors, prepared as above) . Preparation of HIV-l RΝA was performed using 250 μl serum as previously described (Chiodi et al. , J. Clin. Microbiol, 30, p 1768-1771, 1992) . Annealing was performed at room temperature for 10 minutes employing a rotation device. Then beads were washed twice by Washing buffer with LiDS (50 μl) , once with Washing buffer (50 μl) and finally twice with 1 x PCR buffer (50 μl) . RT-PCR was either performed directly on beads or in solution aftr elution of RΝA from the beads. To elute RΝA from the Dynabeads, 10 μl of Elution solution was added into an R ase-free Eppendorf tube with RΝA bound to beads and denaturation was performed by incubating at 65°C for 15 minutes employing rotation. The tube was immediately chilled on ice for 2 minutes, placed in the Dynal MPC and supernatant (10 μl) was transferred to 40 μl of RT-PCR mix. All reagents were used according to manufacturer's recommendations.

Competitive reverse transcription and polymerase chain reaction (RT-PCR) Reverse transcription and outer polymerase chain reactions were carried out in a single-step in 50 μl containing 5 μl of 10 x PCR buffer (100 mM Tris-HCl, pH 8.3 at 25°C, 500 mM KCl, 25 mM MgCl2) and 200 μM of each deoxynucleotide triphosphate (dNTP) with 5 pmol of each primer, 2 units Taq polymerase (Perkin-Elmer, Norwalk, CT, USA) , 0.5 unit MMLV Reverse Transcriptase (Pharmacia, Biotech, Sweden) , 2 μg RNase- free yeast tRΝA (Boehringer Mannheim, Mannheim, Germany) , 10 μl RΝA template and 10 pmol oligo (dT) 2S primer. The temperature profile consisted of reverse transcription: 37°C, 60 minutes linked with outer PCR program: denaturation, 94°C, 5 minutes followed by the cycle program: 95°C, 30 sec; 50°C, 30 sec; 72°C, 30 sec. The inner polymerase chain reaction was carried out in 50 μl PCR buffer containing 5 μl of 10 x PCR buffer (100 mM Tris-HCl, pH 8.3 at 25°C, 500 mM KCl, 25 mM MgCl2) and 200 μM of each deoxynucleotide triphosphate (dΝTP) with 5 pmol of each primer and 1 unit Taq polymerase (Perkin- Elmer, Norwalk, CT, USA) . 5 μl of outer PCR product was transferred to the inner PCR containing biotinylated and fluorescent labelled inner primers. The temperature profile and number of cycles for the inner PCR were identical with the exception that the annealing temperature for the inner amplification was 60°C. Both outer and inner PCR employed 32 cycles (GeneAmp 9600, Perkin Elmer, Norwalk, CT, USA) . All recommended precautions against PCR contamination were taken (Kwok and Higuchi, Nature, 339, p237-238, 1989) , with multiple negative controls included in each PCR run.

Competitive Polymerase Chain Reaction For DNA quantification the DNA competitors were premixed in different fixed ratios before amplification. Ten microliters of competitor mixtures were added together with 10 μl sample to the reaction tube containing the same components as in the RT-PCR step, except the RNA templates, carrier tRNA and oligo(dT)25 primer. The temperature profile was identical, except for the initial step of reverse transcription. Immobilization of PCR products and fragment analysis

The resulting PCR products were immobilized onto 40 μl (10 mg/ml) streptavidin-coated paramagnetic beads (Dynabeads-M280, Oslo, Norway) suspended in binding/washing buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 M NaCl) and incubated for 15 minutes at room temperature. A neodymium-boron permanent magnet (MPC-E, Dynal, Oslo, Norway) was used to collect the beads during supernatant removal, washing procedures and final collection of the dye-labelled strands after elution with alkali (Hultman et al . , 1989, supra) . Ten microliter of the eluted dye-labelled strands were neutralized with 6 μl of 0.1667 M HCl. One μl of the eluted and neutralized strand was mixed with 9 μl of deionized formamide (100%) , containing Dextran Blue 2000 (Pharmacia Biotech, Uppsala, Sweden) , heat-denatured for 5 minutes at 95βC, immediately chilled on ice and loaded on a 6% polyacrylamide gel (Ready Mix, Pharmacia Biotech, Uppsala, Sweden) and electrophoresed on an automated laser fluorescent sequencer (Pharmacia Biotech, Uppsala, Sweden) . An accurate peak area determination cannot be attained when the fluorescent detector is saturated which is indicated by cut peaks in the chromatogram. A 50 bp fluorescent-marked ladder (50-500) was used as a size standard. Quantification and interpretations of the raw data output were facilitated by using fragment Manager software (Pharmacia Biotech, Uppsala, Sweden) .

RESULTS

Two methods for the quantification of HIV-l DNA and HIV-l RNA using multiple competitors were developed, outlined in Figure 1 and Figure 2, respectively. The common feature is the single tube assay format achieved by the co-reverse transcription and/or co-amplification of the sample HIV-l RNA/DNA and multiple competitors. The competitors have been designed to allow internal discrimination. The PCR primers anneal directly adjacent to the polyA stretch of the HIV-l RNA genome in the 3 ' LTR region and to equivalent sequences in the constructed competitors. The resulting PCR products are analysed by fragment size using the internal competitors to create a standard curve employed to quantify the amount of target.

In Example 1 various amounts of HIV-l MN DNA were quantified using this strategy (see also Vener et al . , BioTechniques, 21, p 248-253, 1996) . In that study a premix of fixed amounts of the three DNA competitors of different length was used in semi-nested competitive PCR. The corresponding length of inner PCR products were designed to vary over a short size interval: i.e. competitors no. 1-3; 89 bp, 100 bp and 125 bp, respectively. Importantly, amplification of the target, HIV-l, resulted in a product of 114 bp, thus within the range covered by the competitors.

Optimisation for quantification of DNA sequences

This Example has focused on further optimisation for quantification of HIV-l DNA and then establishment of HIV-l RNA quantification as described below. First, we wanted to investigate if the additional number of inner PCR cycles affected the quantitative results. As suggested by Becker-Andre (1989, supra) , in order to perform this analysis an experiment was carried out with three DΝA competitors and HIV-l MΝ target, which were analysed after 10 to 40 inner PCR cycles. The competitor configuration used was 50:200:1000 copies of competitors no. 1:2:3, respectively, and two separate sets of experiments for different amounts of HIV-l target (MΝ: 500 copies and 1000 copies) were analysed. Results of competitive PCR for 500 HIV-l DΝA copies are depicted in Fig. 13. The presented data clearly show that reliable quantification of HIV-l DΝA can be achieved as illustrated by the unaffected ratios between competitors and target which can be attained irrespective of the number of PCR cycles.

Secondly, to enhance the dynamic range of quantification for viral loads a fourth competitor (136 bp fragment) was constructed by insertion of the HaJo linker (36 bp duplex) at the EcoRV rare site in construct no. 2, as it is depicted in Fig. 5. In this context, different configurations using four competitors were analysed to investigate the dynamic range for chromatographic analysis, since "cut" peaks can not be reliably employed in the construction of the internal standard curve. Figure 14 depicts two alternatives. Panel a shows a configuration of competitors 10:40:200:1000 and HIV-l DNA target corresponding to 500 starting copies. Here competitor no. 4 can not be used in the construction of standard curve due to the "cut" peak. However, by a 5-fold dilution competitor no. 4 can be used while competitor no. 1 is now too low to be detected. In order to achieve the most exact estimation, a more narrow dilution series can be employed depicted in panels c and d. Panel d shows an example in which all 4 peaks are used to create a standard curve.

In vitro transcription of RNA competitors

Since DNA competitors were constructed to contain a poly (A) 30 stretch at the 3 ' -end of the multilinker region, in vitro transcription with T7 RNA polymerase of polyadenylated RNA competitors was possible to create RNA competitors. First, each DNA plasmid was linearised by EcoRI restriction cleavage, purified by phenol extraction and ethanol precipitation and then transcribed. The remaining DNA templates was removed using RNase-free DNase I. RNA transcripts were isolated by phenol extraction, ethanol precipitation and the pellets were redissolved in DEPC-treated water. Thus, four RNA internal competitors were obtained and their sizes estimated by electrophoresis confirmed their calculated total length: no. 1 - 135 bp, no. 2 - 146 bp, no. 3 - 171 bp and no. 4 - 186 bp (data not shown) . Their calculated concentrations were estimated by 2-fold end dilutions experiments.

Sensitivity of solid-phase purification

In order to investigate at which step the RNA competitors can be added, i.e. prior, during or after sample preparation the following experiment was performed. A series of 5-fold dilutions of competitor RNA no. 1, corresponding to 57, 12, 2.3, 0.5 and 0.1 RNA copies, were used. These were then either independently immobilised onto oligo(dT)2s beads and subsequently reverse transcribed on the solid support (Fig. 15, panels 1 a-e) or immobilised onto oligo (dT)25 beads, eluted and subsequently reverse transcribed in solution (Fig. 15, panels 2 a-e) or directly reverse transcribed in solution (Fig. 15, panels 3 a-e), followed by semi- nested PCR. Importantly, the results show that the same sensitivity can be achieved by any of the chosen approaches for quantification.

Solid-Phase RT-PCR using multiple competitors In order to investigate the presence of any bias during immobilisation and/or elution from the solid- phase between the RNA competitors, a premix of competitors with approximately 50 starting copies of each RNA competitor were employed. In Figure 16, panels a and b show (in duplicate) the results of direct solid- phase RT-PCR. Panels c, d depict (in duplicate) the results of RT-PCR of eluted RNAs from the beads and panels e and f present control RT-PCR of the RNA premix directly in solution. The data clearly show that peak profiles and ratios corresponding to different RNA competitors are preserved irrespective of the chosen approach.

Claims

Claims :
1. A method of determining the amount of target nucleic acid in a sample which comprises the steps of : a) adding to said sample a known amount of at least two different competitor nucleic acids at different concentrations, which have at least a portion of the sequence in common with the target nucleic acid, said common sequence comprising a binding site for a complementary primer sequence, b) co-amplifying the target nucleic acid and competitor nucleic acids in the sample by an in vitro amplification reaction using at least one primer, wherein at least one of said primers comprises a region complementary to said common sequence and the amplification products carry a label or means for attaching a label, c) separation of the amplification products, d) assessing the amount of label associated with the amplification products and e) comparison of the amount of label associated with each of the amplified target nucleic acid and amplified competitor nucleic acids to assess the amount of target nucleic acid in said sample.
2. A method as claimed in claim 1 wherein said target nucleic acid is DNA.
3. A method as claimed in claim 1 wherein said target nucleic acid is RNA.
4. A method as claimed in any one of claims 1 to 3 wherein said competitor nucleic acids are DNA.
5. A method as claimed in any one of claims 1 to 3 wherein said competitor nucleic acids are RNA.
6. A method as claimed in any one of claims 1 to 5 wherein additionally the target RNA and/or competitor RNAs are reverse transcribed into cDNA.
7. A method as claimed in claim 6 wherein reverse transcription and amplification is performed by rTth polymerase.
8. A method as claimed in any one of claims 1 to 6 wherein amplification is performed by Taq DNA polymerase.
9. A method as claimed in any one of claims 1 to 8 wherein 3 or 4 competitors are used.
10. A method as claimed in any one of claims 1 to 9 wherein the amplified target and competitor nucleic acids differ in size relative to each other.
11. A method as claimed in any one of claims 1 to 10 wherein the amplified target and competitor nucleic acids are in the range of 50 to 200 bases in length.
12. A method as claimed in any one of claims 1 to 11 wherein the competitor nucleic acids include at least 2 portions of sequence in common with the target nucleic acid.
13. A method as claimed in any one of claims 1 to 12 wherein said target and/or competitor nucleic acids are provided with a means for immobilization to a solid support.
14. A method as claimed in any one of claims 1 to 13 wherein said amplified target and competitor nucleic acids are provided with a means for immobilization by use of at least one amplification primer carrying a means for immobilization to a solid support.
15. A method as claimed in claim 13 or 14 wherein said means for immobilization is a poly A tail.
16. A method as claimed in claim 13 or 14 wherein said mean for immobilization is biotin.
17. A method as claimed in any one of claims 13 to 16 wherein immobilization to a solid support is performed during or after the amplification steps.
18. A method as claimed in any one of claims 13 to 17 wherein said solid support comprises superparamagnetic beads.
19. A method as claimed in any one of claims 1 to 18 wherein said amplified target and competitor nucleic acids are provided with a label or means for attaching a label by use of at least one amplification primer carrying a label or means for attaching a label .
20. A method as claimed in any one of claims 1 to 19 wherein said amplified target and competitor nucleic acids are separated by electrophoresis.
21. A method as claimed in any one of claims 1 to 20 wherein said amplification is performed by PCR.
22. A method as claimed in any one of claims 1 to 20 wherein said target nucleic acid is HIV RNA.
23. A method as claimed in claim 22 wherein said ccmmon sequence occurs in the 3 'LTR region of HIV-l.
24. A method of diagnosis of infection by an organism using the method as defined in any one of claims 1 to 23 wherein said target nucleic acid is any nucleic acid of the organism.
25. A kit for carrying out the method as defined in any one of claims 1 to 24 including at least the following components : a) at least two different competitor nucleic acids, as defined in any one of claims 1, 4, 5, 12, 13, 15, 16 or 23 which have at least a portion of their sequence in common with the target nucleic acid as defined in any one of claims 1, 2, 3, 13, 15, 16 or 22, said common sequence comprising a binding site for a complementary primer sequence; b) at least one primer as defined m any one of claims 1, 14, 15, 16, 19 or 23, wherein at least one of said primers comprises a region complementary to said common sequence; c) a polymerase as defined in claim 1 or 8, and d) buffers for the amplification reaction.
26. A kit as claimed in claim 25 to detect target RNA as defined in any one of claims 3, 22 or 23 wherein additionally said kit includes a reverse transcriptase as defined in claim 1 or 7 and buffers for the transcription reaction.
PCT/GB1996/002376 1995-09-26 1996-09-26 Method for quantifying nucleic acid using multiple competitor nucleic acids WO1997012058A1 (en)

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US9926593B2 (en) 2009-12-22 2018-03-27 Sequenom, Inc. Processes and kits for identifying aneuploidy
US9605313B2 (en) 2012-03-02 2017-03-28 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
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US9932628B2 (en) 2012-07-27 2018-04-03 Gen-Probe Incorporated Dual reference calibration method and system for quantifying polynucleotides

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