WO2001075155A2 - Procedes - Google Patents

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WO2001075155A2
WO2001075155A2 PCT/GB2001/001405 GB0101405W WO0175155A2 WO 2001075155 A2 WO2001075155 A2 WO 2001075155A2 GB 0101405 W GB0101405 W GB 0101405W WO 0175155 A2 WO0175155 A2 WO 0175155A2
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Prior art keywords
amplification
nucleic acid
sample
post
double
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PCT/GB2001/001405
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English (en)
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WO2001075155A3 (fr
Inventor
Walter Fred Bodmer
Rush Spencer Wells
Sylvia Mary Bartlett
Josef Straub
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Imperial Cancer Research Technology Limited
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Priority to AU46678/01A priority Critical patent/AU4667801A/en
Publication of WO2001075155A2 publication Critical patent/WO2001075155A2/fr
Publication of WO2001075155A3 publication Critical patent/WO2001075155A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the present invention relates to the detection of products of nucleic acid amplification reactions, in particular products of a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Nucleic acid amplification methods are well known and are reviewed, for example, in US 5,545,528, incorporated herein by reference. Examples include those using the polymerase chain reaction, QB replicase and ligase chain reaction.
  • NASBA nucleic acid sequence based amplification
  • 3SR nucleic acid sequence based amplification
  • SDA strand displacement amplification; see, for example, Walker et al (1992) Nucl. Acids Res. 20, 1691-1696
  • Rolling Circle Amplification Lizardi et al, (1998) Nat. Genet. 19:3 225-32) are further examples. Methods based on the polymerase chain reaction are particularly widely used because of their simplicity and flexibility.
  • Amplification reactions in particular polymerase chain reactions, may produce the required nucleic acid (specific amplification product).
  • other nucleic acids non-specific amplification products
  • These non-specific amplification products may constitute a significant proportion of the double-stranded nucleic acid present at the end of a reaction.
  • non-specific amplification products formed during early amplification cycles may be preferentially amplified during later amplification cycles. This may happen, for example because the non-specific amplification product is shorter than the required specific amplification product.
  • non-specific amplification products examples are so-called "primer dimers", arising from amplification of a template formed from concatamers of the amplification primers, as well known to those skilled in the art.
  • Primer dimers may typically be less than 80 nucleotides in length and may be present in high copy number in a post-amplification reaction sample.
  • non-specific amplification products may be particularly abundant in post-amplification reaction samples where the template for the specific amplification reaction is absent or present only at low concentration since, during the amplification reaction, there is no specific amplification product competing with the non-specific products for reagents, for example enzyme and/or deoxynucleotidetriphosphates (dNTPs) .
  • dNTPs deoxynucleotidetriphosphates
  • nucleic acid amplification reaction may be detected and/or quantified. Most methods seek to distinguish non-specific amplification products from the specific amplification products. Agarose gel electrophoresis and ethidium bromide staining of the nucleic acid may be employed. Polyacrylamide gel electrophoresis (PAGE) may also be used. Nucleic acids of different sizes may be resolved, allowing specific amplification products to be distinguished from non-specific amplification products of different size.
  • a labelled oligonucleotide capable of hybridising to the specific amplified DNA may be used as a probe.
  • the probe may be labelled with a radionuclide such as 32 P, 33 P and 35 S using standard techniques, or may be labelled with a fluorescent dye.
  • the oligonucleotide probe is fluorescently labelled, the amplified DNA product may be detected in solution (see for example Balaguer et al (1991) "Quantification of DNA sequences obtained by polymerase chain reaction using a bioluminescence adsorbent" Anal. Biochem. 195, 105-110 and Dilesare et al (1993) "A high-sensitivity electrochemiluminescence-based detection system for automated PCR product quantitation" BioTechniques 15, 152-157.
  • PCR products can also be detected using a probe which may have a fluorophore-quencher pair or may be attached to a solid support or may have a biotin tag or they may be detected using a combination of a capture probe and a detector probe.
  • Fluorophore-quencher pairs may be used, particularly for quantitative measurements of PCR reactions (eg reverse transcription PCR (RT-PCR)). Fluorescence polarisation using a suitable probe may also be used to detect PCR products.
  • Amplification refractory mutation system (ARMS) PCR is a useful and routinely used method for single nucleotide polymorphism (SNP) typing (see, for example, EP 0 332,435 and W097/42345 and references therein, incorporated herein by reference).
  • SNP single nucleotide polymorphism
  • PCR-SSP sequence-specific primer
  • the presence or absence of product determines the allele assignment.
  • By careful primer design nearly all point mutations can be amplified, the specificity being determined by the nucleotides placed at the 3' te ⁇ ninal end. This is well known to those skilled in the art.
  • the fluorescence of the dye is enhanced or otherwise altered when bound to the double- stranded nucleic acid, allowing detection and quantification of double- stranded DNA (Glazer et al (1992) Nature 359, 859-861).
  • the dyes may also bind to single-stranded nucleic acid, but may bind less strongly to single-stranded than double-stranded nucleic acid and/or may fluoresce less strongly when bound to single-stranded than double-stranded nucleic acid.
  • US 5,545,528, for example describes the detection of PCR products in solution using intercalating fluorescent dyes.
  • Steurwald et al (1999) Mol Hum Reprod 5(11), 1034-1039 and Hiratsuka et al (1999) Mol Genet Metab 68(3), 357-362 also describe fluorescence-monitored PCR using SYBR Green dye.
  • SYBR Green is used in machines such as the Light CyclerTM, where continual fluorescence monitoring shows real time PCR and also the extent to which the so-called primer dimer formation contributes to the non-specific fluorescence.
  • the fluorescent dye binds not only to the specific amplifcation product but to non-specific products and also to reactants of the polymerase chain reaction, such as excess oligonucleotides.
  • the dyes cannot efficiently discriminate between, for example, the primer-dimers and the specific PCR product as both these entities are double-stranded.
  • the amount of specific amplification product therefore cannot accurately be dete ⁇ nined. This may mean that a false "positive" reading is obtained in a typing assay, due to detection of nonspecific amplification products, when the relevant template is not present and therefore there is no specific product present.
  • the non-specific amplification products may be more abundant in "negative" post-amplification samples than in "positive" post-amplification samples. There is therefore a need for a non-gel based detection system that can distinguish between the specific amplification product and the contaminating smaller, non-specific and template-independent products.
  • Bohling et al (1999) Lab Invest 79(3), 337-345 describes a method for detecting the bcl-l/JH translocation in mantle cell lymphoma in which SYBR Green and fluorescence melting curve analysis is used. Such analysis may be carried out, for example, using a machine such as the DASHTM system from Hybaid. The fluorescence from a double-strand nucleic acid-specific dye is measured along a temperature gradient, allowing the TmS of the double-stranded nucleic acids in the sample to be determined. This may allow the fluorescence arising from the specific amplification product to be determined.
  • Such methods have the disadvantage, particularly in relation to high-throughput use, that specialised and expensive equipment may be required. The requirement, for example, for measurements to be taken with sample temperamres in excess of 80°C may place significant constraints on the equipment that may be used.
  • WO94/24310 describes a method using an intercalating fluorescent dye in which a single-strand-specific endonuclease is used to degrade single stranded nucleic acid molecules and nucleoside phosphates.
  • Okamoto et al (1994) Anal Biochem 221(2), 340-347 describes a method of detecting amplified human leukocyte antigen (HLA) alleles using an intercalating fluorescent dye, in which lambda exonuclease and exonuclease I are added to digest template DNA, partial primer dimers and primers in order to reduce the background fluorescence ie fluorescence not resulting from binding of the dye to the specific amplification product.
  • HLA human leukocyte antigen
  • the present invention provides methods that have these advantages, in which small molecules are added to the post-amplification reaction before determining the presence/absence or quantity of double-stranded nucleic acid present.
  • the problem of background fluorescence caused by short but abundant non-specific amplification products is thereby addressed.
  • Identification of the amplicon (specific amplification product) by a single fluorescence reading step, which may readily be automated, is achieved.
  • the methods may be particularly suitable for use with ARMS-PCR methods for SNP typing, for example in HLA typing.
  • the method may be useful for identifying numerous sequence polymorphisms, such as those within the DR locus of HLA Class II.
  • a first aspect of the invention provides a method for detecting and/or quantifying a specific double-stranded nucleic acid amplification product in a nucleic acid amplification reaction post-amplification sample, comprising the steps of
  • step (1) adding to the post-amplification sample an amount of a small molecule capable of destabilising double-stranded nucleic acid such that under the assay conditions of step (2) any specific product in the post-amplification sample is substantially double stranded and the non-specific product is substantially single-stranded;
  • step (2) assaying the post-amplification sample treated according to step (1) in order to detect and/or quantify any double-stranded nucleic acid present.
  • the double/single stranded nature of the nucleic acid may be determined by methods known to those skilled in the art. For example, the ratio of absorbance at 260nm to absorbance at 280nm (A26o/A28o ratio) may be deteiTnined using methods well known to those skilled in the art. Gel electrophoresis may be used. A fluorescence vs temperature relationship (curve) may be determined, as indicated above, in order to determine that the short products are single-stranded at the temperature selected for the assay of step (2), for example room temperature, as discussed below. It is preferred that a fluorescence vs temperature relationship is used to determine the double/single stranded nature of the nucleic acid.
  • the small molecule may be added to the post- amplification sample in any suitable way; by transfer of an aliquot of the small molecule into a container (for example a well of a microtitre plate) holding the post-amplification sample or by transfer of an aliquot of the post-amplification sample to a container (for example a well of a microtitre plate) holding an aliquot of the small molecule.
  • a second aspect of the invention provides a method for detecting and/or quantifying a specific double-stranded nucleic acid amplification product in a nucleic acid amplification reaction post-amplification sample, comprising the steps of
  • step (2) assaying the post-amplification sample treated according to step (1) in order to detect and/or quantify any double-stranded nucleic acid present.
  • small molecule we include any compounds which have a molecular weight of less than 5000, preferably less than 2000 and more preferably less than 1000, 500 or 100.
  • the high pH buffer molecule CABS (discussed below) has a molecular weight of 235.3.
  • the small molecule does not have enzymic activity, for example exo or endonuclease activity. It will be appreciated that the term “small molecule” does not encompass single-strand-specific endonuclease, lambda exonuclease or exonuclease I.
  • step 1 reduces the amount of double-stranded nucleic acid that is detected in post-amplification samples in which the template for the specific amplification is not present (and therefore the specific amplification product is not present; negative control samples). It is further preferred that the addition of the small molecule does not reduce the amount of double-stranded nucleic acid that is detected in post-amplification samples in which the template for the specific amplification is known to be present (and therefore the specific amplification product is present; positive control samples).
  • a negative control sample may be a post- amplification sample in which a pseudo-template is present ie a nucleic acid that is not a template for the specific amplification reaction.
  • the pseudo-template does not act as a template for amplification of the specific amplification reaction product.
  • the pseudo-template nucleic acid does not comprise nucleic acid sequences complementary to the primer sequences in the orientation and spacing required for amplification of the specific amplification product.
  • the template nucleic acid comprises nucleic acid sequences complementary to the primer sequences in the required orientation (and spacing) for a specific amplification product to be formed in the amplification reaction.
  • the double-stranded nucleic acid is detected and/or quantified using a fluorescent dye.
  • the fluorescent dye binds preferentially to double-stranded nucleic acid, preferably double-stranded DNA, when compared with single-stranded nucleic acid, particularly single-stranded DNA, and/or fluoresces more strongly when bound to double-stranded than to single stranded nucleic acid, for example DNA.
  • a fluorescent intercalating dye it may be classified as an intercalating agent by the Ames test for mutagenicity, or may bind to double-stranded nucleic acid in some other way, for example by binding to the minor groove of the nucleic acid.
  • the sample that is assayed in step (2) comprises a fluorescent dye having the binding properties noted above. Desirable properties of the fluorescent dye are discussed further below.
  • the dye may be added at any stage prior to assay of the double-stranded nucleic acid. For example, it may be provided in the sample before or during the amplification reaction. Preferably it is added to the post-amplification sample, for example at the same time as addition of the small molecule, as required by step (1). Still more preferably, the dye and the small molecule are added to the sample as a single reagent, preferably a solution or suspension, still more preferably an aqueous solution or suspension.
  • Other acceptable solvents will be known to those skilled in the art and may include DMSO (dimethylsulphoxide).
  • step (1) decreases the ratio of (i) fluorescence measurable under the assay conditions of step (2) in a post-amplification sample in which the template for the specific amplification is not present (and therefore the specific amplification product is not present; negative control sample) to (ii) fluorescence measurable under the assay conditions of step (2) in a post- amplification sample in which the template for the specific amplification is known to be present (and therefore the specific amplification product is present; positive control sample).
  • the ratio of background (negative control) fluorescence to positive (positive control) fluorescence is increased.
  • the positive fluorescence may be about twice the background fluorescence, as discussed in Example 1.
  • Addition of a small molecule, as required by step (1) of the method of the invention may increase the ratio of positive to background fluorescence to at least about 2.5: 1, 3: 1, 4: 1, 5:1 , 6:1, 7: 1, 8: 1, 9:1, 10: 1, 12: 1, 15:1 or 20: 1; preferably the ratio of positive to background fluorescence is at least 4:1, still more preferably at least 5: 1 to 8:1.
  • step (1) of the method of the invention increases the ratio of positive to background fluorescence by a factor of at least about 1.5, 2, 2.5, 3, 4, 5, 6, 8 or 10 over the ratio in the absence of the small molecule.
  • addition of the small molecule may increase the ratio of positive to background fluorescence by a factor of 2, from 2:1 to 4: 1.
  • the ratio of positive to background fluorescence indicated above may be measured at 25°C.
  • the ratio may be improved further by measuring the fluorescence with the sample at a higher temperature, for example 42°C. This may reduce the positive signal (for example by about 10% at 42°C), but also decrease the background signal by a greater proportion (for example about 50% at 42°C), thereby improving the signal: noise (positive background) ratio.
  • the small molecule reduces the measurable background fluorescence (negative control) and increases or does not reduce the fluorescence from the specific product (positive control).
  • a further aspect of the invention provides a method for improving background to positive ratio in a method of detecting a specific double- stranded nucleic acid amplification product in a nucleic acid amplification reaction post-amplification sample wherein a small molecule capable of destabilising DNA and/or increasing the pH of the sample is added to the post-amplification sample prior to the detection of double-stranded nucleic acid in the sample.
  • a high pH may preferentially destabilise smaller/imperfect double-stranded nucleic acid molecules, for example non-specific amplification products, when compared with the specific amplification product(s).
  • the pH is too high, the specific amplification product(s) are also destabilised and fluorescence is lost.
  • a still further aspect of the invention provides a method for detecting and/or quantifying a specific double-stranded nucleic acid amplification product in a nucleic acid amplification reaction post-amplification sample, comprising the steps of
  • step (2) assaying the post-amplification sample treated according to step (1) in order to detect and/or quantify any double-stranded nucleic acid present, wherein the double-stranded nucleic acid is detected and/or quantified by measuring the fluorescence from a fluorescent dye in the sample, and wherein the amount of said small molecule added in step (1) is sufficient to increase the ratio of fluorescence measurable in a post-amplification sample in which the specific double-stranded nucleic acid amplification product is present (positive control) to fluorescence measurable in a post- amplification sample in which the specific double-stranded nucleic acid amplification product is absent (background; negative control) by a factor of at least 1.5, 2, 2.5, 3, 4, 5, 6, 8 or 10.
  • the ratio of positive to background fluorescence in the presence of the small molecule is at least 4:1, still more preferably at least 5 : 1 to 8: 1.
  • primers with different sequences may differ in the efficiency with which they amplify the relevant desired specific problem.
  • some primer pairs may produce a weak (ie low-abundance) specific product and a strong (ie high-abundance) non-specific product, or vice versa.
  • the extent of interaction between primers may vary, for example, for different primer pairs.
  • a weak interaction between the primers may lead to a low abundance of non-specific product (primer dimers) whereas a stronger interaction between the primers may lead to a higher abundance of non-specific product.
  • the ratio of background (reflecting the abundance of non-specific product) to positive (reflecting in addition the abundance of specific product) fluorescence depends upon the amplification reaction being performed, ie the primers used, as well as other factors, as known to those skilled in the art, for example the concentration of Mg 2+ ions.
  • Use of the method of the invention may be particularly beneficial in distinguishing "positive” from “negative” results for amplification reactions which yield a low abundance of specific amplification product relative to the amount of non-specific product, ie in which the background fluorescence is high when compared with the positive fluorescence.
  • a higher ratio of "positive” to background fluorescence may be achieved at less cost using methods of the invention than using, for example, fluorescent labelled probes as described in Gelsthorpe et al (1999), cited above.
  • a small molecule capable of destabilising double-stranded nucleic acid is included a small molecule that reduces the melting temperature of double stranded DNA in the post-amplification sample. It will be appreciated that such a molecule may reduce the melting temperature of double stranded DNA such that, at a temperature (for example room temperature, as discussed further below) at which, in the absence of the small molecule, both short non-specific amplification products and longer specific amplification products are double-stranded, the non-specific amplification products are single-stranded and the specific amplification products are double-stranded in the presence of the small molecule. In other words, the melting temperature of the short non-specific amplification products is lowered to below the assay temperature of step (2) whereas the melting temperature of the longer specific amplification products remains above the assay temperature of step (2).
  • a temperature for example room temperature, as discussed further below
  • Examples of such molecules may include formamide, DMSO (dimethylsulphoxide; available from Sigma, for example Cat No D5879), TMAC (trimethylammonium chloride; available from Sigma Cat No T3411).
  • More preferred examples of such molecules are molecules capable of reducing or increasing the pH of the sample, for example a strong base (for example sodium hydroxide) or a high pH buffer.
  • a preferred high pH buffer is CABS buffer (4-[Cyclohexylarnino]-l-butanesulfonic acid; available from Sigma, Cat. No. C-5580).
  • a further preferred high pH buffer is CAPS (2-[cyclohexylamino]-l-butanesulfonic acid). Adding a high pH buffer to the sample may allow more accurate control of the pH in the sample than adding a strong base (for example sodium hydroxide) and may therefore improve reproducibility.
  • CAPS buffer at pH 12.5, 12.7, 13 or 13.5 and concentration of 200 or 250 mM is used.
  • 250mM CAPS at pH 13 is used, to give a final concentration of 125 mM in the reaction mixture, at pH 11.82.
  • Molecules capable of reducing the pH include a strong acid or a low pH buffer. Such molecules are less preferred, because an acidic buffer may increase the background reading.
  • suitable buffers may include MES, BIS-TRIS, ADA or other low pH buffers supplied by Sigma, Calbiochem and/or Fluka.
  • Sodium acetate buffer, for example pH 4.6 may be used, as described in Example 1.
  • a buffer or other small molecule capable of producing a pH of about 3 to 6, preferably about 3 to 5 or 3 to 4 in the post-amplification sample is preferred.
  • the small molecule is added to the sample in the form of a suspension or a solution, preferably an aqueous suspension or solution.
  • TMAC TMAC
  • formamide or DMSO may be used individually or in combination, or in further combination with a high or low pH buffer, acid or alkali.
  • a combination of a molecule which is capable of increasing the pH of the post-amplification reaction with a molecule which is capable of decreasing the pH of the post-amplification buffer is not preferred.
  • a molecule or molecules which are capable of increasing the pH of the post-amplification reaction is used.
  • the small molecule added to the post- amplification sample in step (1) of a method of the invention is a solution of a high pH buffer molecule(s) with a pH of between 11 and 14, preferably between 12 and 13, for example a pH of 12.7 or 13.0.
  • the pH (of sample or buffer solution) is measured at between 18 and 25°C, preferably at 20°C. It may be measured by any suitable means, as well known to those skilled in the art, for example using a pH meter, indicator solution or indicator paper. It is preferred that a pH meter is used. It will be appreciated that the pH of the post-amplification reaction sample need not routinely be measured once conditions have been determined under which the required alkalinity of the sample type is achieved, as discussed further below. It will further be appreciated that the pH of a post-amplification reaction sample after addition of the small molecule may be predicted on the basis of the constituents of the reaction sample (particularly any pH buffering agents) and on the concentration and identity of any buffering agents in a high pH buffer, as well known to those skilled in the art.
  • the PCR buffer is pH 8.6
  • the CABS/SYBR Green reagent is pH 12.7
  • the pH of the final mixture after step (1) is 11.7. This is the pH at which the fluorescence is read in step (2).
  • the optimum amount of destabilising agent or optimum pH of the post-amplification sample after addition of the small molecule may depend on the amplification reaction being performed and the conditions (for example buffer composition) under which it is performed. It may also depend on the temperature at which the assay step
  • the assay step (2) is performed at room temperature, for example between 10 and 25°C, for example at 18 to 22°C, on grounds of convenience and ease of high-throughput operation.
  • room temperature for example between 10 and 25°C, for example at 18 to 22°C, on grounds of convenience and ease of high-throughput operation.
  • other temperatures may be used, for example about 30 to 40°C, for example about 37°C, or about 40 to 45°C.
  • Carrying out the assay step at a slightly elevated temperature may enhance melting of the primer dimers.
  • a particularly preferred temperature may be about 42°C, particularly when using small reaction volumes, for example lO ⁇ l or less, for example 3 ⁇ l.
  • PCR reactions typically contain a buffer, commonly Tris-HCl, at a specific pH. This must be taken into account when dete ⁇ nining how much alkaline denaturant to add.
  • the length of the products also plays a role, with longer products being more resistant to denatoration.
  • known features of the amplification reaction may be used in predicting the optimum types or amounts of destabilising agent and/or optimum pH and how to achieve the desired pH.
  • the specific amplification product is between about 50 and 2000 base pairs (bp) in length, or, in order of preference, between about 60 and 1500 bp, 70 and 1200, 80 and 1000, 90 and 800, 100 and 700, 150 and 600, 200 and 500 bp.
  • bp base pairs
  • a specific amplification product length of between about 150 and lOOObp may be preferred.
  • primer lengths are less than about 50 nucleotides, preferably between about 40, 30, 25, 20 and 15 nucleotides. It is particularly preferred that the primers are between 20 and 25 nucleotides in length.
  • the template (or pseudo-template nucleic acid) nucleic acid may be any nucleic acid, for example genomic or recombinant DNA, or RNA. It may be derived from any source.
  • genomic DNA derived from a patient whose genotype is to be investigated, for example as indicated in Example 1.
  • it may be nucleic acid derived from a pathogen, for example a virus, bacterium or fungus.
  • pathogen for example a virus, bacterium or fungus.
  • the techniques of the invention may be useful, for example in HLA matching/typing, forensics, epidemiology and screening for genetic defects.
  • a post- PCR amplification sample may comprise an aqueous buffer solution compatible with performing PCR, for example as indicated in Example 1 or in Gelsthorpe et al (1999), cited above. It may further comprise a thermostable polymerase, for example Taq polymerase (which may no longer be enzymatically active), residual dNTPs and oligonucleotide primers, template nucleic acid and possibly specific and non-specific amplification products (nucleic acid molecules of various lengths). It is preferred that the small molecule is added immediately before carrying out the assay step.
  • a thermostable polymerase for example Taq polymerase (which may no longer be enzymatically active)
  • residual dNTPs and oligonucleotide primers template nucleic acid and possibly specific and non-specific amplification products (nucleic acid molecules of various lengths). It is preferred that the small molecule is added immediately before carrying out the assay step.
  • this is not essential, ie there may be a delay, for example of between 1 and 24 hours between adding the small molecule and performing the assay step.
  • a shorter delay may be appropriate.
  • SYBR Green particularly with a high pH buffer, for example CABS or CAPS
  • the delay no more than about 3 hours. It is particularly preferred that the fluorescence reading (assay step) is performed without delay once the CABS (or other high pH buffer) is added to the post-amplification reaction sample.
  • high-pH buffer (which may also comprise the dye) may be automated, for example by using a reader such as the BMG Fluostar (Aylesbury, UK) which has an onboard injector so that the addition, mixing and reading can be done with a single machine, within a few minutes for a 96- well plate.
  • a reader such as the BMG Fluostar (Aylesbury, UK) which has an onboard injector so that the addition, mixing and reading can be done with a single machine, within a few minutes for a 96- well plate.
  • a further aspect of the invention provides a method for preparing a post-amplification sample for use in a method of detecting and/or quantifying a specific double-stranded nucleic acid amplification product in a nucleic acid amplification reaction post-amplification sample, comprising step (1) of a method of the invention for detecting and/or quantifying a specific double-stranded nucleic acid amplification product, as indicated above.
  • Suitable fluorescent dyes will be well known to those skilled in the art, and may include SYBR Green, thiazole yellow dimers, oxazole yellow homodimers, for example Yoyo-1 (see, for example US 5,545,528), Hoechst 33258 dye and ethidium bromide or ethidium homodimer (EthD). We have found that the dye SYBR Green is particularly suitable.
  • SYBR Green (catalogue number 7563 or S7567) and other dyes that may be suitable, for example the dye termed PicoGreenTM (catalogue number P7581) may be obtained from Molecular Probes, Inc, (PO Box 22010, Eugene OR97402-0469, 4849 Pitchford Avenue, Eugene OR97402-9165 or Molecular Probes Europe BV, PoortGebouw, Rijnsburgerweg 10, 2333 AA Leiden, Netherlands). PicoGreen may give slightly better results than SYBR Green, but is more expensive.
  • Suitable dyes are described, for example, in US 5,545,528, incorporated herein by reference. As noted in US 5,545,528, dyes with excitation/emission wavelength characteristics that are compatible with performing and analysing the amplification reaction in the same (heat- resistant) microtitre plate may be preferred, particularly for high- throughput operation. Dyes with excitation and emission wavelengths between 400 and 700 nm are preferred. Dyes with a double-stranded DNA partition coefficient of at least lxlO 7 (ie lxlO 7 times more dye bound to the DNA sample than in the surrounding 10% ethanol/ water solution) may be preferred, as such a dye may bind efficiently with the double-stranded nucleic acid.
  • Dyes with a fluorescent response that is linear over a wide concentration range are preferred.
  • the fluorescence signal from binding of the PicoGreenTM reagent to double- stranded DNA may be linear over at least four orders of magnitude, whereas assays using either ethidium bromide, Hoechst 33258 or YOYO- 1 may have a much more limited linear range (Molecular Probes, Inc product information).
  • Suitable dyes may also be described in WO94/24310 and references therein, incorporated herein by reference.
  • the selected dye binds less strongly to single-stranded nucleic acid than to double-stranded nucleic acid and/or exhibits a lower intensity of fluorescence when bound to single-stranded nucleic acid than when bound to double-stranded nucleic acid. It is preferred that the such binding/fluorescence selectivity characteristics of the selected dye are at least as advantageous as those of SYBR Green.
  • the selectivity of SYBR Green for double versus single-stranded DNA may be approximately 15, and for double-stranded DNA versus oligonucleotides may be approximately 50.
  • the amplification reaction and fluorescence detection may be carried out by known techniques in combination with the present invention.
  • the reaction addition of the small molecule and detection of the double-stranded nucleic acid may be performed in solution, for example in a multiwell microtitre plate, for example a 96- well microtitre plate. Suitable heat-resistant microtitre plates, compatible fluorescent dyes and plate-based techniques are described, for example, in US 5,545,528.
  • a solid phase technique may be used.
  • the allele-specific primers may be immobilised on a solid support, for example in a microarray, as known to those skilled in the art.
  • the amplification reaction is carried out across the array using a reaction mixture containing the target DNA and a constant oligonucleotide primer. Suitable solid phase PCR techniques are described, for example, in W096/26291, incorporated herein by reference.
  • the technique is used in high throughput, preferably automated, analysis procedures.
  • procedures in which manipulations of the samples are minimised are preferred.
  • the number of separate reagents added to the post-amplification reaction sample is minimised and/or that the time between starting the amplification reaction and obtaining the final data is minimised.
  • a reagent comprising the small molecule for example high pH buffer, and the fluorescent dye is used.
  • adding the reagent, mixing, and reading can, for example, be performed for a 96- well microtitre plate in about 1 minute.
  • the technique may be performed using 3 ⁇ l PCR reactions performed in a 384-well plate.
  • the technique may be used when accurate quantitation of the amplification products is required. However, it will be appreciated that in some applications, for example SNP typing, a presence/absence (ie positive/negative) determination may be sufficient.
  • the technique may also be used in combination with gel analysis, for example to reduce fluorescence arising from non-specific products so as to improve gel clarity and contrast.
  • Techniques for gel staining using, for example, SYBR Green are well known to those skilled in the art and are described, for example in Molecular Probes, Inc SYBR Green product literature. Such methods may be adapted using the teaching of the present invention.
  • a further aspect of the invention provides a post-amplification reaction sample obtainable by a method of the invention.
  • a further aspect of the invention provides a post-amplification reaction sample comprising a small molecule capable of destabilising double- stranded nucleic acid, and/or having a pH of greater than about 11, preferably greater than about 12 or 13, for example having a pH of about 12.7 or 13, or (less preferably) having a pH of less than about 3, 4 or 5.
  • the post-amplification reaction sample may further comprise a fluorescent dye, as described above, for example SYBR Green or PicoGreen.
  • a further aspect of the invention provides a reagent comprising a fluorescent dye, for example SYBR Green or PicoGreen, wherein the reagent has a pH of greater than about 11 , preferably greater than about 12 or 13, for example having a pH of about 12.7 or 13, or (less preferably) a pH of less than about 3, 4 or 5 and/or (less preferably) comprises a small molecule capable of destabilising double-stranded nucleic acid, for example formamide, DMSO or TMAC. Examples of such reagents are given in Example 1.
  • the reagent comprises a fluorescent dye, as described above, preferably SYBR Green and a high pH buffer molucule, for example a high pH buffer as indicated above, preferably CABS or CAPS buffer.
  • a further aspect of the invention provides a kit of parts comprising (1) a fluorescent dye, as described above, for example SYBR Green and (2) a solution having a pH of greater than about 11, preferably greater than about 12 or 13, for example having a pH of about 12.7 or 13, or (less preferably) a solution having a pH of at least about 3 , 4 or 5 or reagents for preparing such a solution, or (less preferably) other small molecule capable of destabilising double-stranded nucleic acid, for example formamide.
  • Preferences in relation to the kit of parts include the preferences indicated above in relation to the reagent of the invention.
  • the kit may further comprise reagents useful in carrying out an amplification reaction, for example a PCR amplification reaction.
  • the kit may further comprise one or more of the following reagents suitable for use in a PCR amplification reaction: thermostable DNA polymerase (for example Amplitaq Gold from Perkin-Elmer); oligonucleotide primers; dNTPs; PCR buffer (for example as indicated in Example 1); magnesium chloride; positive control template; positive control double-stranded nucleic acid (for example samples of double- stranded nucleic acids of different lengths, for example about 250bp (similar to a specific amplification product) and about 80bp (similar to a non-specific amplification product).
  • thermostable DNA polymerase for example Amplitaq Gold from Perkin-Elmer
  • oligonucleotide primers for example as indicated in Example 1
  • magnesium chloride for example as indicated in Example 1
  • positive control template for example samples of double- stranded nucleic acids of different lengths, for example about 250bp (similar to a specific amplification product) and about 80bp
  • Figure 1 (a) PCR product measured by alkaline SYBR Green fluorescence using sodium hydroxide.
  • D7, D10 and F10 refer to primer pairs.
  • +/- refer to presence of either template that will result in a specific amplicon (ie specific amplification product: +) or presence of template that will not result in a specific amplicon (ie absence of a template that will result in a specific amplicon: -).
  • Figure 2 Effect of 0.2M CABS pH12.7 on the background fluorescence using different primer concentrations.
  • +/- refer to presence of either template that will result in a specific amplicon (ie specific amplification product: +) or presence of template that will not result in a specific amplicon (ie absence of a template that will result in a specific amplicon: -).
  • Figure 3 Effect of annealing temperature during the amplification reaction on fluorescence measured in the post-amplification reaction sample.
  • Figure 4 Effect of template concentration on fluorescence readings and on product analysed by gel electrophoresis with ethidium bromide staining.
  • FIGS. 5 to 7 Class II typing reactions analysed by alkaline SYBR- Green fluorescence. 32 different primer pairs are tested on three different DNA samples.
  • Figure 8 Fluorescence measurements in the presence of different volumes of 0.1N NaOH/SYBR Green reagent (see Example 1). (+) indicates presence of the template for the specific amplicon. (-) indicates the absence of the specific template.
  • Figure 10 Plate layout and primer combinations used in the experiments of Example 1.
  • the system of the present Example relies on the fluorescent properties of the double-strand specific dye SYBR Green, used under buffered alkaline conditions.
  • Allele-specific PCR may be performed by any method.
  • the following protocol is a non-limiting example. PCR reactions were performed on genomic DNA samples extracted from lymphoblastoid cell lines, using primers chosen to produce either positive specific amplifications (ie where the template for the specific amplification reaction is present) or negative non-specific amplifications (ie where the template for the specific amplification reaction is not present).
  • Genomic DNA may be also be prepared from whole blood, for example by a "salting out" method or using a commercial kit intended for preparation of genomic DNA.
  • the optimal annealing temperatures of all the primers were checked using a HybaidTM gradient blockk. A range of annealing temperatures from 58°C to 68°C was chosen for the test primers.
  • the resultant PCR products were checked by gel electrophoresis and by the fluorescence produced in the presence of the SYBR Green CABS pH 12.7 reagent. The results correlated; fainter bands on the gel corresponded with lower fluorescence readings, suggesting that the fluorescence in the presence of CABS pH 12.7 buffer is quantitative.
  • a panel of 32 sequence specific primer mixes was used to amplify 50 DNA samples.
  • the primers were placed on the plate in triplicate and each plate was used to amplify a single DNA sample. Methods used are described in more detail below.
  • a typical PCR reaction composition may be:
  • Template DNA for example 50ng
  • ARMS primers l ⁇ M each in a total volume of 10 ⁇ l.
  • a PCR reaction mixture containing appropriate primers for the samples to be amplified is prepared.
  • the recipes indicated above are for one reaction only and need to be adjusted for multiple samples.
  • the final volume of each sample reaction is 15 ⁇ l.
  • the PCR reaction may be performed in an Advanced Biotechnologies skirted 96 well plate, or similar. For example, thermal cycling may be performed in an MJ Research TetradTM or Hybaid PCR ExpressTM machine using 96 well ThermofastTM (Advanced Biotechnologies) plate. Individual wells may be sealed with a layer of mineral oil.
  • the presence of the PCR product is screened for by adding 15 ⁇ l of SYBR Green/CABS pH 12.7 reaction mixture.
  • the fluorescence of the sample is read, for example on a BMG FluorostarTM or similar. Measurements are taken at an excitation wavelength of 485 nm and emission wavelengths of 538 nm and optionally 590 nm.
  • At least 10 ⁇ l of the following solution may be added with mixing to a lO ⁇ l PCR reaction: NaOH, 0.08N SYBT Green, 1:5000 dilution of stock solution (Molecular Probes)
  • This solution is made by adding 4 volumes of 0.1 N NaOH to 1 volume of a 1 : 1000 dilution of SYBR Green.
  • Some amplification reactions require the addition of more or less of this reagent, depending on the length of the amplification products and their GC content.
  • the addition of 30% more of the above reagent is necessary to achieve the same ratios of positive .-negative fluorescence.
  • the fluorescence in each well may be read using a BMG FluoStar (BMG Lab Technologies) with an excitation filter set at 485 nm and emission filter at 538 nm.
  • the Labsystems Fluoroskan Ascent (Labsystems) system may alternatively be used. All measurements are carried out in the PCR plate after addition of the SYBR Green/NaOH reagent or SYBR Green/CABS (or other high pH buffer) reagent (SYBR Green/high pH reagent solution).
  • the SYBR Green/high pH reagent solution mix and read in a single machine. Addition of the SYBR Green/high pH reagent solution takes approximately 20 seconds, the plate is shaken at 1200 RPM for 10 seconds to mix the solutions, and reading is accomplished in another 30 seconds. The fluorescence values are immediately available on the computer for anlaysis. Alternatively, an eight-channel multipipette may be used to add the SYBR Green/high pH reagent solution.
  • SYBR Green/high pH reagent Other reagent solutions may be used in place of the SYBR Green/high pH reagent, with similar results.
  • Thiazole yellow or ethidium bromide may be used in place of SYBR Green.
  • a solution containing formamide, DMSO or TCMA may be used in place of (or in combination with) the high pH solution.
  • a low pH solution may be used.
  • a buffer of pH 4.6 may be used.
  • the following buffer gave background readings of about 12000 and positive readings of about 40000: 30mM sodium acetate, pH4.6, 280mM NaCl,lmM ZnS04.
  • ATDI alkaline-mediated differential interaction

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Abstract

La présente invention concerne des procédés qui permettent d'établir une distinction entre des produits d'amplification spécifique et des produits d'amplification non spécifique et qui peuvent être plus rapides, plus économiques et/ou plus pratiques à mettre en oeuvre que les procédés existants, notamment par rapport à des applications à haut débit. De petites molécules, telles que des tampons à pH élevé, sont ajoutées à la réaction de post-amplification, avant de déterminer la présence/absence ou la quantité d'acide nucléique à double brin présent. Cette invention aborde le problème de fluorescence de fond, causée par des produits d'amplification non spécifique petits mais abondants et par un excès de réactifs de la réaction d'amplification. L'identification de l'amplicon (produit d'amplification spécifique) est réalisée dans une étape unique de lecture de fluorescence, qui peut facilement être automatisée. Ces procédés conviennent particulièrement à un usage avec des procédés ARMS-PCR, pour un typage SNP, notamment un typage HLA. Le procédé peut être utilisé pour identifier de nombreux polymorphismes de séquence, tels que ceux se trouvant dans le locus DR d'HLA de classe II. Les petites molécules peuvent augmenter le pH dans l'échantillon jusqu'à au moins 11 ou à moins d'environ 5 et/ou peuvent déstabiliser l'ADN à double brin.
PCT/GB2001/001405 2000-04-01 2001-03-29 Procedes WO2001075155A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004061130A1 (fr) * 2002-12-27 2004-07-22 Toyo Boseki Kabushiki Kaisha Methode d'identification d'un polymorphisme nucleotidique
CN111979333A (zh) * 2019-05-23 2020-11-24 石河子大学 一种绵羊多胎主效基因FecB快速可视化检测方法

Citations (1)

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Publication number Priority date Publication date Assignee Title
EP0488243A1 (fr) * 1990-11-30 1992-06-03 Sanwa Kagaku Kenkyusho Co., Ltd. Procédé d'extraction du génome du virus d'un échantillon derivé d'un corps vivant infecté par le virus et procédé de détection du génome

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0488243A1 (fr) * 1990-11-30 1992-06-03 Sanwa Kagaku Kenkyusho Co., Ltd. Procédé d'extraction du génome du virus d'un échantillon derivé d'un corps vivant infecté par le virus et procédé de détection du génome

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Title
VITZTHUM F ET AL: "A QUANTITATIVE FLUORESCENCE-BASED MICROPLATE ASSAY FOR THE DETERMINATION OF DOUBLE-STRANDED DNA USING SYBR GREEN I AND A STANDARD ULTRAVIOLET TRANSILLUMINATOR GEL IMAGING SYSTEM" ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS, SAN DIEGO, CA, US, vol. 276, 1999, pages 59-46, XP001019646 ISSN: 0003-2697 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004061130A1 (fr) * 2002-12-27 2004-07-22 Toyo Boseki Kabushiki Kaisha Methode d'identification d'un polymorphisme nucleotidique
CN111979333A (zh) * 2019-05-23 2020-11-24 石河子大学 一种绵羊多胎主效基因FecB快速可视化检测方法
CN111979333B (zh) * 2019-05-23 2022-06-21 石河子大学 一种绵羊多胎主效基因FecB快速可视化检测方法

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