GB2360088A - Method and kit for determining PCR amplification reaction conditions - Google Patents

Abstract

A method and kit for determining the preferred amplification reaction conditions comprising forming an amplification reaction mixture in a plurality of wells in an apparatus in which said wells are heated individually by supplying electric current to an electrically conducting polymer that forms the wells. Said wells contain one or more reagents which might promote the amplification, other than nucleic acids, polymerase, at least one primer and nucleotides, performing the amplification reaction and determining in which well the most productive amplification reaction has occurred. Preferably different wells may have different temperature/time profiles for each reaction. More preferably a label, either fluorescent or intercalating, may be included to monitor progress of the reaction. Kits to perform the method are also disclosed.

Description

2360088 1 Reaction Methods and Kits The present invention relates to a

method for optimising an amplification reaction, as well as to kits for use in the optimisation.

The polymerase chain reaction or PCR is one of the known procedures for generating large quantities of a particular DNA sequence. It is based upon DNAts characteristics of base 10 pairing and precise copying of complementary DNA strands. Typical PCR involves a cycling process of three basic steps.

Denaturation: A mixture containing the PCR reagents (including the DNA to be copied, the individual nucleotide bases (A,T,G,C), suitable primers and polymerase enzyme) are heated to a predetermined temperature to separate the two strands of the target DNA.

Annealing: The mixture is then cooled to another predetermined temperature and the primers locate their complementary sequences on the DNA strands and bind to them.

Extension: The mixture is heated again to a further predetermined temperature. The polymerase enzyme (acting as a catalyst) joins the individual nucleotide bases to the end of the primer to form a new strand of DNA which is complementary to the sequence of the target DNA, the two strands being bound 25 together.

Each stage of the reaction is typically carried out at a particular temperature. Optimum temperatures and times for the various stages of the vary depending upon factors such as the nature and length of the sequence being amplified, the type of 30 polymerase enzyme involved etc.

In addition, these reactions are generally carried out in the presence of a buffer solution. Other additives such as a source of magnesium ions or other adjuvants such as bovine serum 2 albumin (BSA) may also be included in order to enhance the reaction process.

The precise reaction conditions which work best (providing successful, specific and optimum results) for each PCR reaction need to be determined on an individual basis if the amplification reaction is to be effected successfully. This is particularly important in the case of preparative PCR where the reaction is used to produce quantities of peptides or proteins. High quantities of high specificity product are essential in this case.

In order to determine these in each case, a range of reaction conditions is applied to test runs of the amplification. The conditions which provide the best yield of target product is then selected for future use. Kits containing a range of buffers, adjuvants or additives are available commerically. These maybe combined in a variety of permutations and combinations and various temperature conditions employed. This can be a time consuming and lengthy exercise. In particular because the block heaters used in conventional amplification instruments will apply the same heating profile in terms of the temperatures applied at each stage of the process and the times applied to all the various stages to all the wells in the heater. Therefore, in order to test the effect of the heating profile on the efficiency of the reaction, multiple runs need to be carried out.

WO 98/24548 describes reaction vessels and apparatus comprising an electrically conducting polymer which heats when an electric current is passed through it. Vessels which comprise the polymer as an integral part may provide particularly compact structures.

If several reaction vessels are required for a particular reaction, any electrical connection points can be positioned so that a single supply can be connected to all the reaction 3 vessels or tubes. The reaction vessels may be provided in an array.

Alternatively, each of or each group of reaction vessels may have its own heating profile set by adjusting the applied current to that vessel or group of vessels. This provides a further and particularly important advantage of reaction vessels with polymer over solid block heaters or turbulent air heaters, is that individual vessels can be controlled independently of one another with their own thermal profile. It means that a relatively small apparatus can be employed to carry out a plurality of PCR assays at the same time notwithstanding that each assay requires a different operating time and temperature profile. For example, PCR tests for detecting a plurality of organisms in a sample can be carried out simultaneously, notwithstanding that the nueleotide sequence which is characteristic of each organism is amplified at different PCR operating temperatures and timings.

Such techniques may also be applied in the fields of genetic profiling or diagnosis, where a range of primers to detect a range of genotypes or defects may be applied to samples in wells in apparatus and the different amplification reactions applied simultaneously. In such cases, each of the wells of the apparatus may contain nucleic acid from the same subject so that a full profile may be achieved without risking crosscontamination with other samples.

The applicants have found however that such apparatus and vessels of this type provide an ideal vehicle for optimising the reaction conditions at which particular amplification reactions proceed.

Thus according to the present invention, there is provided a method for determining preferred amplification reaction conditions, said method comprising 4 i) forming an amplification reaction mixture in each of a plurality of wells in apparatus in which said wells are heatable individually by supplying electric current to electrically conducting polymer which either forms or is in close proximity to said well; such that all filled wells contain: (a) a sample of nucleic acid which is the subject of the amplification reaction, (b) a nucleic acid polymerase, and (c) at least one primer capable of hybridising to said nucleic acid, (d) a set of nucleotides; and each well also contains (e) one or more other reagents which might assist in the amplification reaction, provided that different wells or different groups of wells contain different such reagents; ii) simultaneously conducting amplification reactions in each of said wells by supplying electric current to said electrically conducting polymer so as to cause the reagents in each well to pass through a plurality of amplification reaction cycles; and iii) determining in which well the most productive amplification reaction has been effected.

Where a group of wells each contains similar other reagents (e), the electrical current supplied to electrically conducting polymer of each of said wells within a group is suitably controlled to produce a different temperature/ time profile. In this way, not only the effect of variations in the reaction conditions in terms of reagents is obtained, but information regarding the optimum temperature and time profiles of reaction for those conditions may also be obtained.

Suitable other reagents (e) include solutes such as a source of magnesium ions (such as magnesium chloride), buffers (such as Tris buffer and those already found in commercially available optimisation kits), and/or adjuvants (such as formamide, bovineserum albumin (BSA) or single stranded binding proteins) Preferably, each well will be supplied with a source of magnesium ions, a buffer and an adjuvant. The precise nature 5 and combination of these may vary.

In particular, currently available optimisation kits supplied by Sigma comprise a set of 15 different buffers, and 5 different adjuvants. Any of these may be utilised in the invention of the present application. The relative amounts of said other reagents would suitably be in accordance with the recommendations of the current kit. For example, in general from 1-1OmM magnesium ions are added to reaction mixtures.

Generally speaking, it will not be necessary to carry out each and every possible combination of reagents in order to ascertain a set of conditions which work well. Specific combinations may be selected for testing based upon the factorial experimental design principles of the Taguchi method, for example as set out by B.D. Dobb et al., Nucl. Acid. Res. 1994, 22, 18, 3801-3805.

The determination carried out in step (iii) of the method of the invention may be effected in various ways. For example, on completion of a series of amplification cycles, for example from 30 to 50 cycles, products from each reaction may be removed from the wells and separated electrophoretically on a gel. on staining the product in a conventional way, amplification product will be visualised. The conditions which produce the highest yield of product will be apparent from the size and density of the staining, assuming that each well contained similar amounts of nucleic acid sample initially.

In a preferred embodiment however, label means are provided in each reaction mixture, which allows the progress of said amplification to be monitored in situ. Suitable label means are 6 those which produce a visible signal such as a fluorescent signal.

Examples of such label means are well known in the art. They include intercalating dyes, such as SYBRGold TM, SYBRGreen TM, or ethidium bromide, as well as fluorescently labelled probes, which allow for sequence specific detection, which eliminates any inaccuracies due to the presence of non-specific amplification product. In addition, systems are available, such 10 as the TAQMAN TM system, which allow for quantification of the amount of target nucleic acid present in the sample.

In the context of the invention, quantitative methods would provide a check to ensure that optimum reaction conditions were is selected irrespective of whether there were differences, even small ones, in the amount of sample nucleic acid, present in the well initially.

Generic methods utilise DNA intercalating dyes that exhibit increased fluorescence when bound to double stranded DNA species. Fluorescence increases due to a rise in the bulk concentration of DNA during amplifications can be used to measure reaction progress. The increase in fluorescence will vary depending on the efficiency of reaction conditions to effect the amplification. Generally speaking, the greater the fluorescent signal from a particular well in the apparatus used in the method of the invention, the more favoured the reaction conditions used in that well will be.

When generic DNA methods are used to monitor the rise in bulk concentration of nucleic acids amplification can be determined without any time penalty. A single fluorescent reading can be taken at the same point in every reaction. End point melting curve analysis can be used to discriminate artefacts from amplicon, and to discriminate amplicons. Peaks of products can 7 be seen at concentrations that cannot be visualised by agarose gel electrophoresis.

In order to obtain high resolution melting data, the melt experiment must be performed slowly on existing hardware taking up to five minutes. However, by continually monitoring fluorescence amplification, a 3D image of the hysteresis of melting and hybridisation can be produced. This 3D image is amplicon dependent and may provide enough information for product discrimination. Examples of how such techniques can be applied in practice are described for example by M.A. Lee et al, J. Appl. Microbiol. 1999, 87, 218-223.

It has been found that DNA melting curve analysis in general is a powerful tool in optimising PCR thermal cycling. By determining the melting temperatures of the amplicons, it is possible to lower the denaturing temperatures in later PCR cycles to this temperature. Optimisation for amplification from first generation reaction products rather than the genomic DNA, reduces artefact formation occurring in later cycles.

In accordance with the invention, this information may be generated side by side with information regarding the other reaction conditions which result in a good amplification reaction.

Strand specific methods utilise additional nucleic acid reaction components to monitor the progress of amplification reactions. These methods use fluorescence resonance transfer (FRET) as the basis of detection. one or more nucleic acid probes are labelled with fluorescent molecules, a reporter molecule and a quencher molecule. The reporter molecule is excited with a specific wavelength of light for which it will normally exhibit a fluorescence emission wavelength. The quencher molecule is also excited at this wavelength such that it can accept the emission energy of the reporter molecule by resonance transfer 8 when they are in close proximity (e. g. on the same, or a neighbouring molecule) The basis of FRET detection is to monitor the changes at reporter and quencher emission wavelengths. There are two types of FRET probes, those using hydrolysis of nucleic acid probes to separate reporter from quencher, and those using hybridisation to alter the spatial relationship of reporter and quencher molecules.

Hydrolysis probes are commercially available as TaqMan TM probes.

These consist of DNA oligonucleotides that are labelled with reporter and quencher molecules. The probes are designed to bind to a specific region on one strand of a PCR product. Following annealing of the PCR primer to this strand, Tag enzyme extends the DNA with 5' to 31 polymerase activity. Tag enzyme also exhibites 51 to 31 exonuclease activity. TaqMan TM probes are protected at the 3' end by phosphorylation to prevent them from priming Tag extension. If the TaqMan T1 probe is hybridised to the product strand than an extending Tag molecule may also hydrolyse the probe, liberating the reporter from quencher as the basis of detection.

Hybridisation probes are available in a number of guises. Molecular beacons are oligonucleotides that have complementary 5 and 3' sequences such that they form hairpin loops. Terminal fluorescent labels are in close proximity for FRET to occur when the hairpin structure is formed. Following hybridisation of molecular beacons to a complementary sequence the fluorescent labels are separated, so FRET does not occur, as the basis of detection. Pairs of labelled oligonucle ot ides may also be used. These hybridise in close proximity on a PCR product strand bringing reporter and quencher molecules together so that FRET can occur. Enhanced FRET is the basis of detection. Variants of this type include using a labelled amplification primer with a single adjacent probe.

An assay which utilises a combination of a single labelled probe and an intercalating dye is described in International 9 Patent Application No. PCT/GB98/03560.

The use of an intercalating dye and a probe which is singly labelled is advantageous in that these components are much more economical than other assays in which doubly labelled probes are required.

International Patent application No. PCT/GB99/00504 describes assay for detecting the presence of particular nucleic acid sequences which may be adapted to quantify the amount of the target sequence in the sample. In this assay, an amplification reaction is effected using a set of nucleotides, at least one of which is fluorescently labelled. Thus the amplification product has fluorescent label incorporated in it. The reaction is effected in the presence of a probe which can hybridise to the amplification product and which includes a reactive molecule which is able to absorb fluorescence from or donate fluorescent energy to said fluorescent labelled nucleotide. The reaction can then be monitored by measuring the fluorescence of said sample as this will alter during the course of the reaction as more product is formed which hybridises to the probe and gives rise to a FET or FRET interaction between them.

An advantage of the apparatus used in the method of the present invention over a conventional block heater is derived from the fact that polymers which conduct electricity are able to heat rapidly. The heating rate depends upon the precise nature of the polymer, the dimensions of polymer used and the amount of current applied. Preferably the polymer has a high resistivity for example in excess of 10000hm.cm-1. The temperature of the polymer can be readily controlled by controlling the amount of electric current passing through the polymer, allowing it to be held at a desired temperature for the desired amount of time.

Furthermore, the rate of transition between temperatures can be readily controlled after calibration, by delivering an appropriate electrical current, f or example under the control of a computer programme.

The invention further provides a kit for carrying out a method as described above. Suitably the kit comprises (a) a plurality of buffer compositions suitable for effecting amplification reactions; (b) one or more adjuvants suitable for effecting an 10 amplification reaction (c) a disposable apparatus comprising an electrically conducting polymer, a plurality of wells each of which is heatable by supplying electric current to polymer in the region of said well, and electrical connection means which allow each well or groups of wells to be heated individually.

In this way, the wells may be loaded, and then connected to an electrical supply by way of a suitable controller such as a computer controller.

If desired, components (a) and (b) above in dried, frozen or chilled liquid form are predosed into wells in the apparatus. In order to avoid contamination, the well may be covered for example with a removable plastics sheet.

Where label means are to be employed in order to monitor the amplification reactions, these may be included in the kit.

Thus the kit may further comprise an intercalating dye such as those described above.

The invention will now be particularly described by way of example with reference to the accompanying diagrammatic drawings in which:

11 Figure 1 is a schematic view of apparatus used in the method of the invention.

In the apparatus of Figure 1, a plurality of wells (1) are provided in a plate (2) comprising an electrically conducting polymer which has been injection moulded to contain the desired number of wells. Each well is provided with an electrode (3) which allows the content of the well to be heated using a predetermined thermal cycle by passing an electric current. The cycle within each well is controlled by electronic instrumentation (not shown).

The electronic instrumentation controls the temperature and the time at which each amplification reaction is held at the various phases of the amplification reaction.

Amplification primers, nucleotides, polyermase enzymes and the sample nucleic acid are added to each well preferably together with label means as described above and particularly an intercalating dye. In addition, each well or group of wells will contain one or more of a source of magnesium ions (such as MgC1), a buf f er and an adjuvant in various amounts.

Current is then applied to each reaction vessel in a controlled manner such that it proceeds through thermal cycling to effect amplification with various hold temperatures or times and transition rates. Luminescence from each well is monitored to detect the progress of the amplification reaction and to determine whether the amplification has proceeded well.

In it simplest form, preferred reaction conditions will be those present in a well giving the highest level of luminesence specific to the desired product. If necessary the process can be repeated until conditions giving good amplification levels are determined.

12 Thus, the invention provides a rapid and easy means of determining these preferred conditions.

When using apparatus as described above, the Taguchi method also mentioned above may be carried out in stages in the same apparatus. When using a multiwell plate such as a 96 well plate, reagents representing a preliminary selection of say 9 reaction variables may be loaded into some of the wells. Subsequent narrower selections may also be loaded into other 10 wells. However, the selections are not run together but sequentially so that the narrower selections are not run until after the preliminary selection of nine has been completed. Thereafter, only wells containing reagents represented by the narrower selections considered worthwhile are cycled.

once optimised reaction conditions have been determined, the electronic instrumentation is informed of the selection. The heating profile parameters and thus already programmed into the instrumentation and it can be used directly in the assay reaction.

13

Claims (1)

1. Claims

   1. A method for determining preferred amplification reaction conditions, said method comprising i) forming an amplification reaction mixture in a plurality of wells in apparatus in which said wells are heatable individually by supplying electric current to electrically conducting polymer which either forms or is in close proximity to said well; 10 such that all filled wells contain: (a) a sample of nucleic acid which is the subject of the amplification reaction, (b) a nucleic acid polymerase, and (c) at least one primer capable of hybridising to said nucleic acid (d) a set of nucleotides, is and each well also contains (e) one or more other reagents which might assist in the amplification reaction, provided that different wells or different groups of wells contain different such reagents; ii) simultaneously conducting amplification reactions in each of said wells by supplying electric current to said electrically conducting polymer so as to cause the reagents in each well to pass through a plurality of amplification reaction cycles; and iii) determining in which well the most productive amplification reaction has been effected.

   2. A method according to claim 1 wherein a group of wells includes similar said other reagents, and wherein the electrical current supplied to electrically conducting polymer of each of said wells within a group is selected to produce a different temperature/ time profile for said amplification reaction.

   1 4 3. A method according to claim 1 or claim 2 wherein each welf contains a label means which allows the progress of said amplification to be monitored directly.

   4. A method according to claim 3 wherein said label produces a fluorescent signal.

   5. A method according to claim 3 or claim 4 wherein said label means comprises an intercalating dye and/or a labelled probe 10 which is specific for the nucleic acid.

   6. A method according to any one of the preceding claims where said other reagent comprises a source of magnesium ions, a buffer and/or an adjuvant.

   7. A kit for carrying out a method according to any one of the preceding claims, said method comprising (a) a plurality of buffer compositions suitable for effecting 20 amplification reactions; (b) one or more adjuvants suitable effecting an amplification reaction (c) a disposable apparatus comprising an electrically conducting polymer with a plurality of wells each of which is heatable by supplying electric current to polymer in the region of said well, and electrical connection means which allow each well or groups of wells to be heated individually.

   9. A kit according to claim 7 or claim 8 which further comprises label means.

   8. A kit according to claim 7 wherein components (a) and (b) in dried, frozen or chilled liquid form are predosed into wells in the apparatus.

   10. A kit according to claim 9 wherein said label means comprises an intercalating dye.