GB2360087A - Analytical method - Google Patents

Abstract

A method of analysing characteristics of a nucleic acid; said method comprising <SL> <LI>a) subjecting a sample of said nucleic acid to a plurality of different amplification reactions; <LI>b) detecting the temperature at which particular reaction products form duplexes or duplex forms destabilise within said sample, <LI>c) comparing said temperatures with those obtained from the products of similar amplification reactions effected on nucleic acids of known sequence, and <LI>d) determining which sequences are common to the known and sample nucleic acids. </SL> The method is useful in the fields of DNA profiling and in taxonomic classification.

Description

-1 2360087 1 Analytical Method The present invention relates to a method

of analysing characteristics of a nucleic acid based upon the guanidine and/or cytosine content thereof, in particular for use in the taxonomic classification of organisms, or for forensic analysis, and apparatus for use in the method.

Analysis of nucleic acid sequences, for example for forensic purposes, or for taxonomic classification of unknown organisms can be a lengthy process. Generally speaking, the nucleic acid is subjected first to an amplification reaction to increase the quantities available for analysis. The DNA is then subject to cleavage with restriction enzymes. In the case of DNA used in forensic analysis, the enzymes used are those which cut on either side of mini-satellites present in the genome of the host organism. The resulting fragments are then separated by electrophoresis on a gel to provide the DNA fingerprint of DNA profile of the source. This process however, is a complex process which may take several days to complete. Furthermore, the reaction products must be extracted from the vessel for analysis, giving rise to the possibility of contamination or cross-contamination where many samples are being tested at the same time.

In other fields, amplification reactions themselves may be monitored using fluorescence monitoring techniques for example of the polymerase chain reaction (PCR. These techniques include both strand specific and generic DNA intercalator techniques that can be used on a few second-generation PCR thermal cycling devices.

Generic fluorescence PCR methods utilise DNA intercalating dyes that exhibit increased fluorescence when bound to double stranded DNA species. Fluorescence increase due to a rise in the bulk concentration of DNA during amplifications can be used to measure reaction progress and to determine the initial target molecule 2 copy number. Furthermore, by monitoring fluorescence with a controlled change of temperature, DNA melting curves can be generated, for example, at the end of PCR thermal cycling.

These generic fluorescence PCR methods monitor the rise in bulk concentration of nucleic acids 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 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.

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 occuring in later cycles. Melting temperatures of primer oligonucleotides and their complements can be used to determine their annealing temperatures, reducing the need for empirical optimisation.

The applicants have found a method of adapting techniques previously used in melting curve analysis to general nucleic acid analysis, which provides a rapid means of obtaining some characterising data about the nucleic acid.

3 According to the present invention, there is provided a method for analysing characteristics of a nucleic acid; said method comprising a) subjecting a sample of said nucleic acid to a plurality of 5 different amplification reactions; b) detecting the temperature at which particular reaction products form duplexes or duplex forms destabilise within said sample, c) comparing said temperatures with those obtained from the products of similar amplification reactions effected on nucleic acids of known sequence, and d) determining which sequences are common to the known and sample nucleic acids.

The temperature at which various duplexes form or destabilise within a reaction is highly dependent upon the relative guanidine (G) and cytosine (C) content of the sequence. The bonds which form between these bases is relatively stronger than that formed between adenine (A) and tyrosine (T) bases. Consequently, the temperature at which a particular duplex will destabilise or "melt" is highly dependent upon the GC content of the sequence. Furthermore, the percentage GC content of an organisms genome is a recognised signature for determining the affiliations of the organism, such as bacterium, especially at the genus level.

Thus the method provides a rapid in situ method for carrying out analysis, avoiding the risk of contamination.

Suitable amplification reactions comprise polymerase chain reactions (PCR), ligase chain reactions (LCR) or NASBA., but in particular will comprise a PCR reaction.

4 As used herein, the expression 'different amplification reactions" means that different sequences or parts of sequences within a particular sample are amplified. Generally speaking, this will mean that different amplification primers are used in each reaction. The amplification primers are suitably designed such that they amplify sequences which are known to be conserved amongst in the species from which the samples are taken, of in a number of species such as bacteria, fungi or plants, where taxonomic analysis is being carried out.

Suitably, in the method of the invention, the plurality of amplification reactions are effected simultaneously, and most preferably are conducted simultaneously with the amplification reactions effected on the known sequence, so that these can act as a control.

Up to now, carrying out multiple different amplification reactions simultaneously was not possible if different temperatures were required in each vessel. This is because conventional block heaters used to effect thermal cycling could only heat all the multiple reaction wells within the block to similar temperatures.

However, in a particularly preferred embodiment of the present invention, the plurality of amplification reactions are effected in apparatus such as that described and claimed in WO 98/24548.

Apparatus of this type uses electrically conducting polymer to provide means of heating a reaction vessel. Electrically conducting polymers are known in the art and may be obtained from Caliente Systems Inc. of Newark, USA. Other examples of such polymers are disclosed for instance in US Patent No. 5106540 and US Patent No. 5106538. Suitable conducting polymers can provide temperatures up to 3000C and so are well able to be used in PCR processes where the typical range of temperatures is between 300 and 1000C.

By passing an electric current through these polymers, they 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, for example under the control of a computer programme.

Furthermore, rapid cooling can also be assured because of the low thermal mass of the polymer.

In addition, the use of polymer as the heating element in a reaction vessel will generally allow the apparatus to take a more compact form than existing block heaters, which is useful when carrying out chemical reactions in field conditions such as in the open air, on a river, on a factory f loor or even in a small shop.

The reaction vessels may take the form of a reagent container such as a glass, plastics or silicon container, with electrically conducting polymer arranged in close proximity to the container. In one embodiment of the vessel, the polymer is provided as a sheath which fits around the reaction vessel, in thermal contact with the vessel. The sheath can either be provided as a shaped cover which is designed to fit snugly around a reaction vessel or it can be provided as a strip of film which can be wrapped around the reaction vessel and secured.

The polymer sheath arrangement means that close thermal contact is achievable between the sheath and the reaction vessel. This ensures that the vessel quickly reaches the desired temperature without the usual lag time arising from the insulating effect of the air layer between the reaction vessel and the heater.

6 Furthermore, a polymer sheath can be used to adapt apparatus using preexisting reaction vessels. In particular, a strip of flexible polymer film can be wrapped around a reaction vessel of various different sizes and shapes.

Where a sheath is employed it may be advantageous for it to be perforated or in some way reticulated. This may increase the flexibility of the polymer and can permit even readier access by a cooling medium if the polymer is not itself used to effect the cooling.

In another embodiment of the invention, the polymer is provided as an integral part of the reaction vessel. The reaction vessel may be made from the polymer by extrusion, injection moulding or similar techniques. Alternatively, the reaction vessel may be manufactured using a composite construction in which a layer of the conducting polymer is interposed between layers of the material from which the vessel is made or in which the internal or external surfaces of the reaction vessel is coated with the polymer, or again in which the vessel is basically made of the polymer coated with a thin laminate of a PCR compatible material.

Such vessels may be produced using lamination and/or deposition such as chemical or electrochemical deposition techniques as is conventional in the art.

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 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, in that 7 individual vessels can be controlled independently of one anotherwith 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 temperature.

Such an arrangement is particularly preferred for use in the method of the present invention, since the plurality of amplification reactions can be carried out simultaneously, notwithstanding that the operating temperatures of the reactions may be different in each case.

In a particularly suitable apparatus for use in the present invention, the polymer itself is formed, for example, by injection molding, into wells which are integrated into a plate. An electrode is attached to each well, preferably arranged such that even heating of the well can be effected. In particular, the electrodes may be connected at in the region of the base of each well. The thickness of the polymer is suitably as low as possible consistent with structural rigidity and integrity. This reduces the time taken for the polymer to heat to the required temperature as the heat produced by passing the current through the polymer does not have to be distributed throughout a large volume of polymer material.

The electrodes associated with each well may be connected to an individual supply, or several electrodes associated with groups of wells may be connected to different, independently controlled electrical supplies. With this arrangement, the different reactions requiring different temperature stages can be carried out at the same time as each well or group of wells has its own heating element. Furthermore, the cycling reactions can be effected rapidly.

Plates may contain any number of wells, but in order to comply with conventional practice, up to 96 wells may be present in the same plate.

8 When using apparatus of this type in accordance with the invention, each amplification reaction is effected in a well of a multi-well vessel, and each well is heated by supplying current to an electrically conducting polymer which comprises or is 5 arranged to heat said well.

Preferably, wherein the temperature of each well is independently controllable so as to ensure that the particular amplification reaction being carried out in that well will occur. Suitable control means will be computer controllers so that the various reactions may be carried out automatically.

In order to detect the point at which duplexes f orm or destabilise within the reaction mixtures, label means are suitably provided in the reaction mixture. The label is preferably a visible label such as a fluorescent label which are able to signal the formation or destabilisation of said duplexes.

A particularly preferred label means is an intercalating dye such as SYBRGold TM, SYBRGreen TM, or ethidium bromide. When duplexes form during the amplification reaction, dye becomes bound between strands, giving a heightened signal as a result. Thus the signal increases when duplexes are formed in the reaction mixture and decrease when they then destabilise. By monitoring both the temperature in the reaction vessel and the signal from the dye, the temperature at which duplexes form or destabilise can be ascertained. This will be related to the GC content of the particular sequence amplified in the reaction.

Alternatively, the label means may utilise fluorescence energy transfer (FET) as the basis of detection. In this arrangement, the label means comprises two fluorescent molecule components, one of which is able to act as an energy donor and the other of which is an energy acceptor molecule.

9 These components are sometimes known as a reporter molecule and aquencher molecule respectively.

The donor molecule is excited with a specific wavelength of light for which it will normally exhibit a fluorescence emission wavelength. The acceptor molecule is excited at this emission wavelength such that it can accept the emission energy of the donor molecule by a variety of d is t ance- dependent energy transfer mechanisms. A specific example of fluorescence energy transfer which can occur is Fluorescence Resonance Energy Transfer or "FRET". Generally the acceptor molecule accepts the emission energy of the donor molecule when they are in close proximity (e.g. on the same, or a neighbouring molecule) The basis of FET or FRET detection is to monitor the changes at donor emission wavelength. Where the acceptor is also a fluorescent molecule, the acceptor emission wavelengths may also be monitored.

One of these components may be provided on a labelled hybridisation probe which binds to the target sequence at a particular temperature. The other may comprise an intercalating dye as described above. When the probe binds the target strand, intercalating dye is brought into close proximity to the probe and FET takes place. As a result, a change in the fluorescence from the donor and/or acceptor molecule is noted, which is reversed when the probe melts" from the target sequence. Systems which utilise single labelled probes and intercalating dyes are described and claimed in PCT/GB98/03560.

Alternatively, one of the donor or acceptor components of the label means may be included in a nucleotide utilised in the amplification reaction. In this way, the component of the label becomes incorporated into amplification product as it is produced. It is therefore able to undergo energy exchange (FET or FRET) with a labelled probe when that probe binds to the amplification product. In this way, hybridisation of the probe to the sequence, or melting of the probe of the sequence can be detected and the temperature at which this occurs may be monitored. Such an assay is described and claimed in PCT/GB99/00504.

There are particular hybridisation probes which carry both donor and acceptor molecules but which use hybridisation to alter the spatial relationship of donor and acceptor molecules.

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, and this forms the basis of detection.

Pairs of labelled oligonucleotides may also be used. These hybridise in close proximity on a PCR product strand bringing donor and acceptor 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.

Particular probes will bind particular target sequences at characteristic temperatures. Thus by conducting a series of amplification reactions in the presence of different probes and measuring the temperature at which these probes bind or melt from the target sequence, will provide information as to whether or not the target sequence is present in the sample of the target sequence.

The particular type of labelling system which may be employed in any particular set of amplification reactions will depend upon the nature of the sample, the length of any conserved or target sequences etc. For most applications however, an intercalating 11 dye label will be preferred as this provides a simple and cost effective method for determining hybridisation and melt analysis of an amplicon.

In particular, the signal generated when amplification product destabilises will be a preferred measurement, as this provides a clearer signal.

The invention further provides apparatus for use in the method described above. As discussed above, the apparatus suitably comprises comprising a plurality of reaction wells heatable by means of an electrically conducting polymer, a means for generating an electrical current within the polymer and a control means for regulating the amount of electric current passing through the polymer so as to control its temperature.

The control means is suitably an automatic control means such as a computer controlled interface arrangement. By using a programmable controller for the electrical circuit connected to the polymer, a defined heating regime, for example a defined number of cycles of predetermined temperature stages to be established over predetermined time intervals and dwells can be pre-programmed using the apparatus, including employing different temperature and time profiles with different wells in the same apparatus at the same time.

The control means may include a temperature monitoring device such as a thermocouple, which monitors the temperature of the reaction vessel and feeds this information into the control system so that the desired regime of heating and/or cooling is adhered to.

Alternatively, the temperature of the polymer may be monitored directly by measuring its resistivity, for example by arranging the polymer heating element as a resistor in a wheatstone bridge circuit arrangement. This avoids the use of other temperature measurement devices such as thermocouples.

12 Optionally, the apparatus further comprises artificial cooling means such as one or more fans. In addition, fluorescence detection devices such as luminometers may be provided in order to detect duplex formation or destabilisation. These are suitably arranged above the wells so that signals from reagents therein are detected.

In use, wells containing samples under test are suitably conducted in one area of the reaction vessel or plate, and those containing the known samples for comparison are located in a different area of the vessel. In this way, the comparisons may be made directly and quickly.

Other components of the reaction wells will comprise reagents required for the amplification reaction to take place. These are well known in the art, and may include amplification primers, nucleotides, buffers, as well as label means as discussed above.

The method described above is particularly useful in taxonomic classification of unknown organisms. Nucleic acids and in particular DNA from the organism is extracted and placed into a series of test wells. In comparison wells, samples from a range of known organisms are placed. The amplification reaction conditions and the primers used are set such that in each well, a sequence which is known to be characteristic of a particular organism, or to conserved amongst several organisms would be amplified if present. In this way the presence of that sequence in the test organism may be detected. By examining which of these sequences are present, the relationships between the sequences may be determined to allow rapid taxonomic classification.

Similarly in forensic or genetic analysis, a nucleic acid sample from a crime scene or a subject, may be compared with that of suspect or of a perceived relative of the subject. Provided sufficient different amplification reactions are effected to 13 determine whether the sample nucleic acid is from the same. source or related to that of the comparative nucleic acid.

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

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. cycle within each well is controlled by computer (not shown) An area (4) of the plate (2) is designated as a sample area and a separate area is designated as a comparison area (5). A similar set of amplification reaction conditions and primers are effected in wells in the sample area (4) and the comparison area (5).

Sample nucleic acid is placed in each well in the sample area (4) whereas one or more nucleic acids of known origin or sequence are placed individually in wells in the comparison area (5). Amplification primers, nucleotides, buffers or other reagents necessary to carry out an amplification reaction are added to each well together with label means as described above and particularly an intercalating dye.

Current is then applied to each reaction vessel in a controlled manner such that it proceeds through thermal cycling to effect amplification. Luminescence from each well is monitored to detect the melting point of duplexes formed.

14 It is expected that each gene amplified will show a melt temnerature which is similar if the conserved between both the If the sample does not give gene, then it is missing and which the sample nucleic acic sianal but gene is common or highly known and the sample nucleic acid.

rise to a signal for a particular is not present in the organism from is derived. However, if the sample produces a the melting point is different to that of the known sample, then the sample nucleic acid probably contains a polymorphic form of the gene.

By comparing the results for a series of reactions in this way, peaks, relationship at the genomic level between the sample and the known nucleic acid can be determined.

Variations in the intensity of melting point peaks may indicate a lower copy number for the gene in the unknown organism.

is

Claims (14)

Claims

1. A method of analysing characteristics of a nucleic acid; said method comprising a) subjecting a sample of said nucleic acid to a plurality of different amplification reactions; b) detecting the temperature at which particular reaction products form duplexes or duplex forms destabilise within said sample, c) comparing said temperatures with those obtained from the products of similar amplification reactions effected on nucleic acids of known sequence, and d) determining which sequences are common to the known and sample nucleic acids.

2. A method according to claim 1 wherein said amplification reactions comprise polymerase chain reactions (PCR), ligase chain reactions (LCR) or NASBA.

3. A method according to claim 2 wherein the amplification reaction is a PCR reaction.

4. A method according to any one of the preceding claims wherein the said plurality of amplification reactions are effected simultaneously.

5. A method according to any one of the preceding claims wherein the said similar amplification reactions effected on nucleic acids of known sequence are effected simultaneously with the amplification reactions effected on the product.

6. A method according to any one of the preceding claims wherein each amplification reaction is effected in a well of a 16 multiwell vessel, and each well is heated by supplying current toan electrically conducting polymer which comprises or is able to heat said well.

7. A method according to claim 6 wherein the temperature of each well is independently controllable so as to ensure that the particular amplification reaction being carried out in that well will occur.

8. A method according to any one of the preceding claims wherein the reaction mixture includes label means is able to signal the formation or destabilisation of said duplexes.

9. A method according to claim 8 wherein said label means comprises an intercalating dye.

10. A method according to claim 8 wherein said label means comprises a labelled probe.

11. A method according to any one of the preceding claims wherein the temperature measured is that at which amplification product in duplex form destabilises.

12. A method according to any one of the preceding claims which 25 is used in taxonomic classification of organisms.

13. A method according to any one of claims 1 to 11 for use in forensic analysis.

14. Apparatus for use in a method according to any one of the preceding claims.