GB2308188A - Assaying immobilised nucleic acid by primer extension - Google Patents

Description

DETERMINBG NUCLEIC ACIDS The present invention relates to a method of determining the amount of a nucleic acid.

It is desirable to determine the amount of a nucleic acid (e.g. DNA or RNA) for a number of diagnostic purposes. For example, it is desirable to be able to monitor the effectiveness of the treatment (by administration of a drug) by an illness caused by bacteria or virus. The level of bacteria or virus in the patient's body at any particular time after the commencement of treatment is a measurement of the effectiveness of the treatment. One method of determining the level of bacteria or virus present would be to determine the amount (in a blood or other sample from the patient) of a nucleic acid known to be present in the bacteria or virus. A further example in which it is desirable to know the amount of nucleic acid is to determine the levels of protein being expressed in, for example, carcinoma cells.

It is an object of the present invention to provide a method which facilitates the determination of amounts of nucleic acids.

According to the present invention there is provided a method of determining the amount of a nucleic acid, comprising providing the nucleic acid to be determined as a single-strand immobilised on a support, hybridising an oligonucleotide primer to the single-stranded nucleic acid, extending the primer to copy at least a portion of the nucleic acid strand using a nucleotide mixture containing a labelled nucleotide or a labelled primer, detecting the level of labelled nucleotide or labelled primer in the synthesised primer extension product, and comparing the result with calibration data obtained using known amounts of the single-stranded nucleic acid.

Therefore, in the method of the invention, at least part of the sequence of the immobilised nucleic acid is copied to produce the complementary sequence which incorporates labelled nucleotides. The synthesised strand (with labelled nucleotides or primer) may then be detected. The signal obtained from the bound to the support. It is possible to obtain calibration data by using the same procedure but with various known amounts of the nucleic acid immobilised on the support. In this way, a calibration graph may be established from which the concentration of nucleic acid in an unknown sample may be determined.

The solid supports are most preferably provided in a vessel into and from which reagent and washing solutions may be introduced and exhausted. A preferred example of such a vessel is a flow-through column as disclosed in WO-A93/13220 (Tepnel).

The solid supports are preferably of silica (most preferably non-porous silica). The supports may be as disclosed in WO-A-93/13220 which also discloses techniques by means of which nucleic acids may be immobilised on solid supports.

The nucleotide mixture for the copying reaction may, for example, comprise a mixture of dATP, dGTP, dTTP, and dCTP, one of which is present in both a labelled and non-labelled form. During the copying reaction, both the labelled and non-labelled form of the nucleotide will be incorporated into the forming strand. The concentration of the labelled nucleotide or the labelled primer in the nucleotide mixture will determine the strength of the signal obtained on subsequent detection.

The method of the invention may be effected in a number of ways.

In one embodiment, the sample of nucleic acid to be determined is added to the solid supports which are provided with an oligonucleotide to which a singlestranded form of the nucleic acid will hybridise. If the nucleic acid is added to the supports in double-stranded form then it may be de-natured in siX on the supports. The number of immobilised oligonucleotides on the supports should greatly exceed the anticipated number of nucleic acid molecules to be hybridised to ensure that all such molecules are indeed captured. In a subsequent step of this embodiment, a probe is hybridised to the immobilised single-stranded nucleic acid.

A labelled nucleotide mixture is then applied to the support and a primer extension reaction effected (e.g. using DNA polymerase) to copy at least a portion of the immobilised strand thereby producing a labelled primer extension product. This product may then be detected.

In an alternative embodiment of the invention, a single-stranded nucleic acid from the sample under investigation may be captured as described in the previous paragraph. In a subsequent step, the oligonucleotide on the support is used as a primer to form a primer extension product to copy the single-stranded nucleic acid (strictly speaking to produce a complementary strand thereto). The primer extension product is bound to the solid support and the originally immobilised single-stranded nucleic acid is then melted off. The detection method of the invention may then be effected by hybridising a probe to the primer extension product and forming a copy of the latter product with a labelled nucleotide mixture. The labelled product thus obtained may then be detected. The amount of such product equate directly to the amount of the nucleic acid to be determined in the original sample.

For all embodiments of the invention it will be appreciated that calibration data may be obtained using an identical system but with known amounts of the nucleic acid under investigation. It is therefore possible to produce a calibration curve from which the concentration of the specific nucleic acid in an unknown sample may be determined.

In order to improve the sensitivity of the invention, a relatively high amount of the labelled nucleotide may be used. Alternatively or additionally more than one of the nucleotides may be labelled. The label may for example be a radioactive label.

The invention will be further described by way of example only with reference to the accompanying drawings, in which: Fig. 1 illustrates to a much enlarged scale a solid particle with oligonucleotides bound thereto; Fig. 2 schematically illustrates the vessel in which the procedure of the invention may be effected; Fig. 3 illustrates a first embodiment of the method of the invention; and Fig. 4 illustrates a second embodiment of the method.

The procedures shown in the drawings involve the immobilisation of nucleic acid sequences on supports of the type shown in Fig. 1. These supports are of the type described in WO-A-93/13220 and comprise a solid (non-porous) silica particle 1 having on the surface thereof a siloxane matrix to which are bonded a plurality of oligonucleotides 2 (for convenience represented by a straight line).

(For the purposes of clarity only one such oligonucleotide is shown in subsequent figures as being attached to each particle). The oligonucleotides may all be bonded to the support in either the 3'-5' or 5'-3' orientation. For preference, the individual particles 1 are packed together in a vessel 3 which will allow reagent and wash solutions to be introduced into the vessel and drain therefrom as schematically illustrated in Fig. 2. The vessel may for example be a flow-through column and may be incorporated in an apparatus for manipulating nucleic acid sequences as described more fully in WO-A-93/13220.

In the scheme illustrated in Fig. 3, a DNA sample (e.g. obtained from the blood of a patient according to the method described in our co-pending U.K.

Patent Applications Nos. 9314119.0 and 9314175.2) is applied to supports of the type shown in Fig. 1. These supports include an oligonucleotide 2 which will hybridise to a region of one of the strands of the DNA.

In the first step of the procedure, the DNA sample is applied to the supports and de-natured. Hybridising conditions are then applied so as to cause strand 4 of the DNA to become hybridised to the oligonucleotide probes 2.

In the next step, an excess of a probe 5 is added to the supports under hybridising conditions so that the probe becomes hybridised to the single-stranded nucleic acid 4. The probe is one which will "melt off' the strand 4 at a lower temperature than that at which strand 4 will "melt off' the oligonucleotide 2.

In the next step, the particles 1 are washed so as to remove non-hybridised probe.

The next step of the procedure, involves extension of the primer 5 using the strand 4 as a template. For this purpose, a nucleotide mixture (i.e. dATP, dTTP, dCTP, dGTP) is added to the supports together with appropriate enzymes (e.g.

DNA polymerase) as necessary. A fraction of one of the nucleotides is labelled.

The label may for example be a radioactive label or a chromophore. For the purposes of Fig. 3, it is assumed that the nucleotide mixture incorporates labelled dCTP (indicated as dCTP*) although it will be appreciated that any of the other nucleotides may be labelled.

The product of this reaction is a primer extension product 6 incorporating labelled nucleotide at various positions along its length. Although Fig. 3 only shows a few labelled nucleotides having been incorporated in the primer extension product 6, it will be appreciated that the actual number incorporated will depend on (i) the number of G residues in strand 4 (which will be a function of the length thereof), and (ii) the fractional proportion of the dCTP* in the nucleotide mixture.

For a strand 4 having 1,000 base pairs and for the dCTP* fraction being about 10% of the total nucleotide concentration, the number of labelled nucleotides incorporated in the primer extension product 6 might be expected to be about 25.

At the end of the strand synthesis reaction, the nucleotide mixture is drained from the particles which are then washed so as completely to remove all non-reacted labelled nucleotide. Finally the strand synthesis product 6 is melted off strand 4 and passed to a detector. The detected value may then be compared with calibration data to determine the amount of strand 4 present in the original sample.

Fig. 4 illustrates an alternative embodiment of the invention in which the oligonucleotide 2 is shown to be bonded to the support 1 in the 5'-3' orientation (i.e.

5' end bonded to the support). As described for Fig. 3, the nucleic acid strand 4 is captured on the oligonucleotide 2. However in the next step, the oligonucleotide 2 serves as a primer which may be extended to produce a copy of strand 4 (strictly speaking the complementary strand thereof). This copying reaction may be effected, using for example DNA polymerase. The result is a primer extension product 7 which is hybridised to strand 4. In the next step, strand 4 is melted off primer extension product 7 and washed off the supports. It will be appreciated that the amount of primer extension product 7 corresponds with the amount of DNA present in the original sample. In the next step of the procedure of Fig. 4, a labelled probe 5 is hybridised to the extension product 7 to produce an extension product incorporating the labelled primer, and the procedure of steps (e)-(f), and (g) of Fig. 3 repeated (without the addition of the labelled nucleotide) to obtain the concentration of the DNA in the original sample.

It will be appreciated that for the embodiments of each of Figs. 3 and 4, the sensitivity of the technique may be enhanced by increasing the fractional proportion of labelled nucleotide present in the mixture, and/or using more than one labelled nucleotide, and/or using more than one specific primer.

Claims (5)

1. A method of determining the amount of a nucleic acid, comprising providing the nucleic acid to be determined as a single-strand immobilised on a support, hybridising an oligonucleotide primer to the single-stranded nucleic acid, extending the primer to copy at least a portion of the nucleic acid strand using a nucleotide mixture containing a labelled nucleotide or a labelled primer, detecting the level of labelled nucleotide or labelled primer in the synthesised primer extension product, and comparing the result with calibration data obtained using known amounts of the single-stranded nucleic acid.

2. A method as claimed in claim 1 wherein the solid supports are provided in a vessel into and from which reagent and washing solutions may be introduced and exhausted.

3. A method as claimed in claim 1 or 2 wherein the solid supports are of nonporous silica.

4. A method as claimed in any one of claims 1 to 3 wherein the immobilised nucleic acid to which the probe is hybridised is obtained by hybridising singlestranded nucleic acid to an oligonucleotide bonded to the solid support.

5. A method as claimed in any one of claims 1 to 3 wherein the single-stranded nucleic acid to which the probe is hybridised is obtained by the steps of immobilising a target nucleic acid to an oligonucleotide immobilised on the solid support, using the oligonucleotide as a primer to form a copy of at least part of the target nucleic acid strand, and de-naturing the target nucleic acid to leave the primer extension product on the support, to which primer extension product the enzyme labelled probe is hybridised.