GB2283569A

GB2283569A - Quantitative determination of nucleic acid

Info

Publication number: GB2283569A
Application number: GB9422296A
Authority: GB
Inventors: Stephen John Minter
Original assignee: Tepnel Medical Ltd
Current assignee: Tepnel Medical Ltd
Priority date: 1993-11-04
Filing date: 1994-11-04
Publication date: 1995-05-10
Anticipated expiration: 2014-11-04
Also published as: GB2283569B; GB9322723D0; GB9422296D0

Abstract

A method of determining the amount of a nucleic acid comprises providing the nucleic acid to be determined immobilised in a single-stranded form on particulate supports. A probe having a direct or indirect label is then added in excess and hybridised to the single-stranded nucleic acid. Non-hybridised probe is removed, and a representation of the amount of the labelled probe immobilised to the single-stranded nucleic acid is determined. The result is compared with calibration data obtained using known amounts of the single-stranded nucleic acid. The support may comprise non-porous silica.

Description

DETERMINING 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 immobilised in a single-stranded form on particulate supports, hybridising to the single-stranded nucleic acid a probe having a direct or indirect label, washing to remove unbound label, determining a representation of the amount of the labelled probe immobilised to the single-stranded nucleic acid by detection of the label, and comparing the result with calibration data, e.g. obtained using known amounts of the single-stranded nucleic acid.

By the term "direct label" we mean one which is itself capable of detection. Examples of such labels are radiolabels and chromophores. By the term "indirect label" we mean one which must take part in a further procedural step before a detectable product is obtained. An example of an "indirect label" is an "enzymatic label" where reaction with a substrate is required to produce a change which is representative of the amount of label present.

The method of the invention relies on the fact that the amount of labelled probe immobilised on the supports is directly dependent on the amount of nucleic acid on the supports. The signal obtained on detection of the label is also representative of the amount of the nucleic acid on the supports.

It is possible to establish a set of calibration data using known amounts of nucleic acid. From this calibration data the amount of nucleic acid immobilised on the support for an unknown sample may be determined. The washing procedure which is effected ensures that any labelled probe which is non-hybridised to the single-stranded nucleic acid is removed and therefore does not interfere with the determination of the amount of nucleic acid present.

The solid supports are most preferably provided in a vessel into and from which reagent and washing solutions may be introduced and exhausted. A suitable example of such a vessel is a flow-through column as disclosed in WO-A-93l13220 (Tepnel).

The solid supports are preferably of silica (most preferably nonporous silica). The supports may be as disclosed in WO-A-93113220 which also discloses techniques by means of which nucleic acids may be immobilised on solid supports.

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 single-stranded form of the nucleic acid will hybridise. If the nucleic acid is added to the supports in doublestranded form then it may be de-natured in situ 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, labelled probe is hybridised to the immobilised single-stranded nucleic acid and, after removal by washing of non-hybridised probe, the label is detected.If the label is a direct label this may be effected by "melting" the labelled probe of the single-stranded nucleic acid and passing the "melted off" probes to an appropriate detector.

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 then may be effected by hybridising a labelled probe to the primer extension product since the amount of such product equates 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 nucleic acid in an unknown sample may be determined.

Alternatively it is possible to produce a calibration curve obtained using the label system itself which will equate to the amount of labelled primer, and thus amount of nucleic acid.

In order to improve the sensitivity of the invention, it is possible to use two or more different labelled probes each of which hybridises to a different region of the immobilised single-stranded nucleic acid. Thus each strand of the nucleic acid has immobilised thereto two or more probes each of which has the same type of label.

During the subsequent detection step, the greater amount of labels present enhances the sensitivity of the technique.

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 a 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-A93/13220.

In the scheme illustrated in Fig. 3, a DNA sample (e.g. obtained from the blood of a patient) 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 labelled 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 5 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 and therefore leave only the labelled probe which is hybridised to the nucleic acid.

If the label is a direct label (e.g. a radioactive label or a chromophore) then the probe 5 may be melted off the strand 4 and passed to an appropriate detector. Alternatively, the whole of strand 4 may be melted off the support to be passed to a detection, or the probe 5 and strand 4 may be left in place, and the detection carried out in situ.

Fig. 4 illustrates an alternative embodiment of the invention in which the oligonucleotide 2 is shown to be bonded to the support 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 complimentary strand thereof). This copying reaction may be effected using, for example, DNA polymerase. The result is a primer extension product 5 which is hybridised to strand 4.

In the next step, strand 4 is melted off primer extension product 6 and washed off the supports. It will be appreciated that the amount of primer extension product 6 corresponds with the amount of DNA present in the original sample.

In the next step of the process, a labelled probe 5 is hybridised to the extension product 6 and the procedure of steps E and F of Fig.

3 repeated 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 using a second labelled probe which hybridises to the strand 4 (Fig. 3) or strand 6 (Fig. 4) at a region other than that to which probe 5 hybridises.

Claims

1. A method of determining the amount of a nucleic acid comprising providing the nucleic acid to be determined immobilised in a singlestranded form on particulate supports, hybridising to the singlestranded nucleic acid a probe having a direct or indirect label, washing to remove unbound label, determining a representation of the amount of the labelled probe immobilised to the single-stranded nucleic acid by detection of the label, and comparing the result with calibration data.

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 non-porous silica.

4. A method as claimed in any one of claims 1 to 3 wherein the solid supports are provided with an oligonucleotide and the method comprises the steps of hybridising the single-stranded form of the nucleic acid to the oligonucleotide, hybridising labelled probe to the single-stranded nucleic acid immobilised on the oligonucleotide, washing to remove non-hybridised probe, and detecting the label.

5. A method as claimed in any one of claims 1 to 3 comprising the steps of providing solid supports having oligonucleotide bound thereto, hybridising a single-stranded form of the nucleic acid to the oligonucleotide, forming a primer extension product of the oligonucleotide to produce a complementary copy of at least part of said single-stranded nucleic acid, melting off the single-stranded nucleic acid, and hybridising the labelled probe to the primer extension product.