NZ233270A - Diagnosing characteristic dna sequence including preparing a sample of it and applying an amplification technique to it - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £33270 IV4 Q No.: 233270 Date: 10 April 1990 Vf EV NEW ZEALAND O ' PATENTS ACT, 1953 , ] °JUL 1991Z / v COMPLETE SPECIFICATION "A METHOD OF PREPARING SAMPLES CONTAINING NUCLEIC ACID" We, PACIFIC ENZYMES LIMITED, a New Zealand company, of B D O House, 18 London Street, Hamilton, New Zealand, hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- This invention relates to a method of preparing subsamples containing nucleic acid. The invention further relates to a method of amplifying DNA in a subsample and to a method of diagnosis which includes the step of amplifying the DNA contained within a subsample thus prepared to determine whether a selected characteristic DNA sequence is present.

Techniques which involve manipulation of nucleic acid including those which require amplification of DNA are assuming more and more importance in clinical medicine. For example, such techniques have been found to be both generally consistent and accurate in diagnosis of infections such as those caused by human papillomavirus (Manos, H M et. al. , Cancer Cells ]_ 209-214 (1989)) and Chlamydia trachomatis (Dutilh, B, et. al■, Res. Microbiol. 140, 7-16 (1989)).

However, in applying such amplification techniques directly to clinical samples, it has been found that difficulties can arise due to the presence of extraneous protein, lipoprotein and glycoprotein components. This is particularly the case with clinical samples which include blood as it has found that whole blood will inhibit amplification of the DNA in the sample. It has therefore often been necessary to follow extensive and time consuming purification procedures in relation to the samples containing the DNA to be amplified. Such procedures as are commonly employed are described for example by Dutilh et. al. (1989) supra and involve long incubations with proteases, phenol and chloroform extractions, dialysis and ethanolic salt precipitation. These procedures often take from one to two days to complete.

More recently, procedures have been developed which simplify and/or shorten the time of sample preparation by not requiring the DNA to be purified. Examples of these procedures are described in Wright, DK et al; "PCR Protocols" Eds M A Innis, D H Gelfand, J J Sninsky and T J White pp. 153-158, Academic Press, San Diego, California (1990): Kawasaki, E S; "PCR Protocols" (1990) supra; Grimberg, J et al; Nucleic Acids Research, 17, 8893 (1989); and Higuchi, R "PCR Technology: principles and applications of DNA amplification" Ed H Erlich pp. 31-38 Stockton Press, New York (1989). All of these procedures involve incubation of the clinical sample with proteinase K.

However, while these procedures in general and the Higuchi procedure in particular provide a relatively rapid and simple approach to sample preparation, they do have drawbacks. These drawbacks primarily relate to the use of proteinase K and the fact that it requires specific inactivation following completion of the preparative process. This inactivation is generally achieved by incubation of the sample mixture containing proteinase K at 95°C for 10 minutes which adds to the overall time involved in preparing the sample.

It is accordingly an object of the present invention to provide a method of preparing subsamples containing nucleic acid and particularly DNA for subsequent treatment which goes some way towards overcoming the above difficulties, or which at least provides the public with a useful choice.

Accordingly, in a first aspect the invention can be said to consist in a method of preparing a subsample containing nucleic acid for subsequent treatment which includes the step of incubating a sample containing said nucleic acid in the presence of an effective amount of a thermostable protease for a period of time and at a temperature sufficient to substantially degrade any 0 protein, lipoprotein and glycoprotein components present in the sample, said temperature being at least 60°C.

More particularly, the preparative method comprises the steps of: forming a mixture which comprises a sample containing nucleic acid and a thermostable protease; incubating said mixture for a period of time and at a temperature sufficient to allow said protease to substantally degrade any protein, lipoprotein and glycoprotein components present in said sample, said incubation temperature being at least 60°C; and where any of said protease remains active following said incubation, inactivating said protease.

Preferably, the incubated mixture is separated into its solid and liquid components to allow a constant volume of subsample to be obtained.

In the presently preferred embodiment, the mixture is incubated for 70 minutes at a temperature of 75°C.

By "thermostable protease", it is meant a protease which exhibits optimal enzymic activity and stability at temperatures of 75°C or greater, which exhibits minimal en2ymic activity at a temperature of 37°C, and which is autolytically digested upon incubation at 75°C for 60 minutes.

In a further aspect, the invention consists in a method of amplifying nucleic acid comprising the step of amplifying the nucleic acid present in a subsample prepared by a method as defined above.

In still a further aspect the invention consists in a method of detecting the presence or absence of a characteristic DNA sequence in a sample which comprises the steps of: applying an amplification technique to a subsample prepared by a method as defined above so as to amplify an amount of any DNA containing said selected characteristic DNA sequence present in said subsample; and (2) detecting the presence/absence of said selected characteristic DNA sequence.

Preferably, the diagnostic method includes the preliminary step of preparing a subsample by a method as defined above.

Conveniently, the amplification technique is polymerase chain reaction with step (1) comprising the following steps: (i) forming a polymerase chain reaction mixture including said subsample, oligonucleotide primers defining the characteristic DNA sequence, deoxynucleotide triphosphates dATP, dCTP, dGTP and dTTP and a DNA polymerase; (ii) heating said reaction mixture to a first temperature to dissociate said DNA into single strands; (iii) cooling said mixture to a second temperature to allow annealing of the primers to the dissociated strands; (iv) maintaining the reaction mixture at a third temperature to allow polymerisation of complementary strands to occur; and (v) repeating steps (ii)-(iv) a predetermined number of times to amplify said characteristic DNA sequence.

Although the present invention is broadly defined above, it will be appreciated by those persons skilled in the art that it is not limited thereto but that it also includes embodiments of which the following description provides examples. In particular, a better understanding of the diagnostic method of the invention will be obtained by reference to the drawings accompanying the provisional specification in which: ?3i270 Figure 1 shows the ability of the present method to diagnose the presence or absence of cystic fibrosis in a prepared subsample.

Figure 2 shows the ability of the present method to diagnose the presence or absence of papillomavirus DNA in a prepared subsample.

In its first aspect, the present invention provides a method by which subsamples containing nucleic acid, particularly DNA, can be rapidly and easily prepared for subsequent treatment by an appropriate procedure but which does not substantially compromise the consistency and accuracy of the results of the procedure.

Furthermore, the preparative method of the invention is one of general applicability in that it can be used to prepare samples which contain nucleic acid from any source.

The critical step of the preparative method of the invention is the incubation of a sample containing nucleic acid in the presence of a thermostable protease. This thermostable protease is preferably a non-specific protease and can be any protease which has its optimal enzymic activity and stability at temperatures of 75°C or above, which exhibits minimal enzymic activity at a temperature of 37°C, and which is autolytically digested upon incubation at 75°C for 60 minutes. Examples of suitable proteases for use in the method of the invention are the serine proteases obtained from Thermus strains rT-41-A, tok3. RTg and T351 described in Cowan, D A. et. al■, FEMS Microbiology Letters 43. 155-159 (1987) and in Cowan, D A, et. al., Biochimica et Biophysica Acta. 705, 293-305 (1982). A further example of a suitable protease is that obtained from the Desulfurococcus strain TOK^^l ancj described in Cowan, D A, et. al. , Biochem. J. 247, 121-133 (1987).

MZ. PAYti^'i Of i'vui , Mjl5JUL1991 Si -• ' RECEIVE This incubation step must be for a period of time and at a temperature sufficient to allow the protease to substantially degrade the protein, liproprotein and glycoprotein components present in the sample. In terms of the invention, this temperature will always be 60°C or above.

Generally, the higher the incubation temperature is, the shorter the incubation period will be. Accordingly, whilst relatively low temperatures, for example the minimum of 60°C, can be employed in the incubation step for an incubation time of 2 hours, it is preferred that the incubation occur at temperatures of at least 70°C to reduce the incubation time. In practice, a temperature of 75°C and a period of 70 minutes has been found to be convenient.

It should further be noted that it is possible although not preferred for the incubation to be conducted at 94°C for a period of 30 minutes. This shortened incubation can be employed where the sample required for an urgent diagnostic procedure.

The amount of thermostable protease in the incubation mixture will of course be calculated as being sufficient to effect the desired proteolytic degradation in the desired incubation time and at the incubation temperature. Generally, the amount of protease will be up to 0.5 units with 0.3 units being preferred.

It does however remain possible for an amount of protease greater than 0.5 units to be used in the incubation step if it is necessary or desirable to still further reduce the incubation time (eg. to 20 minutes). Where such an excess amount of protease is used, the incubation time will be insufficient to allow autolysis of all the protease. In these circumstances, residual protease should be inactivated as described below. n.z. patent 0\ i\ JAN I®3 received In preferred embodiments, the preparative method includes a preliminary step of forming a mixture to be incubated which comprises the sample containing nucleic acid and the thermostable protease. Moreover, where the sample contains DNA which is to be subsequently treated by the amplification procedure known as polymerase chain reaction (PCR), the mixture will also include an appropriate buffer. Any of the buffers known in the art suitable for use in PCR analysis can be included, with the selection of buffer being made as a matter of routine choice by the ordinarily skilled worker.

Once the desired degree of proteolytic digestion has occurred as a result of protease activity during the incubation step, it is important that the protease be no longer active. As will be appreciated, if residual active protease is left present in the prepared subsample, upon addition of any enzyme such as a DNA polymerase, a restriction endonuclease or a reverse transcriptase during the subsequent treatment procedure the protease will both inactivate and degrade the enzyme. This is to be avoided at all costs.

Most conveniently, the necessary inactivation of the protease will occur by denaturation and self-digestion during the incubation step. For example, where the rT-41A protease is employed in amounts of up to 0.5 units and the incubation step is for 70 minutes at 75°C or for 30 minutes at 94°C, the protease will be inactive at the end of incubation. Accordingly, in the preferred preparative method, no further action is necessary to inactivate the protease.

However, if the time and temperature parameters of the incubation step are selected such that the protease remains active The incubation step is then performed as above. j 21 JAN ^ following incubation or if an excess amount of protease is used, it will be necessary for the residual protease to be inactivated at that time. This can be achieved by, for example, the addition of a suitable reagent. An example of such a reagent is ethylene glycol bis(B-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) as this does not inhibit the enzymes likely to be used in subsequent treatment of the subsample.

Once the mixture has been incubated and the protease inactivated, a separation step is preferably performed. This step is included as it allows a constant volume of subsample to be taken from the mixture for subsequent treatment which is desirable in terms of obtaining consistent and accurate results.

The separation step may involve techniques such as filtration or sedimentation. However, while such techniques are possible it is preferred that centrifugation be employed.

The nucleic acid present in a subsample prepared in accordance with the method described above can then be subjected to any one of a number of conventional procedures. For example, the DNA can be prepared for separation by pulse field gel electrophoresis or for use in a DNA sequencing procedure. The mRNA can be employed in a conventional cloning procedure using reverse transcriptase such as is generally described in Maniatis (1982) infra. However, in the preferred alternative, the DNA can be prepared for subsequent amplification.

Where the DNA is to be amplified, the amplification step can be performed employing any amplification technique known in the art. An example of such a suitable technique is the PCR procedure. This procedure is described in Saiki, R K et. al.. Science. 230 1350 (1985) and involves two oligonucleotide primers that flank the DNA sequence to be amplified and repeated cycles of heat denaturation of \ Z. PATENT \ 21 JAN the DNA, annealing of the primers to their complementary sequences, and the extension of the annealed primers with DNA polymerase.

These primers are hybridised to opposite strands of the target sequence and are oriented so DNA synthesis by the polymerase proceeds across the region between the primers, effectively doubling the amount of the DNA sequence present. Moreover, since the extension products are also complemetary to and capable of binding primers, each successive cycle essentially doubles the amount of DNA synthesised in the previous cycle. This results in the exponential accumulation of the specific target segments, (approximately 2n where n is the number of cycles), which accumulation of specific target segments can then be detected.

Preferably the PCR amplification procedure employed involves the formation of a reaction mixture which includes the subsample, oligonucleotde primers defining the DNA sequence selected for amplification, deoxynucleotide triphosphates and a DNA polymerase. The DNA polymerase is included in the reaction mixture to enable polymerisation of complementary strands to occur. The DNA polymerase used is preferably also thermostable and capable of withstanding the temperature variations to which the reaction mixture is subjected. An example of a suitable DNA polymerase is the "Amplitaq" DNA polymerase marketed by Cetus Corporation.

Following formation of the polymerase chain reaction mixrure. the next step in the method comprises heating the reaction mixture to a temperature at which the DNA in the mixture is dissociated into single strands. Generally, this dissociation occurs at temperatures in excess of 90°C with a temperature of 94°C being commonly ? "■ Following dissociation of the DNA into single strands, the reaction mixture is cooled to a second temperature at which annealing of the oligonucleotide primers to the complementary sequences of the single strands can occur. While this temperature will vary depending on the DNA sequence selected to be amplified, it is common for the temperature to be in the range of 45°C-65°C.

Upon annealing of the primers to the single strand DNA, the reaction mixture is maintained at a third temperature to allow polymerisation of complementary strands to occur. The temperature at which the reaction mixture is maintained in this step is of course dependent upon the DMA polymerase used and in particular on the activation temperature of the polymerase. For some DNA polymerases, this temperature may be the same as the temperature at which the annealing of the primers to the dissociated strands occurs. However, it is common that the third temperature is greater than that at which the annealing occurs. By way of example, where the preferred Amplitaq polymerase is employed, the polymerisation reaction takes place with the reaction mixture maintained at a temperature of 72°C.

Upon completion of the polymerisation of complementary strands, the steps of dissociation, annealing and polymerisation are repeated. The number of times these steps are repeated will depend on the degree of amplification desired for the selected DNA sequence. Conveniently, the dissociation, annealing and polymerisation steps are repeated at least 30 times.

The above amplification procedures are suitable for automation. For example, there are various thermocycling devices commercially available which would be suitable for use in the present method. One such suitable apparatus is the Perkin-Elmer Cetus DNA Therrnocycler.

Once amplified, the selected DNA sequence can be recovered for subsequent use if this is desired.

In a further aspect, the invention provides a diagnostic method which employs both the preparative and amplification procedures outlined above. The diagnostic method is to detect the presence or absence of a selected characteristic DNA sequence in a particular sample.

As indicated, the first step of the diagnostic method involves the application of an amplification technique to a subsample prepared by the preparative method of the invention so as to amplify the amount of any DNA containing the selected characteristic DNA sequence present in the subsample. Conveniently, the PCR procedure is employed to amplify the DNA.

The second and final step of the diagnostic method of this invention is performed following the completion of the amplification step. This final step involves the detection of the presence or absence in the reaction mixture of the characteristic DNA sequence which has been selected. If the selected sequence is present in amplified quantities in the reaction mixture, the clinical sample from which the DNA amplified was obtained is positive.

The procedure adopted to detect the presence or absence of the selected characteristic DNA sequence in the reaction mixture can be any of those known in the art. Conveniently, the procedure adopted is gel electrophoresis performed for example on ethidium bromide stained 21 agarose gel.

Other suitable procedures employ the technique of Southern blotting (Maniatis et. al., Molecular Cloning : A Laboratory Manual, Cold Spring Harbour (1982)}. An electrophoresis gel is run as above and the DNA transferred to nitrocellulose filters and incubated. A labelled DNA probe is then added to the filters to allow hybridisation between the probe and a region of the DNA within the selected characteristic sequence. The filters are then autoradiographed and any hybridisation which has occurred detected.

A further procedure which can be used to detect the presence or absence of an amplified sequence involves digestion of the DNA in the reaction mixture with one or more restriction enzymes. The digested mixture is then run on an electrophoresis gel to determine whether the expected digestion products are present.

Aspects of the present invention will now be described with reference to the following non-limiting examples.

Example 1 ; Preparation of A Subsample for Amplification A 1 p.1 sample of blood was added to 30 |il of PCR buffer (50 mM KC1, 10 mM Tris pH 8.3, 2 mM MgCl2, 0.012 gelatin). 1 p.1 of a thermophilic protease (0.3 units) extracted from Thermus rT-41-A (Cowan et. al., FEMS (1987) supra) were added and the mixture incubated at 75°C for 60 minutes under mineral oil.

Following incubation, the mixture was centrifuged for 2 minutes at 15,000 g and a 1-5 p.1 subsample was removed for analysis by PCR.

Example 2 : Screening for Cystic Fibrosis by PCR Amplification A DNA sequence characteristic for cystic fibrosis was selected from published information (Kerem, B-S et. al■, Science 245, 1073 (1989)). The selected characteristic sequence was 93 base pairs in length and defined by the following primers: N.Z. PAngST omes •15JUL1991 v RECEIVE (1) 5'-GTTTTCCTGGATTATGCCTGGGCAC-3* (2) 5'-GTTGGCATGCTTTGATGAACGCTTC- 3 *.

For PCR amplification, a 1 |jl1 subsample prepared as in Example 1 was mixed with 50 p.1 of PCR buffer prepared as described, 200 mM of each of deoxynucleotide triphosphates dATP, dCTP, dGTP and dTTP 100 ng of each of the primers, and 0.5 units of Amplitaq polymerase (Perkin-Elmer Cetus). Mineral oil was then added to the reaction mixture thus formed.

The reaction mixture was initially subjected to 3 minutes incubation at 94°C (initial strand dissociation) followed by 45 cycles of 60 seconds at 94°C (denaturation), 45 seconds at 62°C (primer annealing) and 60 seconds at 72°C (polymerase extension) using thermocycling apparatus.

The presence/absence of the 93 bp amplified product was detected by direct gel analysis performed as follows.

A 7 |xl aliquot of the reaction mixture was subjected to electrophoresis at 65 volts on a 2Z agarose gel containing ethidium bromide and the DNA visualised by UV fluorescence.

Negative and positive controls were included in each reaction in the form of samples containing no DNA and samples containing purified human DNA, respectively.

The results are shown in Figure 1. As shown, the selected 93 bp fragment was present in amplified amounts, indicating a positive result for cystic fibrosis.

Example 3 : Detection of Papillomavirus by PCR Amplification Samples of cervical tissue obtained from patients being screened for genital warts were homogenised. A 1 jjlI sample of homogenised cervical cells was then prepared for PCR amplification using the procedure of Example 1.

A DNA sequence characteristic for papillomavirus (PVP) was selected as described in Manos, M M et■ al■. Cancer Cells 7, 209 (1989). The selected characteristic sequence is 450 bp in length derived from the 3' end of LI gene from PVP and defined by the following primers: (1) 5'-GCA(C)CAGGGA(T)CATAAC(T)AATGG-3 * (2) 5'-CGTCCA(C)AA(G)A(G)GGAA(T)ACTGATC-3'.

A reaction mixture was formed as described in Example 2 but with a 1 |il PVP subsample and the PVP primers replacing the cystic fibrosis subsample and primers.

The reaction mixture was initially subjected to three minutes incubation at 94°C followed by 45 cycles of 60 seconds at 94°C, 60 seconds at 50°C and 60 seconds at 72°C using thermocycling apparatus .

As in Example 2, the presence/absence of the 450 bp amplified product was detected by electrophoresis on a 21 agarose gel containing ethidium bromide.

Negative and positive controls were included in each reaction in the form of samples containing no DNA and samples containing Caski cells infected with PVP, respectively.

The results are shown in Figure 2. As shown, the selected 450 bp fragment was present in amplified amounts, indicating a positive result for PVP.

Thus, in accordance with this invention there is provided a method for preparing subsamples containing nucleic acid for subsequent treatment which can be performed in less time than present procedures without compromising the results obtainable. In particular, the use of the thermostable proteases in the preparative method allows the proteolytic digestion to proceed at higher temperatures and therefore at a faster rate. Further, the vulnerability of the thermostable proteases to autolytic degradation and their minimal activity at 37°C means that a specific protease inactivation step is usually unnecessary. This in itself represents a considerable advantage over present preparative methods.

It will be appreciated by those persons skilled in the art that the above description is provided by way of example only and that the invention limited only by the scope of the following claims.

Claims (24)

WHAT WE CLAIM IS:

1. A method of preparing a subsample containing nucleic acid which comprises incubating a sample containing said nucleic acid in the presence of a thermostable protease for a period of time and at a temperature sufficient to substantially degrade any protein, lipoprotein and glycoprotein components present in the sample, said temperature being at least 60°C.

2. A method as claimed in claim 1 wherein the nucleic acid is DNA.

3. A method as claimed in claim 1 wherein the nucleic acid is RNA.

4. A method as claimed in claims 1-3 wherein the incubation is at a temperature of 60°C for a period of about 2 hours.

5. A method as claimed in claims 1-3 wherein the incubation is at a temperature of 75°C for a period of about 70 minutes.

6. A method as claimed in claims 1-3 wherein the incubation is at a temperature of 94°C for a period of about 30 minutes.

7. A method as claimed in claim 1 which comprises the steps of: forming a mixture which comprises a sample containing nucleic acid and a thermostable protease; incubating said mixture for a period of time and at a temperature sufficient to allow said protease to substantially degrade any protein, lipoprotein and < ~ Of glycoprotein components present in said sample, said' ,nn« 21 JAN w incubation temperature being at least 60°C; and deceived where any of said protease remains active following said incubation, inactivating said protease.

A method as claimed in claim 7 including the further step of separating the incubated mixture into its solid and liquid components to allow a constant volume of subsample to be obtained.

A method as claimed in claim 7 or claim 8 wherein any of said protease remaining active following said incubation step is inactivated by addition of an appropriate amount of ethylene glycol bis(B-aminoethyl ether)-N,N1-tetraacetic acid (EGTA).

A method as claimed in any one of claims 7 to 9 wherein the nucleic acid is DNA.

A method as claimed in any one of claims 7 to 9 wherein the nucleic acid is BNA.

A method as claimed in any one of claims 1 to 11 wherein the thermostable protease is the serine protease of Thermus strain rT-41-A.

A method as claimed in any one of claims 1 to 11 wherein the thermostable protease is selected from the TOK3, RT6, and TOK12S1 proteases.

A subsample containing nucleic acid which has been prepared by a method as claimed in any one of claims 1 to 13. f W ■ PATEN"; <■ 21 JAN

A method of amplifying nucleic acid which comprises the step of amplifying the nucleic acid present in a subsample as claimed in claim 14.

A method as claimed in claim 15 wherein the amplification is of a selected DNA sequence within said DNA by polymerase chain reaction (PCR).

A method as claimed in claim 16 which comprises the steps of (i) forming a polymerase chain reaction mixture including said subsample, oligonucleotide primers defining said selected DNA sequence, deoxynucleotide triphosphates dATP, dCTP, dGTP and dTTP and a DNA polymerase; (ii) heating said reaction mixture to a first temperature to dissociate said DNA into single strands; (iii) cooling said mixture to a second temperature to allow annealing of the primers to the dissociated strands; (iv) maintaining the reaction mixture at a third temperature to allow polymerisation of complementary strands to occur; and (v) repeating steps (ii)-(iv) a predetermined number of times to amplify said selected DNA sequence.

DNA which has been amplified by a method as claimed in any one of claims 15 to 17.

A method of detecting the presence or absence of a characteristic DNA sequence in a sample which comprises the steps of: (1) applying an amplification technique to a subsample as claimed in claim 14 so as to amplify the amount of any DNA containing said characteristic DNA sequence present in said - ao - "70 subsample; and (2) detecting the presence/absence of said characteristic DNA sequence.

20. A method as claimed in claim 19 wherein the amplification technique is the polymerase chain reaction (PCR). -s

21. A method as claimed in claim 20 wherein step (1) comprises: (i) forming a polymerase chain reaction mixture including said subsample, oligonucleotide primers defining said characteristic DNA sequence, deoxynucleotide triphosphates dATP, dCTP, dGTP and dTTP and a DNA polymerase; (ii) heating said reaction mixture to a first temperature to dissociate said DNA into single strands; (iii) cooling said mixture to a second temperature to allow annealing of the primers to the dissociated strands; (iv) maintaining the reaction mixture at a third temperature to allow polymerisation of complementary strands to occur; and —^ (v) repeating steps (ii)-(iv) a predetermined number of times to amplify said characteristic DNA sequence.

22. A method of preparing a subsample containing nucleic acid ^ substantially as herein described with reference to any example thereof.

23. A method of amplifying DNA as claimed in claim 15 substantially as herein described with reference to any example thereof. L - 21 - 70

24. A method of detecting the presence or absence of a characteristic DNA sequence as claimed in claim 19 substantially as herein described with reference to any "N example thereof. .j^J/Thoir authorisad Agent • J. PARK & SUN /&/ • J ?(JU 0 i patent office 21 JRN1993 ' received