IL97686A

IL97686A - Method for the specific multiplication of nucleic acid templates and for the determination of nucleic acids

Info

Publication number: IL97686A
Application number: IL9768691A
Authority: IL
Original assignee: Boehringer Mannheim Gmbh
Priority date: 1990-03-31
Filing date: 1991-03-26
Publication date: 1994-12-29
Also published as: ZA912357B; NO911240D0; AU7381791A; JPH05211873A; AU631304B2; KR910016919A; EP0451591A1; PT97194A; FI911557A; CA2039388A1; KR940010863B1; NO911240L; IL97686A0; DE4010465A1; NZ237578A; FI911557A0; IE911034A1

Abstract

A method for the specific amplification and for the detection of nucleic acid templates by hybridisation of two nucleic acid primers onto this template, formation of a template-complementary nucleic acid with these primers and transcription of the nucleic acid which is formed to give a plurality of template-analogous nucleic acids, characterised in that the template is hybridised with two primers which are arranged in the same orientation, contain sequences complementary to the template and of which the second primer carries at the end opposite to the first second primer a transcription initiation site and a double-stranded sequence to which an RNA polymerase can bind, the two primers hybridised in this way are covalently linked by filling in the gap between them to give a template-complementary nucleic acid, transcripts of this template-complementary nucleic acid are formed, and these transcripts are detected where appropriate in a manner known per se. [EP0451591A1]

Description

METHOD FOR THE SPECIFIC MULTIPLICATION OF NUCLEIC ACID TEMPLATES AND FOR THE DETERMINATION OF NUCLEIC ACIDS Method for the specific multiplication of nucleic acid templates and for the determination of nucleic acids.

The subject matter of the invention is a method for the specific multiplication of nucleic acid templates and a method for the determination of nucleic acids.

Methods for the specific multiplication of nucleic acid templates are described for example in EP-A 0 200 362. There it is proposed that the nucleic acid to be detected is multiplied by an in vitro system. For this at least one primer is added to the sample for each nucleic acid single strand to be multiplied. Starting from the primer a nucleic acid strand is formed by an enzymatic elongation reaction which is complementary to each of the nucleic acid single strands. This reaction can be carried out several times^one after the other whereby the newly formed nucleic acid strands can also be multiplied. A disadvantage of this method is that a heating step is necessary between each multiplication step .

In EP-A 0 329 822 a method is described for multiplying specific nucleic acid templates by hybridization of two nucleic acid primers to this template, template-dependent formation of DNA with these primers and transcription of this DNA into a multitude of nucleic acids which are analogous to the template. A disadvantage of this method is in particular that the method requires long incubation periods at relatively high temperatures. In addition the method is only suitable for the multiplication of DNA after complicated pre-treatment of the template.

The object of the present invention was to eliminate the aforementioned disadvantages of the state of the art and to provide a simpler, particularly sensitive method for the multiplication and for the specific detection of nucleic acid templates.

The subject matter of the invention is a method for the specific multiplication of nucleic acid templates by hybridization of two nucleic acid primers to this template, formation of a nucleic acid complementary to the template with these primers and transcription of the nucleic acid formed into a multitude of nucleic acids analogous to the template, which is characterized in that, the template is hybridized with two primers which are arranged in the same orientation, which contain sequences complementary to the template and of which the second primer carries, at the end facing away from the first primer, a transcription- initiation site and a double-stranded sequence to which a R A polymerase can bind, the two primers hybridized in this way are covalently linked to form a nucleic acid complementary to the template by filling up the gap between them and transcripts (nucleic acids which are analogous to the template) are formed of this nucleic acid complementary to the template.

In a preferred embodiment the transcripts which are formed are used again as templates for hybridization with the primers and the multiplication cycle is repeated. In a further preferred embodiment furt er primers can be used, in particular to improve the specificity, which have a region complementary to the template which is different to that of the primer used in the first cycle.

In the sense of the invention nucleic acid templates are understood as DNA and RNA originating from prokaryotes or eukaryotes. These also include viral and bacterial nucleic acids as well as nucleic acids from viroids. They can be single-or double-stranded. They can be episomal nucleic acids, such as plasmids, or genomic chromosomal nucleic acids. The nucleic acids can be characteristic for a particular organism or a group of organisms.

The nucleic acids can also be used as a crude lysate or purified (e.g. by phenol extraction or guanidine/isothiocyanate gradient centrifugation) .

Modified nucleic acids can, however, also be used such as nucleic acids cut by means of restriction enzymes or nucleic acids modified by exonuclease treatment. In the case of RNA, cDNA can be produced beforehand. It has proved to be advantageous if the nucleic acids are in the form of single strands or are converted into a single-stranded form before carrying out the reaction.

A primer is understood as a nucleotide sequence which contains sequences complementary to the nucleic acid template and can hybridize by this means with the template under stringent conditions (e.g. Anal. Biochem. 138 (1984) 267-284).

A nucleic acid complementary to the template is understood as a nucleic acid which is complementary to the template in at least one section. A nucleic acid analogous to the template is understood as a nucleic acid which has been produced by means of the multiplication method and, as a result, at least one section corresponds to the complementary sequences to the primers and to the filled up gap sequence, in which the sequence is homologous to the template sequence.

A nucleic acid fragment which contains a complementary sequence to the template with preferably 15 - 40, particularly preferably 16 - 25 bases is used as the second primer. A transcription initiation site and a double-stranded sequence to which a RNA polymerase can bind follows this single-stranded sequence containing the complementary sequence. The double-stranded sequence has preferably a length of 17 - 100 bases, particularly preferably 7 - 50 bases. Both strands of the double-stranded part can either be present in an open form or the two ends which face away from the sequence complementary to the template can be connected via a further nucleic acid sequence which preferably has a length of 5 - 100, particularly preferably 5 - 10 bases and preferably represents a polynucleotide.

In a preferred embodiment the section of the second primer sequence which is complementary to the template begins with a phosphorylated 5· end at the end facing towards the first primer. It is equally preferred that the 3' end facing the first primer ends with a dideoxynucleotide which is particularly preferably complementary to the transcription initiation site or to the last (3') nucleotide of the promoter section.

Suitable double-stranded sequences to which a RNA polymerase can bind are for example described in Melton et al., NAR 12 (1984) 7035-7056, Pfeiffer, and Gilbert W. , Protein Sequences and DNA Analysis 1 (1988) 269-280.

The first primer contains sequences complementary to the template which enable a hybridization of the primer to the template under stringent conditions (see above) .

Those conditions are preferred under which the primer only binds to primer-specific sequences in the template nucleic acid.

The length of the complementary regions of the primer is preferably 15 - 40, in particular 16 - 25 bases.

In a further preferred embodiment one of the two primers contains a covalently-bound partner of a biological binding pair at its end which faces away from the other primer. In addition, or as an alternative, the transcription of the nucleic acids complementary to the template into the nucleic acids analogous to the template can be carried out using mononucleotides which contain a bound partner of a biological binding pair, instead of the unmodified mononucleotides. The nucleic acids complementary to the template or/and the transcripts which are formed can then be removed from the reaction solution and immobilized via the other binding partner which is immobilized on a carrier. The nucleic acids immobilized in this way can then be detected for example by methods which are familiar to one skilled in the art.

Examples of suitable binding pairs are biotin-streptavidin or avidin, hapten-antibody, antigen-antibody, concanavalin-antibody, sugar-lectin or complementary nucleic acids. Complementary nucleic acids having a length of 5 to 100, preferably 10 - 30 bases are preferably used.

In a preferred embodiment the method is used for the multiplication of specific DNA templates and specific RNA templates. If the DNA template is present as a double-strand, the template is preferably converted before hybridization into the single-stranded form by methods which are well-known to one skilled in the art.

In a preferred embodiment the nucleic acid to be multiplied or to be determined is converted before hybridization with the primers into a modified template by treatment with restiction endonucleases or exonucleases.

In a further preferred embodiment the hybridization between template and DNA complementary to the template is stopped before production of the transcript. This is preferably carried out with a RNaseH. RNaseH from E. coli or from calf thymus is particularly preferably used.

The concentration of the RNaseH is preferably 0.5 - 2 U/reaction volume.

The multiplication is preferably carried out at 30 -37 °C for 30 min to 2 hours. The reaction volume is preferably chosen as 25 - 100 μΐ. The concentration of the primer is preferably 0.3 - 3.5 μπιοΐ/ΐ in each case.

In order to fill up the gap between the primers a ligase and reverse transcriptase are preferably used. T4 ligase is particularly preferably used as the ligase. The concentration of the ligase is preferably between 1 and 10 U/reaction volume. The gap can also be closed by addition of a suitable oligonucleotide and linkage with ligase. When ligases are added a cofactor, for example ATP for T4 ligase or NAD+ for E. coli ligase, must be added.

The concentration of the reverse transcriptase is preferably 0 - 30 U/test volume. MoMLV or AMV reverse transcriptase is preferably used as the reverse transcriptase.

The production of the transcripts is carried out by addition of RNA polymerase. A phage-coded RNA polymerase, such as for example T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase is preferably used. The concentration of the polymerase is preferably 10 -100 U/reaction volume.

The invention also provides a method for the detection of nucleic acid templates by hybridization of two nucleic acid primers to this template, formation of a nucleic acid complementary to the template with these primers and transcription of the nucleic acid formed into a multitude of nucleic acids analogous to the template, characterized in that the template is hybridized with two primers which are arranged in the same orientation, which contain sequences complementary to the template and of which the second primer carries, at the end facing away from the first primer, a transcription- initiation site and a double-stranded sequence to which a RNA polymerase can bind, the two primers hybridized in this way are covalently linked to form a nucleic acid complementary to the template by filling up the gap between them, transcripts are formed from this nucleic acid complementary to the template and these transcripts are detected in a known manner (e.g. Molecular Cloning 1982, Eds. Maniatis et al., p. 199-206).

The preferred embodiments of the method of detection are analogous to those of the multiplication method.

The subsequent purification, or the detection of the transcription products formed, can be carried out as described above by an immobilization and subsequent detection with suitable methods. It is equally suitable to separate the transcription products by gel electrophoresis, to stain the RNA and to visualize either directly or by Northern transfer and subsequently to hybridize with labelled probes specific for a target sequence. Equally suitable are dot-, blot/slot-, blot-hybridization methods with labelled probes specific for a target sequence and labelling of the transcription products with one or several NTPs which are labelled for example radioactively, fluorescently or with enzymes.

The products can be made directly visible in dot-, slot-or Northern blot by incorporating 32P-labelled or nonradioactive^ labelled NTPs. The incorporation of digoxigenin or biotin (cf. WO 89/06698)' can be used for the direct detection using an antidigoxigenin antibody.

Fig. 1 shows the nucleotide sequence of the first primer used in Example 1.

Fig 2 shows two variants for the second primer.

Fig. 3 shows a preferred variant of a detection method for nucleic acids according to the present invention.

The following examples and the figures elucidate the invention further: Example 1 Production of R A templates The plasmid pSPT18 (sequence cf. WO 89/06698) is used for the production of transcripts of the neomycin resistance gene (neo) . The neomycin gene (an amino glycoside-3 ' -phosphotransferase II) is inserted into this plasmid as described in Beck et al, Gene 19 (1982) 327 - 336. Using the resultant plasmid pSPT18neo transcripts of the gene in the sense orientation can be produced using SP6 RNA polymerase. The plasmid pSPT18neo is linearized with Bgll. RNA transcripts which are employed as templates in Example 2 are produced from this linearized plasmid by in vitro transcription as described in Biochemicals for Molecular Biology, Boehringer Mannheim (1987) page 38 - 40.

Example 2 RNA amplification a) Production of the primers 1 and 2 The sequence for the primer 1 (DNA oligonucleotide of 24 nucleotides, Fig. 1) is complementary to a region of the mRNA of the neo gene which according to Beck et al corresponds to the nucleotides 2008 - 2031 of the DNA sequence. The sequence of primer 2 (also 24 nucleotides, Fig. 2) corresponds to. the nucleotide positions 1937 - 1960 of the neo gene. In addition primer 2 contains the minimum necessary double-stranded sequence of the promoter for the RNA polymerase of bacteriophage T7 (T7 RNAP, sequence cf. Fig. 2) -(Uhlenbeck et al., Nature, 328 (1987) 596 - 600) . In addition primer 2 contains an AT-rich loop region which stabilizes the partial double strand. Two functional variants of the primer sequence are shown in Fig. 2 (ddA or ddG at the 3' ends). Primer 2 is phosphorylated at the 5' end, as described for example in Maxam and Gilbert in Methods in Enzymology Volume 60 (1980) p. 499 and Nucleic Acids Research 3 (1976) 863. In addition ddA or ddC are added at the 3 ' end as also described there.

Amplification reaction Reaction mixture: 40 mmol/1 Tris HC1 (pH 8 at 37°C) , 10 mmol/1 DTT, 1 mmol/1 spermidine, 0.01 % Triton X 100, 8 % polyethylene glycol, 20 mmol/1 MgCl2, 1 mmol/1 ATP, 2 mmol/1 each NTPs 1 mmol/1 each dNTPs, 500 nmol/1 primer 1, 1 jumol/l primer 2, 5 U/reaction volume T4 ligase, 40 U/reaction volume T7 RNAP, 20 U/reaction volume reverse transcriptase and 1 U/reaction volume RNaseH.

The non-enzymatic substances used are pre-treated before use with 0.1 % diethylpyrocarbonate analogous to Maniatis (see below) pages 7.3 - 7.4.

The sample (RNA fragment according to Example 1 or dilutions thereof) is added to a reaction vessel of 50 μΐ volume and the vessel is filled up with the reaction mixture.

The preparation is incubated for two hours at 37 °C, the RNA produced is precipitated with ethanol, separated in a RNA gel, dyed and transferred onto a nylon membrane as described in Molecular Cloning, 1989, editors Sambrook et al., CSH, pages 7.43 - 7.51. The membrane-bound RNA can be detected with neo-specific probes which are labelled with digoxigenin (produced according to "DNA Labelling and Detection", Boehringer Mannheim GmbH, 1989, pages 27 - 28 or according to WO 89/06698).

Claims

97686/2 - 13 - CLAIMS ;

1. A method for the specific multiplication of nucleic acid templates comprising the steps of a) hybridizing a template with a first and a second primer which are arranged in the same orientation, wherein said primers contain sequences complementary to the template and said second primer carries, at the end facing away from said first primer, a transcription-initiation site and a double-stranded sequence to which an RNA polymerase can bind, b) covalently linking said primers hybridized to said template by filling in the gap between them to form a nucleic acid complementary to said template, c) forming transcripts from said nucleic acid complementary to the template, and d) repeating steps a) - c) using the transcripts formed in step c) as a template, wherein the transcripts of step c) are directly incorporated into step a).

2. A method for the detection of nucleic acid templates comprising the steps of a) hybridizing a template with a first and a second primer which are arranged in the same orientation, wherein said pi liners contain sequences complementary to the template and the second primer carries, at the end facing away from the first primer, a transcription-initiation site and a double-stranded sequence to which an RNA polymerase can bind, b) covalently linking the two primers hybridized to the template by filling in the gap between them to form a nucleic acid complementary to the template, c) forming transcripts from said nucleic acid complementary to the template, d) repeating steps a) - c) using the transcripts formed in step c) as a template, wherein the transcripts of step c) are incorporated into step a) and e) detecting said transcripts. ·

3. The method according to claim 1 or 2, wherein said template and nucleic acid complementary to said template are separated before formation of the transcripts.

4. The method according to claim 3, wherein said separation of said template and nucleic acid complementary to said template occurs by Rnase H digestion. 9 7686 / 2 - 1 4 -

5. The method according to claim 1 or 2, wherein said template is DNA.

6. The method according to claim 1 or 2, wherein said template is RNA.

7. The method according to claim 5 or 6, wherein said template is converted into single-stranded form before hybridization with said primers.

8. The method according to claim 7, wherein said template is a double-stranded DNA template which is cleaved with at least one restriction endonuclease and converted into single-stranded form.

9. The method according to claim 1 or 2, wherein said double-stranded sequence of said second primer has a length of 17 to 100 complementary bases.

10. The method according to claim 9, wherein said complementary bases of said double-stranded sequence are linked together via a nucleic acid fragment of 5 to 100 bases. 11· The method according to claim 9, wherein said second primer has a phosphorylated 5' end and the 3' end of the double-stranded sequence facing the first primer ends with a dideoxynucleotide. TS