Note: Descriptions are shown in the official language in which they were submitted.
Method for the specific multiplication of nucleic
acid templates and for the determination of nucleic
acids.
i
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.
Method for the specific multiplication of nucleic
acid templates are described for example in
EP A 0 200 362, Mullis et al., filed March 27, 1986
in the name of Hoffmann-La Roche AG. 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.
En EP-A 0329 822, Davey et al., filed August 26,
1988 in the name of Cangene Corporation, 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 transcrip-
tion 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
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required long incubation periods at relatively high
temperatures. In addition -the method is only
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suitable for the multiplication of DNA after complicated
pre-treatment of the template.
The present inven-tion seeks 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 aontain
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 and transcripts (nucleic acids which
are analogous to the template) are formed of this
nucleic acid complementary to the template.
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In a preferred embodiment the transcripts which are
formed are usecl again as templates for hybridization
with the primers and the multiplication cycle is
repeated. In a further preferred embodiment additional
primers can be used, in particular ~o improve the
specificity, which have a region complementary to the
template which is di~ferent to ~hat region of the
primer used in the first cycle.
In the sense of the invention nucleic acid templates are
understood as DNA and RN~ 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
characteri~tic 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
cas~ 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 se~uences complementary to the nucleic acid
template and can hybridize by this means with
a portion of the
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template under stringent conditions (e.g. Anal. Biochem.
138 (19~4) 267-284).
A nucleic acid complementary to the template is
understood as a nucleic acid which is complementary to
the template i~ at least one section. A nucleic acid
analogous to the template i5 understood as a nucleic
acid which has been produced by means o~ 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
se~uence to the template with pxeferably 15 - 40,
particularly preferably 16 - 25 bases i9 used as the
second primer. A transcription initiation site and a
double-stranded sequence to which a RNA polymerase can bind
is contiguous to this single-stranded sequence containing
the complementary sequence. The double-stranded sequence
has preferably a length of 17 - LOO bases, particularly
pre~erably 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
~urther nucleic acid sequence which pre~erably 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
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2~39388
complementary to the transcription initiation sike or to
the last (3') nucleotide of the promoter sequence.
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 complementaxy 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 inco~porated a 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.
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Examples of suitable binding pairs are biotin-
~treptavidin 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 khe method is used for the
multiplication of speci~ic DNA templates and specific
RNA templates. If the DNA template is present as a
double-strand, the template is pxeferably converted
before hybridization into the single-stranded ~orm 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 template and
DNA complementary to the template are separated
before production of the transcript. This is
preferably carried out with a RNase~. 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 multiplicakion is preferably carried out at 30 -
37C for 30 min to 2 hours. The reaction volume is
preferably chosen as 25 - 100 ~1. The concentration of
the primer is preferably 0.3 - 3.5 ~mol/l in each case.
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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 o~ 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 O - 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 3~ RNA polymerase. A phage-coded RNA
polymerase, such as for example T7 ~NA polymerase, T3
RNA polymerase or SP6 RNA polymerase is preferably used.
The concentration of the polymerase i5 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 orientatlon, which contain
sequences complementary to the template and of
which the second primer carries, at the end facing
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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 kemplate by ~illing 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 ~982, Eds. Maniatis et al., p. 199-206).
The preferred embodiments of th~ method of detection are
analogous t~ 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 ths RN.~ 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 non-
radioactively labelled NTPs. The incorporation of
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digoxigenin or biotin (cf. WO 89/06698) can be used for the
direct detection using a biological binding partner direc-
-ted against digoxigenin or biotin, for example an anti-
di~oxigenin an-tibody.
Fig. 1 shows the nucleotide sequence of the first primer
used in Example 1.
Fig 2 shows two variants ~or the second primer.
Fig. 3 shows a preferred variant of a detection method
for nucleic acids according to the present invention.
-- 10 --
The following examples and the figures elucidate the
invention further:
Example
Production of RNA -templates
The plasmid pSPT18 (sequence cf. W0 89/06698, Holtke et al.,
filed January 12, 1989 in the name of ~oehringe~ M~elm GmbH) 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 pol~nerase. The plasmid pSPT18neo
is linearized with BglI. RNA transcripts which are
employed as templates in Éxample 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
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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 nuc]eotides 2008 -
2031 o~ the DNA sequence. The sequence of primer 2
(also 24 nucleotides, Fig. 2) corresponds to the
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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 c~. 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 ddG are added at the 3' end as also
described there.
Amplification reaction
Reaction mixture:
40 mmol/l Tris ~ICl (pH 8 at 37C),
10 mmol/l DTT,
1 mmolll spermidine,
0.01 ~ Triton X 100,
8 % polyethylene glycol,
20 mmol/l MgCl2,
1 mmol/l ATP,
2 mmol/l each NTPs
1 mmol/l each dNTPs,
500 nmol/l primer-1,
1 ~mol/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.
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The non-enzymatic substances used are pre-treated be~ore
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 ~1 volume and the vessel is filled up with the
reaction mixture.
The preparation is incubated for two hours at 37c, the
RNA produced is precipitated with ethanol, separated in
a RNA gel, dyed and trans~erred onto a nylon membrane as
described in Molecular Cloning, 1989, editors Sambrook
et al., CSH, pages 7.43 - 1.51. 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 W0 89/06698).
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