 |
Description  |
|
|
the invention relates to a method for detecting the presence and, if
necessary, characterizing a nucleic acid or a sequence of the latter in a
specimen which can contain it. It relates also to the reactants necessary
for the application of this method. Finally it relates also to the
application of such a method, among other possible applications, to the
rapid in vitro diagnosis of the presence in a biological specimen, derived
notably from a human or animal host, of particular nucleic particles, for
example infectious in nature, or again the integrity or not of this or
that particular gene belonging to the normal genetic patrimony of the
host.
It is not necessary to dwell on the extraordinary richness in various
nucleic acids which any biological specimen can contain, for example
blood, which it is possible to sample from any living creature. It is also
the same regarding different sequences, for example, of numerous genes
which any particular nucleic acid may contain in this specimen; whence the
immense difficulties that the genetician may encounter at the level of the
detection or characterization of certain nucleic acids in a specimen,
difficulties which also arise as soon as there is a question of
characterizing the presence of certain fragments, for example of genes,
contained in these nucleic acids.
The characterization of a particular nucleic acid or of particular
genes--for example for the study of the organization of genetic sequences
of DNA which contains them--hence involves the production previously from
the medium studied, of a fraction enriched in this nucleic acid. To this
end, there have already been proposed enrichment techniques exploiting
hybridation reactions between the nucleic acid or the gene sought and a
probe, to the extent that the latter was available and when the hybrids
formed could then be separated from the medium, for example by
differential sedimentation in a solution subjected to
ultra-centrifugation.
Such probes have already been described: they are generally constituted by
ribonucleic acids (RNA, DNA), such as the RNA obtained in the course of
the genetic transcription of the structural genes contained in the
desoxynucleic acids (DNA) of the cellular organisms from which they
originate, these RNA being then capable of being themselves "translated"
into proteins capable of being coded by these structural genes. It is
known that these RNA have sequences of nucleotides complementary to those
of the DNA from which they are derived, this complementarity being
manifested by the capacity possessed by these RNA to form mixed hybrids
with corresponding sequences of these DNA previously denatured, inasmuch
as the latter were initially bi-catenary, for example after incubation in
a high ionic strength medium and at a high temperature or in a basic
medium.
It has been suggested to have recourse, for marking the hybrids formed, to
radioactive labeling either of the genes themselves, or of the RNA probes.
These techiques are however difficult to put into practice and, in
addition, do not always enable satisfactory localization of the genes
concerned in their DNA.
It is with the object of permitting easier localization of the genes under
study in the DNA containing them, and of promoting a method of obtaining
fractions enriched in predetermined segments of DNA from these same DNA
that Hanning et al. proposed a physico-chemical detection technique for
these genes, consisting of chemically modifying the RNA probe, by fixing
biotin groups to the latter, through bridges formed by groups derived from
cytochrome C and fixing physical marks visible with the scanning
(electron) microscope, to the DNA, after hybridization with the probe,
formed by submicroscopic spheres having diameters of about of 60 nm,
notably based on poly(methacrylate), previously modified chemically and
coupled in covalent manner to avidin molecules (notably in the articles
entitled "A New Method of in situ Hybridization", Chromosoma (Berl.) 53.
107-117 (1975), Springer-Verlag 1975 and "A Method for Gene Enrichment
Based on the Avidin-Biotin Interaction. Application to the Drosophila
Ribosomal RNA Genes", Biochemistry, Vol. 16, No. 7, 1364-1369, 1977).
In fact, the incubation of hybrids modified with biotin in the presence of
submicroscopic spheres modified with avidin permits the "labeling" and
makes locatable the positions of the desired genes in the DNA which
contains them, with respect to the overall structure also visible in the
electronic microscope of this DNA, due to the fact of the very powerful
non-covalent interactions which are then produced between the sites
remaining free of the biotin and of the avidin.
This method however is hardly applicable to the purposes of rapid detection
of the presence or absence of such genes and of such DNA in a biological
specimen derived from a human or animal host, for example with the object
of establishing rapid diagnosis either of the disease with which the host
may possibly be afflicted, or of the integrity or not of a gene or of a
DNA sequence, for example, in this host.
The invention arises from the conversion of the Manning et coll. method,
which conversion leads to techniques of detection, even of
characterization, capable of being applied in the absence of expensive
equipment, by persons having only little laboratory experience.
The method of detection according to the invention of the possible presence
or of the characterization of a sequence or particular fragment of nucleic
acid, notably of a gene, even of the whole nucleic acid in a complex
sample of nucleic acids, by contacting the sample, if necessary after
prior denaturation of the nucleic acid under study, with a probe
comprising a complementary nucleic acid, capable of being hybridized with
the nucleic acid sequence of the nucleic acid sought, is characterized in
that the reagent or probe used is a probe modified chemically by coupling
or for its coupling with an enzyme prior or subsequent to the
hybridization reaction, the possible presence of nucleic acid sequence or
of the nucleic acid sought being revealable by the action of the
thus-transformed product of the probe and of the sequence or of the
nucleic acid sought, on an enzyme substrate.
Advantageously, the enzyme is selected according to its capacity to act on
a chromogen substrate, which permits the measurement by optical or similar
analysis, of the conversion ratio of the substrate, which ratio is then
correlatable with the presence or not of the nucleic acid sequence or of
the nucleic acid sought in the initial sample.
In a preferred embodiment of the application of the invention, the probe is
modified by a chemical group capable of forming a stable complex with the
enzyme or a molecule itself bound stably to the enzyme. Advantageously,
the above-said chemical group and the above-said molecule are respectively
constituted by biotin and avidin or vice versa, the enzyme being itself
advantageously constituted by .beta.-galactosidase.
It is self-evident that the chemical modification must be such that it does
not prevent the possible subsequent hybridization of the probe with the
DNA sequence or fragment sought.
It will immediately appear to the specialist that this technique permits a
rapid determination of the presence or not in a biological sample of the
gene or DNA fragment corresponding to the probe used, and this even in the
presence of a considerable amount of other nucleic acids. This is
particularly so due to the fact of the amplification at the level of
detection which is obtained by the action of the enzyme fixed to the
hybrid on the substrate brought into its presence. It is even possible,
after sufficient purification of the hybrid, to obtain an indication as to
the concentration in the DNA sought in the biological samples studied or
as to the distribution ratio of the gene sought in a purified DNA, by
measurement of the enzymatic activity observed.
Starting from a nucleic acid sample to be studied, it is possible to first
carry out the hybridization, then the coupling reaction between the
chemically modified and hybridized probe, on the one hand, of the enzyme,
on the other hand, to then proceed with the separation or the degradation
of the possible excess of non-hybridized probe and of the excess enzyme
which has not reacted with the probe, before carrying out the above
mentioned measurement.
As an alternative, the separation or degradation of the possible excess of
non-hybridized probe may be carried out before the coupling reaction
between the chemically modified and hybridized probe, on the one hand, and
the enzyme, on the other hand.
The specific probe can be constituted by any specific RNA or DNA either
single strand (mono-catenary), or denaturated previously by techniques
known per se, if it relates to a DNA (or an RNA) initially double strand.
When the chemical modification of the probe is carried out by means of
biotin, it is possible to resort to the technique described by Manning et
coll. in the already mentioned publications, through cytochrome C, notably
in the proportion of one molecule of biotin on the average for about 100
nucleotides.
Advantageously, recourse is then had for labeling the hybrid by the enzyme,
to the product resulting from the coupling of avidin and the enzyme,
notably .beta.-galactosidase, by the Avrameas method ("Immunochemistry",
1969, 6, 43-52).
It goes without saying that it is possible to resort to other chemical
modifications of the probe and, if necessary, of the enzyme, to effect
their coupling, preferably after the hybridization reaction, and that it
is possible to reverse the modifying agents of the probe and of the enzyme
respectively.
Other pairs of modifying agents of the probe on the one hand, and of the
enzyme, on the other hand, may also be used. By way of example, the
following pairs are mentioned, the first of these agents being preferably
used for the chemical modification of the probe and the second for the
chemical modification of the enzyme. For example, the probe may be
modified, by a known method, by metallic ions (mercury for example) and
the development is done by means of an enzyme having hydrosulphide groups
(--SH), or coupled to a support including such groups.
By way of example, which is of course non-limiting, of an experimental
procedure which may be applied in the case where the sample to be analyzed
is constituted by a blood specimen of some milliliters, it is possible to
operate as follows:
The blood cells are first lysed and the DNA is extracted therefrom by a
conventional technique.
A small amount of the DNA obtained, for example comprised between 1 and 100
.mu.g, is denatured by 0.1 to 0.3N soda, the solution then being
neutralized and brought back to pH 7.
To the solution obtained, the probe corresponding to the DNA fragment or to
the DNA sought is then added in the proportion of about 1 .mu.g of probe
per 100 .mu.g of denatured DNA (the amount of soda to be used is a
function of the proportion of DNA sought in the specimen to be analyzed).
The solution is then completed with salts for conferring on the medium a
high ionic force, at least 0.3M NaCl, in the presence of 50% formamide and
a chelating agent at low concentration, preferably in small volume. The
hybridization can then be carried out at ordinary temperature for 1 to 40
hours (generally overnight). It is also possible to use the technique
already described by Manning. As an alternative, any other hybridization
technique can also be resorted to, for example, that described by KOHNE et
al in "Biochemistry" (1977) (16, 5329-5341), at ordinary temperature in a
phenol emulsion.
Avidin coupled to an enzyme such as .beta.-galactosidase is then added to
the medium under conditions permitting the coupling of the biotin of the
probe with the free groups of the avidin of the coupling compound of the
avidin and the enzyme.
The non-hybridized reagent is then separated from the hybridized reagent by
conventional techniques, such as precipitation with polyethylene glycol,
passage over gel, for example that of the type named SEPHAROSE,
ultra-centrifugation, etc.
As an alternative it is also possible to carry out the separation of the
non-hybridized probe before the coupling of the avidin bearing the enzyme
with the biotin groups coupled to the hybridized probe with the DNA.
The enzyme possibly fixed and consequently the possible effective
hybridization of the probe with the DNA studied may be visualized or
detected by placing in contact with the medium a substrate of the enzyme,
notably that constituted by orthonitrophenol galactoside (ONPG).
It is self-evident that the experimental conditions once well-fixed, it is
possible to determine a measurable activity threshold, for example, by a
colorimetric or fluorographic technique, beyond which it is possible to
conclude in the presence in the treated sample of DNA or of the DNA
fragments sought.
The following description of a test carried out in the laboratory has
simply the purpose of illustrating the manner in which the process
according to the invention may be put into practice, it being obviously
understood that the modifications at the level of techniques, according to
the nature of the biological specimen studied and of that of the DNA or of
the DNA fragment sought, are within the evident scope of the technician
skilled in the art.
Experiments were carried out on the model consisting of detecting the
presence of a mouse DNA by hybridization of this DNA with a mouse
ribosomic RNA used as a probe.
Mouse DNA (100 .mu.g per 100 .mu.l of aqueous solution) is denatured by
addition of soda (10 .mu.l of 1M NaOH). 10 minutes later, the solution was
brought back to pH neutral by the addition of 10.mu.l of 1.5M acid sodium
phosphate NaH.sub.2 PO.sub.4.
1 .mu.g of ribosomic RNA labeled with biotin by means of cytochrome C,
prepared by the technique of Wanning & Coll., is added to the denatured
DNA solution. The volume was adjusted to 160 .mu.l with water. 40 .mu.l of
a solution having a concentration of mineral salts equal to twenty times
that of the solution called SSC (abbreviation of the English expression
"standard saline citrate") and 200 .mu.l of redistilled or deionized
formamide was then added to the medium. It is recalled that the SSC
solution is an aqueous solution of 0.15M sodium choloride, 0.015M sodium
citrate, at pH 7.0.
The mixture was incubated until the next day at ordinary temperature, then
dialyzed at 4.degree. C. against a solution having a double concentration
of the SSC solution, then for 8 hours against 500 ml of a phosphate buffer
at pH 7.0 containing phosphate at a concentration of 0.1M, sodium chloride
at a concentration of 1M and ethylene-diamine-tetrasodium acetate (EDTA)
at a concentration of 0.01M. The latter dialysis is then repeated twice,
each time for 8 hours.
The solution thus-obtained was treated with pancreatic ribonuclease for 1
hour at ordinary temperature, to obtain a final concentration of 10 .mu.g
per ml of ribonuclease, this treatment permitting the degradation of the
non-hybridized RNA.
To the medium obtained was then added a solution of cytochrome C (1 mg per
ml) and 1 microliter of a solution containing 1 mg per ml of avidin and 2
mg per ml of .beta.-galactosidase, of which 1 molecule of
.beta.-galactosidase in seven is coupled with avidin. It is mixed and the
solution is then left to stand at 4.degree. C. for 4 hours. The medium was
then diluted to 10 ml with the phosphate dialysis buffer and the solution
obtained is subjected to ultracentrifugation for 1 hour at 35,000 rpm (in
a BECKMAN ROTOR SW 41 centrifuge). The DNA and the hybridized RNA are to
be found in the centriguation culot, as well as the avidin
.beta.-galactosidase bound to this RNA. The supernatant liquor contains
the non-hybridized RNA degraded by the ribonuclease and the unbound avidin
.beta.-galactosidase.
The culot is collected and resuspended in 10 ml of buffer. It is
recentrifuged and the culot is taken up again in 0.5 ml of buffer (tube
No. 1) and the activity of the .beta.-galactosidase on the ONPG substrate
is assayed by the technique described by Miller ("Experiments in bacterial
genetics, 1972, Cold Spring Harbor Laboratory", Cold Spring Harbor, N.Y.
U.S.A.), by measurement of the eptical density of the medium at 420 m.mu.,
after incubation of the medium at 37.degree. C. for 30 minutes or more.
Controls are prepared under conditions strictly identical with those which
have been described above, except that in a first case the initial
addition of ribosomic RNA (tube No. 2) was omitted and in the other case
the addition of mouse DNA (tube No. 3) was omitted.
The results of the three assays carried out are shown in the table below:
______________________________________
Results of the assay (optical
Contents density at 420 mu after 30
Tube No,
DNA RNA minutes at 37.degree. C.
______________________________________
1 + + 0.45
2 + - 0.14
3 - + 0.15
______________________________________
The signs + and -, respectively in the columns under the headings DNA and
RNA, signify the presence or absence either of DNA, or of RNA, in the
initial medium.
As can be observed on examining this table, the optical density measured in
tube No. 1 (containing the hybrid) is very significantly greater than the
optical densities measured in the control tubes.
The experimental model which has just been described therefore illustrates
the conditions under which the possible presence of a desired DNA or DNA
fragment may be detected, to the extent that a probe complementary to this
DNA or to this RNA fragment is available by resorting to a simple
technique requiring neither very complicated laboratory equipment nor a
particularly experienced technician.
The invention is applicable particularly advantageously to in vitro
diagnosis operations of the presence, for example in a biological sample
(blood sample, specimen of stools, etc.) of various viruses, such as those
named Herpes, Epstein Barr, virus Pox, cytomegalo, etc. In the same way,
the invention may be applied to the diagnosis, for example, of specific
chromosomic anomalies.
It is also applicable to the realization of bacterial diagnoses, in
particular in the case where individuals are bearers of pathogenic genes,
both expressed and non-expressed (or latent).
It will appear naturally to the specialist, in the case of investigating an
infectious DNA, that it is possible to conclude rapidly as to the healthy
character of the treated biological specimen, and having regard to the
nucleic acid or the fragment of nucleic acid sought, in the absence of
induction observe on the chromogenic substrate, or at least an over-shoot
of the activity threshold, either predetermined experimentally, or by
comparison with controls free of the virus.
Conversely, the absence of action observed with respect to the chromogenic
substrate, notably beyond the above-mentioned threshold, can, in the other
type of application, envisaged above by way of example, translate the
presence of an anomaly of the chromosomic anomaly sought, in the absence
of observed total or partial hybridization, between the probe and the DNA
studied.
It is advantageously possible to place, for example, at the disposal of
medical analysis laboratories, "kits" containing all of the essential
reactants for the application of the process according to the invention.
These kits can, in particular, contain a sampling of probes corresponding,
for example, to the DNA of the virus or bacteria, of conventionally sought
pathogenic viruses or bacteria, or even of probes relating to particular
genes which should normally be contained in biological specimens, notably
blood specimens, under test.
In this regard, the invention relates hence to a "kit" characterized in
that it comprises:
at least one specific probe formed from RNA or a single RNA strand,
characteristic of a nucleic acid sequence or of a nucleic acid to be
sought, this probe being modified chemically for its coupling with an
enzyme,
said enzyme, if necessary, modified so as to be able to be coupled with
said probe,
a substrate, notably a chromogene, specific to the enzyme,
the reactants necessary for the lysis of the cellular medium to be studied,
notably a blood medium, and for the extraction of nucleic acids from the
cells of this medium.
As has already been observed in the foregoing, it is advantageous to
constitute the modified probe by a probe to which biotin is bound, the
modified enzyme being then constituted by the enzyme itself, for example
.beta.-galactosidase, coupled to avidin.
The invention relates also moreover, by way of novel industrial product, to
the coupling product of an enzyme (of which the action may be revealed
with respect to a substrate, notably chromogenic) and of a probe (RNA or
single strand DNA), either directly, or through a coupling agent. It
relates also again to the coupling product of the enzyme and of at least
one chemical molecule, the whole then being capable of being coupled in
its turn with a probe (RNA or DNA), if necessary modified, itself capable
of being hybridized with a DNA or a DNA fragment. By way of examples of
such novel industrial products, may be mentioned the coupling products of
a probe (RNA or DNA) with an enzyme, such as .beta.-galactosidase, or
again coupling products of avidin or of biotin with such an enzyme.
Of course, the invention may be applied in other fields of application,
notably for the labeling of certain DNA fragments in well-known genetic
experiments seeking to establish the genotype of the DNA concerned. In
particular, the invention may be applied to the determination of the
incorporation or not of a particular DNA fragment in experiments of
genetic sorting comprising for example operations of transforming DNA from
an infected cell with a foreign DNA containing the DNA fragment concerned
or on the contrary operations of transduction including the incorporation
of a DNA fragment concerned, normally contained in the DNA of the cell, in
the DNA of the virus used for the infection of the cell, etc., to the
extent that, of course, a probe constituted by the RNA fragment or DNA
complementary to the sought nucleic acid fragment is available.
As is self-evident and as emerges already besides from the foregoing, the
invention is in no way limited to those of its modes of application and
embodiments which have been more especially envisaged; it encompasses on
the contrary all modifications, notably those where recourse is had to
modifications of the probe which may enable the enzymatic assay of the
hybrid and modifications relating to the formation and/or purification of
the hybrids, to the labeling or the chemical modification of the DNA
studied itself, under conditions which have been described above, the RNA
probe not being the subject of any particular labeling; such an inversion
of the reactants may be envisaged, for example in the case of a DNA
including numerous examples of repetetive genes, that it is desired to
isolate from the whole DNA, in the form of a hybrid with a probe, after
fragmentation of the DNA concerned by conventional techniques. It is
self-evident that these equivalents are included within the field of
protection defined by the claims.
By way of yet another modification, it is possible to have recourse to a
process consisting of marking the hybrid formed by the desired DNA and the
probe, by means of an anti-hybrid antibody, coupled to an enzyme such as
.beta.-galactosidase.
* * * * *
|
|
 |
|
 |
Description |
|
