A novel process is disclosed utilizing a subtraction hybridization technique for producing DNA hybridization probes of high specific activity or cDNA subtraction libraries, useful in connection with the cloning of differentially expressed genes. A preparation of single stranded cDNA derived from transcript mRNA of a target cell source is subjected to subtraction hybridization using excess single stranded "driver" nucleic acid from a reference cell source so that all the cDNA having a nucleotide sequence complementary to transcript mRNA of the reference cell source is subtracted by annealing with the "driver" nucleic acid to form duplex molecules. The strands of these duplex molecules are then chemically cross-linked by treatment with an aziridinylbenzoquinone interstrand cross-linking agent, and the remaining unsubtracted single stranded unique cDNA derived solely from the target cell source is processed in the presence of the chemically cross-linked duplex molecules to provide labelled probe material or a subtraction cDNA library, using random priming and a DNA polymerase lacking exonuclease activity and inactive with respect to the cross-linked duplex molecules.

FIELD OF THE INVENTION 
The present invention relates to the field of molecular biology and is 
particularly concerned with the technique known as subtraction 
hybridization. This is a technique often used in connection with methods 
for detecting and identifying differences in gene expression between 
related tissue cells, such as may perhaps arise in cells that have 
undergone some genetic modification. The technique can be especially 
useful for facilitating production of specific screening probes usable for 
example in conjunction with gene cloning procedures for isolating the gene 
sequences involved in such differential gene expression, and in preparing 
so-called subtraction libraries in which DNA derived from differentially 
expressed genes is enriched. The invention also specifically concerns a 
novel process using a subtraction hybridization method for producing 
labelled DNA hybridization probes of high specific label activity. 
BACKGROUND OF THE INVENTION 
Subtraction hybridization as commonly used in association with cloning of 
cDNA derived from mRNA extracted from particular cells that are under 
investigation is most useful, as already indicated, for developing or 
producing hybridization probes that can be utilised as screening agents to 
detect or locate DNA, in clone colonies or cDNA libraries for example, 
related to genes that are differentially expressed as compared with genes 
of other cells that exhibit different gene expression characteristics. 
This technique may, for example, be used in cancer research for comparing 
the gene products of tumour tissue cells with those of corresponding 
normal tissue cells in order to study the genetic changes that have 
occurred at the nucleic acid level. Probes obtained using this technique 
which are specific to DNA whose expression characteristics are modified by 
such genetic changes may be useful not only for carrying out genetic 
screening in connection with cDNA cloning, but also as diagnostic tools. 
In a typical procedure for applying this technique of subtraction 
hybridization to investigate differences in the active genes of a certain 
sample of test or target cells, e.g. from tumour tissues, as compared with 
the active genes of a sample of reference cells, e.g. cells from 
corresponding normal tissue, total cell mRNA is extracted (using 
conventional methods) from both samples of cells. The mRNA in the extract 
from the test or target cells is then used in a conventional manner to 
synthesise corresponding single stranded cDNA using an appropriate primer 
and a reverse transcriptase in the presence of the necessary 
deoxynucleoside triphosphates, the template mRNA finally being degraded by 
alkaline hydrolysis to leave only the single stranded cDNA. In one 
particular version of the technique, especially relevant to the present 
invention, care is taken to avoid unwanted synthesis of any second strand 
cDNA in this initial stage. The single stranded cDNA thus derived from the 
mRNA expressed by the test or target cells is then mixed under hybridizing 
conditions with an excess quantity of the mRNA extract from the reference 
(normal) cells. The latter is herein generally termed the subtraction 
hybridization "driver" since it is this mRNA or other single stranded 
nucleic acid present in excess which "drives" the subtraction process. As 
a result, cDNA strands having common complementary sequences anneal with 
the mRNA strands to form mRNA/cDNA duplexes and are thus subtracted from 
the single stranded species present. The only single stranded DNA 
remaining is then the unique cDNA that is derived specifically from the 
mRNA produced by genes which are expressed solely by the test or target 
cells. 
From this point onwards, to complete the subtraction process and use the 
single stranded unique cDNA, for example for producing labelled probes 
that may perhaps then be used for detecting or identifying corresponding 
cloned copies in a cDNA clone colony (labelling of such probes is 
frequently introduced by using labelled deoxynucleoside triphosphates in 
synthesis of the cDNA), it has hitherto generally been necessary first 
physically to separate out the common mRNA/cDNA duplexes, using for 
example hydroxyapatite (HAP) or (strept)avidin-biotin in a chromatographic 
separation method, after which one or more repeat rounds of the 
subtraction hybridization may be carried out to improve the extent of 
recovery of the desired product. 
This same need physically to separate out the duplex molecules generated in 
the subtraction hybridization stage has moreover remained even in many 
variations or modifications that have been used or proposed in respect of 
the basic subtraction hybridization scheme outline above, for example 
variations or modifications in which cDNA derived from mRNA of both cell 
sources is first synthesized and possibly amplified by a cloning or 
polymerase chain reaction (PCR) procedure, followed by using one of the 
cDNA mixtures (after denaturation where the cDNA is double-stranded) as 
the "driver" for carrying out the subtraction hybridization. The known and 
published methods for separating out the duplex molecules from the single 
stranded unique cDNA product, such as the above-mentioned hydroxyapatite 
or (strept)avidin-biotin chromatographic separation methods, however, are 
not entirely satisfactory, often leading to incomplete separation of the 
duplexes and a significant loss of unique cDNA or potential probe 
material. Such defects can be especially serious when the genetic material 
of interest provides only mRNA transcripts at a low abundance. Also, these 
known methods usually involve a fairly complex procedure requiring 
considerable experimental manipulation throughout, and particularly in 
producing radiolabelled probes the handling of radioactive material 
throughout a number of different stages introduces additional 
complications and hazards. 
SUMMARY OF THE INVENTION 
Accordingly, it is an object of the present invention to provide an 
improved subtraction hybridization process which permits use of a 
simplified procedure and which can enable at least some of the 
disadvantages or problems associated with the methods hitherto known, such 
as the disadvantages or problems indicated above, to be overcome. 
It is a further specific object of the invention to provide a novel 
procedure for carrying out a subtraction hybridization process which, for 
at least many end uses, requires no physical separation of the duplex 
molecules, generated during the subtraction hybridization stage, from the 
unique cDNA product. 
It is a further object of at least some embodiments of the invention to 
provide an improved process for producing efficiently DNA hybridization 
probes of high specific activity from target tissue cells having genes 
that are differentially expressed as compared with the genes of reference 
tissue cells, effective even when the product of such differential gene 
expression is of low abundance. 
It is yet another specific object of at least some embodiments of the 
invention to provide a subtraction hybridization process for producing DNA 
hybridization probes which enables multiple probes to be prepared from the 
same batch of subtracted material. 
A yet further object of at least some embodiments of the invention is to 
provide an improved subtraction hybridization process for use in preparing 
subtraction cDNA libraries in connection with differential gene expression 
screening investigations. 
These, and further objects and features of the invention, will become more 
clearly apparent from the description hereinafter contained. 
The present invention provides basically a subtraction hybridization 
process in which the nucleotide strands of the duplex molecules generated 
in carrying out the subtraction hybridization are chemically cross-linked 
and stabilized without affecting remaining non-subtracted single stranded 
unique cDNA present in the reaction mixture, using for this purpose an 
aziridinylbenzoquinone interstrand cross-linking agent. 
Thus, in applying the invention to a subtraction hybridization process 
wherein single stranded cDNA derived from transcript RNA of a target cell 
source is reacted under hybridizing conditions (through one or more 
cycles) with excess single stranded hybridization driver nucleic acid 
derived from transcript RNA of a reference source so as to cause 
substantially all said cDNA having a nucleotide sequence complementary to 
RNA that is common to both said sources to become bound in duplex 
molecules such that the only single stranded cDNA then remaining is that 
having a sequence complementary to transcript RNA that is specific to the 
target cell source, that is, unique cDNA derived solely from said target 
cell source, the reaction mixture is treated with an 
aziridinylbenzoquinone interstrand cross-linking agent effective 
selectively to chemically cross-link the nucleotide strands in the duplex 
molecules, thereby increasing the stability of these molecules, without 
affecting the remaining single stranded unique cDNA. 
It has been found that after carrying out the subtraction hybridization 
process substantially as specified above, the non-subtracted single 
stranded unique cDNA that remains can then be subjected to a labelling 
operation without complications arising from the continued presence of the 
chemically cross-linked duplex molecules if a complementary DNA strand 
incorporating labelled nucleosides is synthesised from this non-subtracted 
single strand cDNA which provides a template, using a DNA polymerase which 
lacks exonuclease activity and which is inactive with respect to said 
duplex molecules. Upon termination of the labelling reaction, the double 
stranded product can then be denatured, for example by boiling, to release 
the single strand length or lengths of labelled DNA from the template 
strand, ready for use as a probe or probes. Consequently, the basic novel 
subtraction hybridization process is extendable to produce labelled DNA 
hybridization probes in a very convenient and advantageous manner. 
DNA polymerases with the required characteristics specified above for the 
labelling operation are available commercially, such as for example that 
supplied under the Registered Trade Mark "Sequenase" by United States 
Biochemical of Cleveland, Ohio, U.S.A. which not only lacks exonuclease 
activity but is also unable to utilise RNA as a template for synthesis. 
The labelling method employed is preferably a random priming method using, 
in conjunction with the DNA polymerase, a random oligonucleotide primer 
mixture and the necessary deoxynucleoside triphosphates of which at least 
one species carries the labelling. Complete Random Primed DNA labelling 
kits suitable for this purpose, including the Sequenase.RTM. DNA 
polymerase and including a recommended protocol for use thereof, are also 
supplied by the same Company, United States Biochemical (Product No. 
70150). In most cases, a radioactive label is preferred with a radioactive 
deoxynucleoside triphosphate being used in the labelling operation. 
In carrying out the subtraction hybridization process in accordance with 
the invention, in order to ensure that substantially all the free cDNA 
derived from transcript RNA (or more specifically mRNA) common to both the 
target cell source and the reference source is hybridized and subtracted 
by the driver nucleic acid, the initial round of subtraction hybridization 
may be repeated if required one or more times, using each time a fresh 
quantity of the driver nucleic acid. Also, it will be appreciated that the 
final batch of subtracted material may, if desired, be divided into two or 
more portions, thereby enabling a corresponding multiple number of probe 
preparations to be made at different times, as and when required. 
Furthermore, since the labelling operation for producing probes is not 
carried out until after performing the subtraction hybridization, the 
reaction mixture containing the subtracted material can in any case be 
stored at a suitably low temperature so that, if desired, the labelling 
operation can be delayed and carried out later at such time that the probe 
is required for use. This is especially important when using radioactive 
labelling material as the amount of manipulation of radiolabelled DNA is 
reduced and the effects of decay in probe activity during the subtractive 
hybridization are eliminated. 
Another major advantage arises from the fact that the main series of 
operations can generally all be carried out in the same single reaction 
vessel if desired. 
The aziridinylbenzoquinone interstrand cross-linking agent used should be 
capable, under the conditions existing in the reaction mixture, of 
promoting cross-linking of substantially all the duplex molecules present, 
i.e. effectively 100% cross-linking, and should be capable of bonding to 
short but specific nucleotide sequences in DNA strands. Preferably, it is 
a compound of the formula 
##STR1## 
in which R is selected from H, alkyl, thiol, alcohol and halide. When R is 
alkyl, thiol or alcohol it is preferred that it should not contain more 
than six carbon atoms, the size of the group being limited by a need to 
avoid unduly impairing favourable physical properties such as solubility. 
Especially satisfactory cross-linking may be established when R is methyl, 
ethyl or propyl, or one of the corresponding lower alkyl thiols or 
alcohols. However, the most preferred compound is the symmetric compound 
2,5-diaziridinyl-1,4-benzoquinone (DZQ) which, under reducing conditions 
(giving the reduced hydroquinone form) at neutral pH, was reported by J. 
A. Hartley et al, (1991) Biochemistry, 30, 11719-11724, as being able to 
produce 100% interstrand cross-linking, primarily between GC pairs in 
5'-TGC-3' sequences, in DNA nucleic acid duplexes. 
The subtraction hybridization process of the present invention can usefully 
be used, as hereinafter explained, where the single stranded hybridization 
driver nucleic acid is cDNA derived, via synthesis and denaturation of 
double stranded cDNA (amplified possibly by PCR), from mRNA of the 
reference source. However, in general the single stranded hybridization 
driver nucleic acid is preferably RNA provided directly, or through an RNA 
cloning amplification process (see later), by the mRNA of the reference 
source so that the duplex molecules produced on hybridization are in fact 
RNA/DNA duplexes. Somewhat unexpectedly, it has been found that the 
aziridinylbenzoquinone interstrand cross-linking agents referred to above 
are effective and can be used with both DNA/DNA duplexes and RNA/DNA 
duplexes, although a rather higher concentration is usually found to be 
required for use with a given concentration of RNA/DNA duplexes than is 
needed for use with a similar concentration of DNA/DNA duplexes in order 
to achieve the most satisfactory results. 
According to a refinement or development of the basic process outlined 
above for producing probes, the labelling operation may alternatively be 
carried out while the unsubtracted unique cDNA and the cross-linked duplex 
molecules are all immobilised on a solid phase support instead of being 
free in the reaction mixture. Thus, in carrying out this modified version 
of the process, the first strand cDNA may be synthesized initially using a 
biotinylated primer, the subtraction hybridization and chemical 
interstrand cross-linking operations are carried out as before, and then, 
before labelling, a solid phase support material coated with avidin or 
streptavidin is introduced into the reaction mixture. Since all the cDNA, 
both the non-subtracted single stranded cDNA and the subtracted cDNA 
incorporated in the cross-linked duplex molecules, will carry biotin 
terminal groups which bind to the avidin or streptavidin of the solid 
support material, all the molecules containing such cDNA are effectively 
immobilised. It has been found that the previously described selective 
labelling operation of the unique cDNA, using for example the preferred 
random priming technique, can then still be carried out, without 
interference in respect of the cross-linked duplexes, while both molecular 
species remain bound in situ to the support. The temperature can then be 
raised by heating, e.g. to about 90.degree. C., for a short time in order 
to denature the double-stranded DNA formed in the labelling operation, 
thus releasing the labelled strand or strands lengths providing the probe 
material while leaving the original unsubtracted unique cDNA template 
strand in place. After decanting or filtering off the solution containing 
this labelled single strand probe material, the labelling operation may 
thereafter be repeated as many times as desired in order to continue 
producing further probe material, all from the same cDNA template 
originating from a single batch of subtracted material. 
A particularly convenient form of solid support for use in this modified 
version of the process is provided by magnetic beads or microspheres such 
as, for example, those which are commercially available already coated 
with streptavidin from Dynal Limited (UK) under the Trade Mark Dynabeads 
M-280 Streptavidin. Such magnetic beads can readily be manipulated within 
the reaction mixture by using external magnets, thereby considerably 
facilitating handling and filtering or separation operations. 
This above-described modified version of the process in which cDNA from the 
target cell source is immobilised on a solid phase support through 
interaction between suitable binding groups such as biotin and 
complementary receptor material such as (strept)avidin is generally 
advantageous when the hybridization driver nucleic acid is an RNA derived 
from the reference source. It can be especially important, however, if in 
carrying out the invention it is desired to produce labelled DNA 
hybridization probes using cDNA derived from the reference source as the 
subtraction hybridization driver because in that case there arises an 
additional requirement to separate or distinguish the excess single 
stranded driver cDNA that remains after the subtraction hybridization from 
the unsubtracted unique single stranded cDNA, in order to permit the 
selective labelling of the latter. As will be appreciated, such separation 
can readily be achieved when all the unsubtracted unique single stranded 
cDNA is immobilised and bound to a solid support material, as above 
described, simply by decanting or filtering off the solution containing 
the residual excess driver cDNA before releasing the labelled probe 
material from the solid support, and preferably before even commencing to 
carry out the labelling operation. Alternatively, to achieve separation 
from the unsubtracted unique single stranded cDNA before labelling of the 
latter, a cDNA subtraction hybridization driver may itself carry the 
biotin binding group instead of the cDNA derived from the target cell 
source so that the solution which is decanted or filtered off then 
contains the unique single stranded cDNA ready for labelling whilst the 
excess single stranded driver cDNA (and cDNA duplexes) remains behind, 
immobilised on the solid support. This is not a preferred procedure, 
however, since there is a greater risk of excessive or unnecessary loss of 
probe material and the full benefits of the invention are unlikely to be 
fully realised. 
In addition to the production of DNA hybridizing probes, the subtraction 
hybridization process of the present invention is also useful for 
preparing cDNA subtraction libraries, for example by using the single 
stranded unique cDNA product for cloning in suitable vectors and 
transformant host cells. For cloning, it will of course generally be 
necessary to convert this single stranded cDNA into a double stranded 
form, but this may be achieved by again carrying out a random priming 
operation, again using Sequenase.RTM., exactly as when producing probes 
except that no labelling need be incorporated and the double stranded 
product will not require to be denatured although it may need ligase 
treatment and also the addition of linkers to create suitable restriction 
sites for insertion and cloning in the chosen vector. Such cloning 
operations can still be carried out in the presence of the chemically 
cross-linked RNA/DNA duplex molecules since (a) the latter, with the 
interstrand chemical cross-linking, would not be susceptible to cloning, 
and (b) the addition to an RNA/DNA duplex of restriction site linkers, 
necessary for insertion into the vector, would be very inefficient 
compared with addition to a DNA/DNA duplex. Alternatively, however, if the 
subtraction hybridization is carried out with the duplexes and unique 
single stranded cDNA immobilised on a solid phase support, as in the 
modification of the main process herein described, then after carrying out 
a first stage of random priming as in producing probes, using 
Sequenase.RTM. but without labelling, and denaturing to release the 
newly-synthesised single stranded DNA, the solution containing the latter 
may be decanted or filtered off. This solution can then be subjected to a 
further round of DNA synthesis using a DNA polymerase which, at this 
stage, may be either Sequenase.RTM. again or a Klenow fragment DNA 
polymerase in order to provide the required double stranded DNA for 
cloning.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT OF THE INVENTION 
By way of further description of the invention, and to illustrate the 
preferred manner of putting it into practice, a specific test example will 
now be more fully described. 
EXAMPLE 
This test example relates to preparation of a high specific activity 
radiolabelled DNA hybridization probe adapted for use in detecting and 
identifying expression of the O.sup.6 -methylguanine methyltransferase 
gene (O.sup.6 -MMT) in eukaryotic cells treated with the alkylating 
antitumour drug mitozolamide. 
In this example, RJKO cells (a known Chinese hamster lung fibroblast cell 
line) were used which normally express RNA transcripts for the protein 
.alpha.-actin. As disclosed by Morten and Margison (1988) Carcinogenesis, 
9, 45-49, treatment with mitozolamide induces in such RJKO cells 
expression also of the O.sup.6 -MMT gene, but the mRNA transcripts from 
the latter are in relatively low abundance as compared with those from the 
.alpha.-actin gene. 
Materials Used 
Other than standard laboratory reagents, these included: 
RJKO cells kindly supplied by Dr. G. P. Margison (co-author of the 
above-mentioned paper); 
"Superscript".TM. reverse transcriptase (a ribonuclease H.sup.- recombinant 
form of Moloney murine leukaemia virus reverse transcriptase) from Gibco 
BRL, Paisley, Scotland; 
"Sequenase II".RTM. T7 DNA polymerase from United States Biochemical 
(Cleveland, Ohio, U.S.A.); 
2,5-diazaridinyl-1,4-benzoquinone (DZQ). 
Preparation of DZQ 
This compound is not readily available commercially, but methods of 
synthesis of DZQ and other aziridinylbenzoquinones have been previously 
described in various publications (see, for example, Chou, F., et al 
(1976) J. Med. Chem. 19, 1302., Dzielendziak, A., & Butler, J (1989) 
Synthesis, 643, Dzielendziak, A., et al, (1990) Cancer Res. 50, 2003 and 
Petersen, S., et al (1955) Angew. Chem. 67, 217). For the purpose of this 
example, a quantity of DZQ was synthesised as follows: 
Sublimed benzoquinone (2.25 g) was suspended in tetrahydrofuran (20 ml) 
dried over molecular sieves (both compounds obtained from Aldrich Chemical 
Co. Ltd., Gillingham, Dorset, U.K.). Working in a fume cupboard and taking 
appropriate precautions to reduce toxicity and explosion hazards, 3 ml of 
aziridine (obtained from Serva Feinbiochemica GmbH & Co. KG, of P.O. Box 
No. 105260, Carl Benz Strasse 7, D-6900 Heidelberg, Germany) in dry 
tetrahydrofuran (5 ml) was then added to this suspension, and the reaction 
mixture was stirred for 20 minutes on ice, followed by filtering and 
drying under vacuum. The target product thus obtained was finally 
recrystallized by adding an excess of ethanol (dried over sodium metal). 
Yield of DZQ--at least 0.5 g. 
Other analogues within the scope of the invention may be prepared by 
similar methods, using the appropriate benzoquinone derivative. 
Culture and Treatment of RJKO Cells and Isolation of mRNA Therefrom 
The RJKO cells were cultured and one sample thereof was treated with the 
alkylating drug mitozolomide substantially as described by Morten and 
Margison in their above-mentioned paper of which the content is 
incorporated herein by reference. A second sample was left untreated to 
provide a control or reference cell source, the treated sample 
constituting the test or target cell source. 
Using conventional techniques, total RNA was extracted and purified from 
both cell samples by means of the cytoplasmic lysis method of Favoloro, J. 
R., Treisman, R. & Kamon, R. (see Methods in Enzymology (1980) 65, 718), 
and from this polyadenylated mRNA (Poly A.sup.+ mRNA) was selected and 
purified using oligo dT cellulose chromatography as described by Aviv, H. 
and Leder, P. (1974) Proc. Natl. Acad. Sci. USA 69, 1408. Again, the 
content of both the two above-mentioned papers are incorporated herein by 
reference. To ensure that the Poly A.sup.+ mRNA preparations were free of 
DNA, they were digested with DNase. This was accomplished by resuspending 
the RNA and DNase I buffer (0.1M sodium acetate, 5 mM magnesium sulphate, 
pH 5.0) in a volume of about 50 .mu.l. 10 units of ribonuclease free DNase 
I (supplied by Boehringer Mannheim, Cat. No. 776785) was added and the 
mixture incubated for 15 minutes at 37.degree. C. Following this the DNase 
I was inactivated by two extractions with phenol/chloroform (50:50) and 
the mRNA was precipitated with ethanol. 
Procedure 
(a) Synthesis of First Strand cDNA 
Poly A.sup.+ mRNA, (3-5 .mu.g), free of DNA, from the mitozolamide treated 
cells was processed to synthesize corresponding first strand cDNA copies 
using unlabelled deoxynucleoside triphosphates and "Superscript".TM. 
reverse transcriptase in accordance with the manufacturer's recommended 
protocol. The choice of this particular reverse transcriptase enzyme, in 
preference to other avian or murine reverse transcriptase enzymes, was 
important because it is free of RNase H which degrades RNA in DNA/RNA 
hybrids and which, as a result of premature degradation of the parental 
mRNA template strand under synthesizing conditions, could lead to 
miscellaneous priming of exposed first strand cDNA and hence excessive 
second strand cDNA synthesis. In order to carry out the method of the 
present invention satisfactorily, wherein originally isolated mRNA is used 
as the driver in the subtraction hybridization stage as described below, 
it is essential that the cDNA produced should be substantially wholly 
first strand cDNA since only the first strand cDNA can be subtracted. 
Second strand cDNA cannot be so subtracted as it would have no 
complementary counterpart in the subtracting driver mRNA utilised in this 
process, and hence an RNase H free reverse transcriptase enzyme should be 
used. 
After completing the first strand cDNA synthesis, the RNA template strand 
was removed by alkaline hydrolysis (0.5M NaOH at 55.degree. C. for 15 
min.), and the first strand cDNA was recovered by ethanol precipitation 
following passage of the reaction mixture through a Sephadex G50 
(Registered Trade Mark) spin column, prepared as described for example in 
Molecular Cloning: A Laboratory Manual, Maniatis, T., Fritsch, E. F. & 
Sambrook, J. (1982),--Cold Spring Harbor Publication ISBN 0-87969-136-0, 
p. 466. The above-mentioned ethanol precipitation was accomplished by 
adding salt to a concentration of 0.1M, followed by adding three volumes 
of ethanol. The mixture was then cooled on dry ice for about 10 minutes, 
followed by centifugation in a bench top microcentrifuge for 10 minutes at 
13,000 rpm. 
(b) Subtractive Hybridization 
Approximately 500 ng of the first strand cDNA obtained in the last stage 
was then hybridized to excess DNA free Poly A.sup.+ mRNA (10 .mu.g) 
isolated (as previously described) from the normal RJKO cells (reference 
cell source) that do not express the O.sup.6 -MMT gene, i.e. not subjected 
to the treatment with mitozolamide. In general, a 5-50 fold excess of this 
driver mRNA over the mRNA from the target cell source should be used for 
this hybridization. 
The hybridization was carried out for approximately 20 hours at 68.degree. 
C. in a total volume of 10 .mu.l containing 0.5M NaCl, 25 mM hepes buffer 
(pH 7.5), 5 mM EDTA (ethylenediaminetetraacetic acid) and 1% SDS (sodium 
dodecylsuphate). This mixture was then diluted five fold with sterile 
distilled water, and the RNA-cDNA complex was precipitated by the addition 
of three volumes of ethanol. The precipitate, containing the cDNA-RNA 
duplex hybrids and free low abundance unsubtracted single strand cDNA, was 
dissolved in 50 .mu.l of 25 mM Tris-HCl (pH 7), 1 mM EDTA, 5% DMSO 
(dimethylsulphoxide), together with 2 mM ascorbic acid, and was incubated 
at 68.degree. C. for 3 minutes to remove hairpin structures from the 
single stranded cDNA. 
The incubation temperature was then lowered to 45.degree. C. and 
2,5-diaziridinyl-1,4-benzoquinone (DZQ) in fresh dry DMSO was added to a 
relatively high concentration of 200 .mu.M. Still at neutral pH, the 
reaction mixture was left for 20 minutes at the same temperature, a time 
sufficient under the reducing conditions produced by the ascorbic acid for 
GC base pairs of the RNA-cDNA hybrids to become effectively chemically 
cross-linked by the DZQ. The nucleic acid material containing the 
unsubtracted cDNA product was then precipitated (three vols. ethanol, 0.1 
vol. 3M sodium acetate) and, optionally, a second round of hybridization 
was carried out following addition of a further 10 .mu.g of DNA free Poly 
A.sup.+ mRNA from the untreated RJKO cells. This rehybridization stage was 
carried out as before, again for 20 hours, as above described. 
(c) Radiolabelling 
The subtracted probe was produced by a second strand synthesis and 
labelling operation carried out, still in the presence of the RNA-cDNA 
duplex hybrids, directly on the unsubtracted cDNA product (i.e. 
substantially wholly single stranded cDNA unique to the treated RKJO 
cells) which provided a template, using random priming with a mixture of 
all possible hexanucleotides as the primer mixture and incubating in the 
presence of a mixture of deoxynucleoside triphosphates, including 100 
.mu.Ci of [.alpha..sup.32 P] dCTP, 3000 Ci/mMol, with 6 units of Sequenase 
II DNA polymerase substantially as recommended by the manufacturers. The 
incubation was carried out for 20 minutes at room temperature before 
terminating the reaction (e.g. by adding EDTA), and the product was then 
denatured by boiling to release the single stranded labelled DNA material 
for use as a probe. As previously indicated, use of the Sequenase DNA 
polymerase, rather than say Klenow fragment DNA polymerase, was important 
for the selective radiolabelling herein described. This is because the 
Sequenase DNA polymerase lacks any exonuclease activity and, most 
importantly, is unable to utilise RNA (e.g. in the cross-linked duplexes) 
as a template during the radiolabelling. 
In general, it was not necessary to remove unincorporated deoxynucleoside 
triphosphates or surplus driver RNA remaining in the reaction mixture. 
Results 
For testing, subtracted probes thus produced were applied in conjunction 
with Southern blotting to electrophoresed samples of .alpha.-actin and 
O.sup.6 -MMT cDNA cloning vector inserts (.alpha.-actin insert obtained 
from plasmid 91 described by Minty, A. J. et al. (1981) J. Biol. Chem. 
256, 1008-1014, and the O.sup.6 -MMT insert obtained from the 1.1 Kb clone 
described by Rafferty, J. A. et al. (1992) Nucleic Acids Research 20, 
1891-1895). The results of the Southern blotting on preparations of equal 
quantities (100 ng) of these inserts using a subtracted probe (produced 
after only one cycle of subtraction hybridization) and, for comparison, 
using an unsubtracted probe (made with the same amount of cDNA), are seen 
in the autoradiographs reproduced in FIG. 1 in the accompanying drawing. 
Using autoradiograph densitometry (performed by video image analyser, U.V. 
Products Version II, Cambridge, U.K.), the results of using the 
unsubtracted probe (B) show that the .alpha.-actin gene had an observed 
signal approximately 120-150 fold higher than that from the O.sup.6 -MMT 
level of expression. In contrast, the results of using the subtracted 
probe (A) show that the .alpha.-actin signal had been reduced to half that 
of the O.sup.6 -MMT gene cDNA which was maintained and indicated an 
overall enrichment of 240-300 fold in the relative response from the 
O.sup.6 -MMT gene after only one round of subtraction hybridization. 
As already indicated, major advantages of this chemical cross-linking 
subtraction hybridization technique applied to the production of probes as 
herein above described include the fact that: 
1) the subtracted probe does not need to be radiolabelled until use, 
thereby reducing manipulation of labelled DNA and also probe decay during 
the subtractive hybridization; 
2) more than one probe preparation can be made from one batch of subtracted 
material; 
3) the technique does not necessarily include physical separation of 
cDNA-RNA hybrids from unique cDNA's thereby improving efficiency and 
reducing losses of material that occur as a consequence of the extra 
manipulation necessary with other procedures. 
A possible disadvantage of the technique may appear to reside in the fact 
that it requires at least 10 .mu.g of poly A.sup.+ mRNA, at least from the 
reference cell source when using this mRNA directly as the subtraction 
hybridization driver. This could be a limiting factor for some 
applications. However, this apparent disadvantage can be overcome if need 
be by generating an additional quantity of equivalent RNA through use of a 
directional cloning vector such as the lambda phage cDNA cloning vector 
described by Pallazzolo & Meyerowitz (1987) Gene 52, 197, whereby sense or 
antisense RNA can be synthesised from a cDNA population. 
For constructing such a vector, an initial synthesis of double stranded 
cDNA from the cell mRNA source is carried out using a primer adapter, e.g. 
5' XbaITTT 3', so that all the cDNA's have a restriction enzyme site (XbaI 
in the quoted example) at the 5' end. After methylation with a DNA 
methylase if necessary to protect against linker site cleavage, linkers 
providing a further restriction enzyme site (e.g. EcoRI) are ligated to 
the ends. Opposite ends of the cDNA molecules may then be selectively 
cleaved to permit ligation into the appropriately cut phage vector in 
between different RNA polymerase initiator promoter sites (e.g. SP6 and T7 
of the lambda phage) on either side of the insert cloning site. One or 
other of these polymerase initiator promoter sites may then be selectively 
removed by restriction site digestion, allowing the other to be used to 
synthesize sense or antisense cDNA by in vitro transcription, all as 
described in the above-mentioned paper by Palazzolo and Meyerowitz of 
which the content is incorporated herein by reference. 
The invention also includes all novel and inventive features and aspects 
herein disclosed, either explicitly or implicitly and either singly or in 
combination with one another, and the invention is not to be construed as 
being limited by the illustrative examples or by the terms and expressions 
used herein merely in a descriptive or explanatory sense, it being 
recognised that the scope of protection is defined and limited only by the 
claims which follow. In particular, it must also be pointed out that 
insofar as the terms "target cell source" and "reference (cell) source" 
are used in the present specification in the context of denoting abnormal 
tissue cells and normal tissue cells respectively, on the assumption that 
the abnormal tissue cells are expressing genes not expressed in the normal 
tissue cells, in some cases abnormal tissue cells may be characterised by 
a failure to express genes that are expressed by the normal tissue cells. 
In this event, in carrying out the invention, the normal tissue cells 
should therefore be regarded as being the "target cell source" and the 
abnormal tissue cells would be regarded as being the "reference cell 
source" from which the subtraction hybridization driver nucleic acid would 
be derived.