Method of localizing nucleic acids bound to polyamide supports

A method of detecting and localizing nucleic acids on polyamide supports which comprises contacting said supports with a suspension of colloidal metal particles whereupon the metal particles bind to the polyamide support and form a colored background against which the location where nucleic acids are present become visible as lighter spots. The method enables the mere qualitative localization of the nucleic acids and the quantitative determination thereof following art-known procedures.

Detection of unlabeled nucleic acids, e.g. after electrophoretic separation 
in agarose or polyacrylamide gel system, is routinely performed using 
ethidiumbromide staining and ultraviolet illumination. However, thus far, 
no useful method for the detection of unlabelled nucleic acids on 
polyamide (i.e. nylon based) transfer membranes is available. Such 
membranes are routinely used for the transfer and hybridization of nucleic 
acids. Consequently, in the present state of the art, a thorough 
evaluation of the transfer process is often difficult and unreliable since 
no direct comparison of the hybridization pattern with the complete 
nucleic acid pattern of the preparation can be made. Such can easily be 
done following the method according to the invention which combines a 
remarkable ease of operation with an excellent sensitivity. 
Nucleic acids as referred to herein are meant to include both ribonucleic 
acids (RNA) and desoxyribonucleic acids (DNA). Polyamide supports to which 
the method according to the invention can be successfully applied include 
any type of polyamide material to which nucleic acids can be fixed either 
directly (dot spot) or by transfer from gel supports by the so-called 
blotting technique. In the specialized literature, blotting of DNA is 
usually referred to as "Southern" and blotting of RNA as "Northern" 
blotting. 
Examples of polyamide or nylon based membranes to which the method can be 
successfully applied include the polyhexamethylene adipamide polymers 
which are available from various manufacturers and under various 
tradenames. 
Metals which may be used in the present technique are essentially those of 
which stable colloids can be prepared and which under the relevant pH 
conditions are positively charged. Examples of such metals are gold, 
platinum, silver and copper, gold being preferred. The particle size of 
the colloidal metal particles may vary within wide limits but is 
preferably comprised between 1 and 100 nm. The appropriate pH is 
preferably the pH at which binding of the colloid to the polyamide 
membrane is maximal, i.e. when the colloidal metal particles and the 
membrane have opposite net charges. Adjustment of the pH can be achieved 
in any of the usual ways. Addition of a stock buffer to about a 10 mM 
final concentration to the colloidal metal particles is a preferred 
method. The appropriate concentration of the colloidal metal particles is 
one that gives a uniform and easily discernable colouration of the 
polyamide background within practical incubation times (from few minutes 
to about one day). It can be obtained by appropriately choosing the proper 
concentrations of the starting materials with which they are prepared, or 
by dilution or concentration by art-known methods. 
Compared with the presently available techniques, the method according to 
the invention has important advantages. 
The method allows fast and sensitive detection of nucleic acids, fixed on 
widely used nylon based membranes. This can be advantageous, when 
comparing hybridization patterns on autoradiographs with a complex banding 
pattern of the preparation. Direct superposition of both images allows 
direct localisation of the hybridizing fragments. Moreover, the staining 
allows quantitative and qualitative evaluation of the transfer process, 
without the need to include radiolabelled markers. Since staining is 
roughly proportional to the amount of nucleic acid fixed, one can make an 
estimation by comparing with a standard dilution series stained in 
parallel. 
In view of its sensitivity and ease of operation, the method according to 
the invention has a wide field of applications. In principle it can be 
used in any circumstances where localisation of nucleic acids on nylon 
based membranes is desired. 
Such is, for example, currently done in connection with nucleic acid 
sequence determinations which is largely based on the splitting of DNA or 
RNA chaines with specific enzymes, separation of the fragments formed by 
electrophoretic techniques and subsequent identification of the separated 
fragments, e.g. by means of hybridization. Nucleic acid sequence 
determinations are becoming more and more important as a tool in genetic 
engineering research and development. 
The method may also find application in the field of clinical diagnosis, 
more particularly, when the presence of certain specific nucleic acids in 
biological specimens are indicative for certain pathological conditions, 
e.g. genetic aberration. 
The invention is further illustrated by the following examples which are 
not intended to limit the scope thereof.

EXAMPLES 
Materials and Methods 
Reagents: 
All reagents were of the highest available quality. Bovine serum albumine 
(BSA) (fraction V) was obtained from Boehringer Mannheim. Plasmid DNA, 
used in these experiments, was purified by two rounds of CsCl purification 
and was essential free of RNA. Anionic stabilized colloidal gold solution 
(Auro-dye) was from Janssen Chimica (Belgium) and had a particle size of 
20 nm mean diameter (for description see Moeremans et al. in Jour. Anal. 
Biochem. 145, (1985) p.315-321.) 
Membranes: 
In our experiments, we routinely use Pall Biodyne A membranes. The method 
has however, also been tested on Gene Screen membranes (NEN). 
Dot spot procedure: 
Samples of solutions (usually 1 .mu.l), containing different concentrations 
of nucleic acids, were applied on the filter and air dried. Subsequently, 
the filters were either baked (80.degree. C., 1 hour) or treated with 
denaturing solution (0.5M NaOH; 1.5 M NaCl) and neutralizing solution (3M 
Na acetate, pH 5.8) for five minutes each, prior to baking. Denaturation 
and neutralization were performed by placing the filters (side of 
application upward) onto a piece of Whatman 3 MM paper, saturated with the 
relevant solution. 
Southern transfer: 
Was performed essentially as described by Maniatis, T., Fritsch, E. F. and 
Sambrook, J. ((1982) Molecular cloning: A Laboratory Manual, Cold Spring 
Harbor, New York, Cold spring Harbor Laboratory, p. 383-386). After 
separation of the DNA in agarose gel (1.4%) and staining with 
ethidiumbromide, the gel was incubated in denaturing solution (30 min) and 
subsequently in neutralizing solution (30 min). The gel was then blotted 
dry and placed onto sheets of Whatman 3 MM paper, saturated with 
20.times.SSC (3M NaCl, 0.3M Na citrate, pH 7.0) and soaked into 
20.times.SSC solution at two opposing ends. A piece of Biodyne A membrane 
was placed on top of the gel and covered with dry paper towels. Transfer 
was allowed to proceed overnight. The filter was then removed and air 
dried before baking (80.degree. C., 1 hour). 
Staining procedure 
Auro-dye: 
After the baking, the membranes are briefly washed in 0.2% Tween 20 
solution (3 times, 5 min) and then incubated in the Auro-dye solution with 
the application side down. The solution is agitated gently for several 
hours, until the membrane is stained intensely red. Overnight staining is 
recommended, when possible. It may also be necessary to replace the 
Auro-dye solution after a few hours. Finally the membrane is washed with 
water and dried. It was found that 0.1 .mu.g of DNA could easily be 
detected when applied in 1 .mu.l of solution (covering about 3-4 
mm.sup.2). When RNA was used, about 0.5 .mu.g of ribosomal RNA could be 
detected when applied in a similar way. It was also shown that the 
sensitivity for detection on Gene-screen membranes was comparable with 
that on Pall Biodyne A, both for RNA and DNA.