EP0183822A4

EP0183822A4 - Catalyzed nucleic acid hybridization using enzymatic reagent.

Info

Publication number: EP0183822A4
Application number: EP19850903113
Authority: EP
Inventors: Edward J Zapolski; Douglas M Gersten; Robert S Ledley
Original assignee: NATIONAL BIOMEDICAL RESEARCH FOUNDATION; NAT BIOMEDICAL RES FOUNDATION
Current assignee: NATIONAL BIOMEDICAL RESEARCH FOUNDATION; NAT BIOMEDICAL RES FOUNDATION
Priority date: 1984-06-01
Filing date: 1985-05-31
Publication date: 1988-08-29
Also published as: JPS61502282A; WO1985005685A1; EP0183822A1

Abstract

A hybridization method for treating target DNA fragments to identify known genetic sequences comprises the following steps: disposing the target DNA fragments on a carrier, either a dried gel film or a blotting matrix to which the target DNA fragments are transferred by the southern blotting technique or a variation thereof; forming a hybridization reagent consisting of a radioactive-labelled probe DNA and catalyzing enzymes and cofactors; immersing the carrier in the hybridization reagent for a given period of time; washing the carrier in at least one wash solution; exposing an x-ray film to the carrier; and developing the x-ray film to detect and locate the radioactive-labelled probe DNA. The catalyzing enzymes utilized in forming the hybridization reagent include RecA protein and SSB DNA binding protein in the presence of ATP.

Description

Description Catalyzed Nucleic Acid Hybridization Using Enzymatic Reagent Technical Field The invention generally relates to a hybridization method for treating target DNA fragments to identify known genetic sequences, and more particularly, a hybridization method which is catalyzed by use of an enzymatic hybridization reagent formed, inter alia, of RecA protein, single- stranded DNA binding protein (SSB) , and adenosine triphosphate (ATP) . Background Art

Nucleic acid hybridization technology, used to identify the presence of known genetic sequences in a particular sample, is a necessary element in many processes of modern biotechnology, and in almost all aspects of genetic engineering. Under appropriate conditions, single strand nucleic acids that are complementary (homologous) unite to form duplex strands. This process of hybridization is generally accomplished by utilizing conditions whereby duplex nucleic acid molecules unwind ("melt") and slowly reanneal to natually form the classical double stranded helix. Because of the rigid specificity (only strands of complementary base sequence will pair) , the process is used to seek and identify regions of DNA (target material) that are complementary to a specific single strand of DNA which is employed as the probe.

Of the available techniques for hybridization, the Southern blot approach is especially valuable because information about the size of the polynuσleotide fragments containing the sequences of interest is also obtainable. See Southern, E.M. , J. Mol. Biol., 98:503-517 (1975). In most common practice, DNA fragments are separated according to size by electrophoresis in agarose gel, denatured to single strands by treatment with alkali, and neutralized. They are then transferred to a membrane or to specially treated paper matrix by blotting. The transfer may be expedited -by electrophoresis. The nucleic acid fragments are then fixed to the matrix by baking. The bound matrix containing the nucleic acids is then exposed to a prehybridization solution of noncomplementary DNA in order to mask extraneous DNA binding sites on the matrix, thereby reducing background. Hybridization is then performed, and is accomplished by annealing the matrix bound target DNA with probe DNA in various formulations of media and at temperatures which can be as high as 65-70^βC. Some formulations contain up to 50% dimethylformamide, or other dangerous substances, to help lower the DNA melting temperature so that hybridization can occur by annealing.

Southern's original study (cited above), as can be seen from the above protocol, described a very time-consuming procedure which, in addition, tends to transfer different size fragments from 'the electrophoresis gel to the blotting matrix at different rates. See Southern, E.M. , Meth. Enzmol. _r 68:152-176 (1979). Consequently, many variations on the basic theme, which variations seek to overcome these aspects of the method, have been reported.

One of the most promising of such modifications is hybridization in situ. first described by Shinnick et al. See Shinniσk et al, Nucl. Acid. Res.. 2:1911-1929 (1975). In this procedure, hybridization is performed directly in the electrophoresis gel without transfer to another matrix. A further modification has recently been reported in which the electrophoresis gel is dried down, and hybridization is performed using the dried gel. See Purrello et al, Analyt. Biochem.. 128:393- 397 (1983). Currently practiced DNA hybridization procedures that are accomplished either in situ in agarose gels or after transfer to another matrix require the following steps and minimum times: (1) Denaturation with alkai - i hour (2) Neutralization - i hour

(3) Optional transfer to another matrix by blotting (6 hours) or electrophoresis (2 hours)

(4) Prehybridization - 1 hour (5) Hybridization - 6-8 hours, but most likely 16-20 hours (6) Stringent Washing - 2 hours Thus at least 12 hours, and more likely 24 hours, are devoted to hybridization by annealing. That hybridization can occur on a dried gel

(i.e. at the gel surface) implies, at most, only a limited requirement for the probe material to migrate into the interior of the gel. Therefore, there should be sufficient target DNA at the gel surface to permit adequate hybridization. With the latter point in mind, it should be noted that, because of the time involved in steps (1), (2), (4) and (5) above, as well as the inherent inefficiency of blotting or electrophoresis transfers in step (3), some loss of target material is inevitable, and this represents a significant problem in prior art methodologies.

The prior art techniques in this area of technology are represented by the following U.S. patents, which are of background interest: 4,302,204;

4,358,535; 4,395,486; 3,930,956; and 4,342,833. The following articles are also of backgroun interest:

Radding, "Recombination Activities of E. coli RecA Protein", Cell. 25:3-4 (July 1981); Sedgwick, "Roles of RecA Revealed", Nature,

287:676-677 (October 23, 1980);

Weinstock et al, "ATP-Dependent Renaturation of DNA Catalyzed by the RecA Protein", Proc. Natl. Acad. Sci. USA, vol. 76, no. 1, pp. 126- 130 (January 1979);

Radding et al, "Kinetics and Topology of Homologous Pairing Promoted by Escherichia coli RecA- gene Protein", Cold Spring Harbor Sy p. Quant. Biol.. 45:385-390 (1980); Wu et al, "Formation of Nascent

Heteroduplex Structures by RecA Protein and DNA", Cell. 30:37-44 (August 1982);

Cox et al, "On the Role of Single-Stranded

DNA Binding Protein in RecA Protein-Promoted DNA strand Exchange", The Journal of Biological

Chemistry, vol. 258, no. 4, pp. 2577-2585 (February

25, 1983) ; and

Potter et al, "Genome Fusion", Cold Spring Harbor Svmp. Quant. Biol.. 45:371-383 (1980). Disclosure of Invention

The present invention generally relates to catalyzed hybridization utilizing an enzymatic hybridization reagent. More particularly, the invention relates to

■a hybridization method for treating target DNA fragments to identify known genetic sequences, the method involving the disposition of the target DNA fragments on a carrier, the formation of a hybridization reagent consisting of a radioactive- labelled probe DNA and catalyzing enzymes, the immersing of the carrier in the hybridization reagent for a given period of time, the washing of the carrier in at least one wash solution, the exposure of X-ray film to the carrier, and the developing of the X-ray film to detect and locate the radioactive- labelled probe DNA, thus identifying a known genetic sequence. The present invention relates to the discovery of the fact that two proteins from the bacterium E. coli. specifically the RecA protein and the single-stranded DNA binding protein (SSB) , in the presence of adenosine triphosphate (ATP) , stimulate the transfer of single-stranded DNA to homologous duplexes in solution. The RecA protein will pair homologous molecules of DNA by a mechanism that differs kinetically from the thermal reannealing of complementary DNA single strands. It will pair homologous single strands of DNA to form the duplex DNA, or it will promote the exchange of a single- strand DNA with duplex-strand DNA. The SSB DNA binding protein binds cooperatively to single-strand regions of DNA to increase the conversion of single- strand DNA into double-strand DNA mediated by RecA protein.

Both of these proteins have been used in the prior art to study the mechanisms of DNA recombination and repair. In addition, the SSB DNA binding protein has been used in electron microscopy to locate single-strand regions of DNA. However, while the biochemical activity of both of these proteins has been documented in the prior art, their use in DNA hybridization is unknown in the prior art. Moreover, the use of these two proteins when the target DNA is disposed on a matrix or carrier, rather than a solution, is also unknown in the prior art.

Thus, the subject invention describes a suitable, novel, rapid ^' enzymatic alternative to physicochemical denaturation/renaturation for hybridization on a matrix, which alternative can bypass the fixation, denaturation, neutralization, lengthy incubation, high temperature and stringent washing steps of the prior art, as described previously.

In accordance with the inventive method, preferably, the carrier or matrix comprises a gel film on which target DNA fragments are placed. However, as also described below, target DNA fragments can be transferred from such a matrix to another matrix by use of the Southern blotting technique, or variations thereof, and the hybridization method can proceed accordingly. The invention calls for the use of an enzymatic hybridization reagent consisting of the following components: 4ON microgra s of RecA protein; 28N micrograms of SSB DNA binding protein; 28ON micrograms of adenosine 5-triphosphate, disodium salt; 100N micrograms of Bovine Albumin; 6N micromoles of Tris; and 4N micromoles of magnesium chloride; where N = the number of milliliters of hybridization reagent. Therefore, it is a primary object of the present invention to provide an enzyme-catalyzed hybridization method for treating target DNA fragments to identify known genetic sequences. It is an additional object of the present invention to provide an enzymatic hybridization reagent formed, inter alia, of RecA protein, SSB DNA binding protein, and ATP.

It is an additional object of the present invention to provide a hybridization method which is capable of treating target DNA fragments to identify known genetic sequences in very rapid fashion relative to prior art techniques.

It is an additional object of the present invention to provide a hybridization method involving curtailed loss of DNA due to less diffusion and fewer step .

It is an additional object of the present invention to provide a hybridization method^' in which the unhybridized, remaining radioactive-labelled probe DNA is reusable in subsequent enzymatic procedures.

The above and other objects of the invention, as will hereinafter appear, and the nature of the invention, will be more clearly understood by reference to the following description and the appended claims. Best Mode for Carrying Out The Invention

The invention will now be more fully described. The target DNA fragments to be treated and a radioactive-labelled probe DNA, to be used (together with certain catalyzing enzymes also disclosed below) in forming the hybridization reagent, were formed as follows: Target DNA: Lambda DNA - Hind III digestion fragments (Bethesda Research Labs Inc., Gaithersburg, Maryland) were placed into sample wells (1.0 or 0.5 micrograms total DNA) , and were separated according to their molecular weight by horizontal electrophoresis in agarose gel (0.6% w/v) , affixed to a glass plate of 15 x 15 cm. in size, and the agarose gel was dried to provide target DNA.

Probe DNA: Radioactive labelled probe DNA was prepared by nick translation of 1.0 micrograms lambda DNA using a reagent kit (BRL Inc. , Gaithersburg, Maryland) and 50 micro-Ci of deoxycytidine-5'-[ oc - ³²P] triphosphate (Amersham Corp., Arlington Heights, Illinois). The enzymatic hybridization reagent (5 milliliters thereof) was formed as follows: the probe DNA;

200 micrograms of RecA protein (P-L Biochemicals, Div. of Pharmacia, Inc., Milwaukee, Wisconsin) ;

140 micrograms of SSB DNA binding protein (Worthington Diagnostic Systems, Inc. , Freehold, New Jersey) ;

1400 micrograms of adenosine 5' triphosphate, disodium salt (Sigma Chemical Company, St. Louis, Missouri) ;

500 micrograms of Pentex Bovine Albumin (Miles Laboratories, Inc., Elkhart, Indiana);

30 micromoles of Tris; and 20 micromoles of magnesium chloride

(MgCl₂), ph of 7.4. Of course, greater or lesser amounts of the reagent may be formed by proportionately adjusting the amounts of the various components. As a first example, the dried gel film (15 x 15cm) , containing the target DNA fragments, was immersed into a solution containing 0.5M NaOH, 3M NaCl for 15 minutes, then into 0.5M Tris, 0.3M NaCl for 15 minutes, and then into the hybridization reagent (5 ml) containing the labelled probe DNA, enzymes and co-factors. It was incubated at 37^βC for one hour. The glass plate bearing the film was washed by gentle agitation a total of 6 times for 5 mins. each wash. Four washes were 50ml 2X SSPE-1% SDS, and the final washes were 50ml of 0.1X SSPE-1% SDS. Note: SSPE includes (per liter) 8.7 gm. NaCl, 1.38 gm NaH₂P0₄ • H₂0, and 0.37 g . EDTA (diosodiu salt) ; ph 7.4. The location of ³²P-labelled probe DNA which was enzymatically hybridized to undenatured target DNA was determined by radiofluorography developed after 24 hours exposure to X-ray film at -70^βC. As a second example, in lieu of performing hybridization enzymatically for target DNA in agarose gel film, the target DNA was transferred to nitrocellulose membrane by the Southern blotting technique and fixed by baking. Enzymatic hybridization was then performed exactly as described above (starting with immersion into the hybridization reagent) .

It should be noted that, whereas some of the probe DNA is used up in this procedure, the enzymes in the reagent are not used up.

The following advantages were obtained by employment of the method and reagent of the present invention: (1) Time for treatment of the target DNA fragments was cut from the 12-24 hours usually associated with the prior art techniques to only 2 hours. (2) Employment of the inventive technique resulted in curtailed loss of DNA, relative to that lost during prior art techniques; this curtailed loss of DNA results from less diffusion and fewer steps in the inventive technique. (3) In the inventive technique, the unhybridized, remaining radioactive-labelled probe DNA is reusable in subsequent enzymatic procedures, and this is of particular advantage in view of the expense involved in procuring/forming the probe DNA. (4) The .inventive technique presents the further advantage of employment of mild conditions; for example, incubation of the dried gel film and target DNA fragments, immersed in the hybridization reagent containing the labelled probe DNA> enzymes and co-factors, takes place at 37^βC. for one hour).

(5) The hybridization method of the invention is characterized by increased sensitivity in that the enzymatic procedure employed therein is possibly more efficient than the thermal annealing technique employed in the prior art.

(6) Finally, the procedure employed in accordance with the inventive hybridization technique is characterized by simplicity, especially in view of the reduced number of steps involved therein. While preferred forms and embodiments have been described above in illustrating the invention, it is to be clearly understood that various changes in detail may be made without departing from the spirit and scope of this disclosure.

Claims

AMENDED CLAIMS

[received by the International Bureau on 23 September 1985 (23.09.85); original claims 1-17 replaced by new claims 1 -17 (3 pages)]

1. A hybridization method for treating target DNA fragments to identify known genetic sequences, comprising the steps of: (a) disposing the target DNA fragments on a carrier;

(b) forming a hybridization reagent from a radioactive-labelled probe DNA and other components including RecA protein and single-stranded DNA binding protein used as catalyzing enzymes;

(c) immersing the carrier in the hybridization reagent for a given period of time;

(d) washing the carrier in at least one washing solution; (e) exposing x-ray film to the carrier;

(f) developing the x-ray film to detect and locate the radioactive-labelled probe DNA; and

(g) correlating the developed x-ray film to the identification of known genetic sequences. 2. The method of claim 1, wherein said carrier comprises a gel film.

3. The method of claim 2, wherein step (a) comprises the placement of said target DNA fragments into sample wells on said gel film and the separation of said target DNA fragments according to weight by electrophoresis.

4. The method of claim 2, wherein step (a) includes the affixation of the gel film to a glass plate, said step (d) comprising washing the gel film and the glass plate in said at least one wash solution.

5. The method of claim 4, wherein the gel film and the glass plate are initially washed in 50 ml. of 2X SSPE - 1% SDS. 6. The method of claim 4, wherein the gel film and the glass are finally washed in 50 ml. of 0.1X SSPE - 1% SDS.

7. The method of claim 1, wherein said other components of said hybridization reagent consist of the following quantities for each milliliter of said enzymatic hybridization reagent: 40 micrograms of RecA protein; 28 micrograms of single-stranded DNA binding protein; 280 micrograms of adenosine 5' triphosphate, disodium salt; 100 micrograms of Bovine Albumin; 6 micromoles of Tris; and 4 micromoles of magnesium chloride.

8. The method of claim 1, wherein, in step (b) said radioactive-labelled probe DNA is obtained by nick translation of 1.0 micrograms of lambda DNA and 50 micro-Ci deoxycytidine-5'-[ o - ³²P] triphosphate.

9. The method of claim 8, wherein step (e) comprises exposing the x-ray film for 24 hours at -70^βC.

10. The method of claim 1, wherein step (e) comprises exposing the x-ray film for 24 hours at -70^βC

11. The method of claim 1, wherein said carrier comprises a blotting matrix.

12. The method of claim 11, wherein step (a) comprises transferring said target DNA fragments to said blotting matrix by a blotting technique.

13. The method of claim 12, wherein said blotting technique is a Southern blotting technique.

14. The method of claim 1, wherein said step (d) comprises initially washing the carrier in 50ml. of 2X SSPE - 1% SDS. 15. The method of claim 1, wherein said step (d) comprises finally washing the carrier in 50 ml. of 0.1X SSPE - 1% SDS.

16. An enzymatic hybridization reagent formed from a radioactive-labelled probe DNA and other components including catalyzing enzymes, said other components consisting of the following quantities for each milliliter of enzymatic hybridization reagent: 40 micrograms of RecA protein; 28 micrograms of single-stranded DNA binding protein; 280 micrograms of adenosine 5' triphosphate, diosodium salt; 100 micrograms of Bovine Albumin; 6 micromoles of Tris; and 4 micromoles of magnesium chloride. 17. The reagent of claim 16, wherein said radioactive-labelled probe DNA is obtained by nick -translation of 1.0 micrograms of lambda DNA and 50 micro-Ci deoxycytidine-5'-[oc - ³²p] triphosphate.