DE19856391A1 - Method for the non-radioactive detection of membrane-bound nucleic acids and test kit - Google Patents

Abstract

The present invention relates to a novel method for non-radioactive detection of membrane-bonded nucleic acids, including nucleic acids that, for instance, contain single nucleotide polymorphisms (SNP's), DNA arrays (cosmide, yeast, artificial chromosones (YAC's), bacterial artificial chromosones (BAC's), cDNA's, PCR fragments, oligonucleotides), RNA arrays and all nucleic acid fragments that are tranfered from gels (agarose or PAA) to membranes, including genomic DNA/ plasmid DNA fragments (southern) and mRNA's (northern). The invention also relates to a test kit to carry out said method.

Description

The present invention relates to a new method for non-radioactive detection of membrane-bound Nucleic acids, including nucleic acids, e.g. B. "Single Nucleotide Polymorphisms "(SNP's) contain DNA arrays (Cosmide, Yeast artificial chromosomes [YACs], Bacterial artificial chromosomes [BACs], cDNAs, PCR fragments, Oligonucleotides), RNA arrays and any Nucleic acid fragments made up of gels (agarose or PAA) Membranes have been transferred, including genomic DNA / Plasmid DNA fragments (Southern) and mRNAs (Northern), too are understood, as well as a test kit for performing the Procedure.

To a defined extent, arrays are preferably on one Membrane arranged samples of individual nucleic acids.

Identification of exchanges of individual bases within The DNA (SNP's) is for research into the causes complex diseases, e.g. B. of high blood pressure as well as for diagnostic purposes of great importance (Lander, E.S. (1996): Science 274, 536-539, Collins, F.S. et al. (1997) Science 278, 1580-1581, Marshall, E. (1997): Science 277, 1752-1753). By sequencing the areas of interest these exchanges can be recognized.

Conventionally, the sequence is enzymatic Chain termination method determined in which the sequencing products by incorporating radioactive or not radioactively labeled nucleotides are labeled. in the Connection to sequencing and gel electrophoretic The sequence can be separated by evaluating the Sequence gels are determined. The direct marking of the  However, sequencing products have the disadvantage that only one processed only DNA fragment per sequencing approach can be.

The multiplex technique (see EP-A 0 303 459) overcomes this Disadvantage due to the simultaneous sequencing of several DNA molecules. The resulting mixtures of sequencing products are separated by gel electrophoresis, onto a Transfer nylon membrane and fixed there. By Hybridization of this membrane with one for each DNA fragment specific probe can be successively on the same membrane read the sequences of several DNA fragments become. However, there is a major disadvantage of this method in that the detection of different DNA fragments through the use of radiolabelled probes happens. Automation of the entire process for further increase in throughput is therefore not feasible.

A non-radioactive is also known Detection method by Richterich and Church (Richterich, p., Church, G.M. (1993) Methods Enzymol. 218: 187-222), but has the disadvantages not to be suitable for successive hybridizations and the hybridizations are traditionally not exclusive be carried out at room temperature.

The object of the present invention is therefore a easy to perform non-radioactive process establish that, on the one hand, the sensitivity of radioactive Maintains process or even improves it and on the other one enables immediate, direct, successive detection.

The invention is implemented according to the claims. Surprisingly, this problem can be solved by a method in all steps at room temperature can. The method according to the invention is for non- radioactive detection of membrane-bound nucleic acids  (NA) suitable, in which one known per se Process obtained NA bound to a membrane with a special hybridization buffer, the Tris-HCl, Contains Tris-Base, NaCl, Triton X-100, SDS and Block Reagent and then the bound NA was detected. Not to The scope of the invention includes the detection of Multiplex sequencing products EP 303 459 (G. Church).

Preferably membrane-bound DNA is detected.

The method is preferably characterized by the following steps:

• 1. Pre-hybridization of a method known per se obtained DNA-containing membrane preferably in a Dish, with a special hybridization buffer,
• 2. Hybridization of the membrane with a 5'-biotinylated Probe in the same buffer,
• 3. washing the membrane several times with buffer I which contains PBS (Na ₂ HPO ₄ , NaH ₂ PO ₄ , NaCl, pH approx. 7.3), SDS and block reagent,
• 4. Incubation of the membrane with streptavidin-alkaline Phosphatase conjugate in buffer I,
• 5. washing the membrane once with buffer I, then repeated washing with buffer II, the PBS and SDS contains, as well as repeated equilibration at pH 9.75 Buffer III, which contains Tris-HCl and diethanolamine,
• 6. after equilibration transfer the membrane into a Substrate solution consisting of CDP-Star (Tropix) and buffer III composed, and possibly multiple recordings and Placing the membrane in order to distribute it evenly Substrate solution,
• 7. after a short drip fixation of the membrane, eg. B. on a piece of plastic, and cover, preferably with one Transparent film,
• 8. Expose, preferably with an X-ray film or one CCD camera,  
• 9. after exposure, return of the membrane and repeated Stripping with preheated buffer IV, EDTA and SDS contains
• 10. Wash once with buffer V, the Tris-HCl and NaCl contains.

The evaluation is carried out according to conventional methods such as e.g. B. the principle of "skilled pattern analysis".

The most important step in the above The procedure is Hybridization with a special hybridization buffer, which enables multiple successive hybridizations and a sensitivity is achieved that the radioactive detection is equal or partially improved. The method according to the invention therefore does not radioactive detection of nucleic acids, e.g. B. "Single Nucleotide Polymorphisms "(SNP's) contain a large one Progress. In addition, the Avoid harmful use of radioactivity become. Furthermore, by summary, otherwise time-consuming intermediate steps and by optimizing the Compositions of the solutions and the necessary ones Washing parameters consistent results can be achieved creating the basis for the future development of a automated detection machines could be created.

The test kit according to the invention for non-radioactive detection of nucleic acids, e.g. B. "Single Nucleotide Polymorphisms" (SNP's) contains the following solutions:

Hybridization buffer: 5 l

500 ml 10 x TNT
1250 ml 20% SDS
10 g block reagent (Boehringer Mannheim, DIG-Kit)
3250 ml H ₂

O (bidest)

10 × PBS: 5 l

516.2 g Na ₂

HPO ₄

132.6 g NaH ₂

PO ₄

198.7 g NaCl
H ₂

O ad 5 l pH: approx.7.3

Buffer I: 5 l

10 g block of reagent
250 ml 10x PBS
125 ml 20% SDS
4625 ml H ₂

O (bidest), heat in the microwave for 8 min, stir until block reagent is dissolved

Buffer II: 5 l

250 ml 10x PBS
125 ml 20% SDS
4625 ml H ₂

O (bidest)

10 × buffer III: 2 l

64.4 g TrisHCl
1770 ml H ₂

O (bidest)
210.4 g diethanolamine pH: 9.75 (adjust!)

Buffer IV: 5 l

4700 ml H ₂

O (bidest)
50 ml 0.5 M EDTA, pH 8.0
250 ml 20% SDS

Buffer V: 1 l

920 ml H ₂

O (bidest)
30 ml 5 M NaCl
50 ml 1 M TrisCl, pH 8.0

10 × TNT: 1 l

44.4 g TrisHCl
25.6 g TrisBase
73 g NaCl
818 ml H ₂

O (bidest)
100 ml TritonX-100 pH: approx.8.0

Embodiment

All steps are carried out at room temperature.

• 1. Membrane with the DNA side up in a bowl 180 ml hybridization buffer (28 mM Tris-HCl / 21 mM Tris base / 125 mM NaCl / 1% Triton X-100/5% SDS / 0.2% block Prehybridize for 1 h. Rocky: 12 ° / 0.5
• 2. Discard the pre-hybridization solution. 5'-biotinylated Probe for 5 min. at max. Centrifuge and spin immediately at a final concentration of 10 pmol / ml to 100 ml Give hybridization buffer (200 µM / 5 µl). Membrane for 1 h hybridize. Rocky: 12 ° / 0.5
• 3. Remove the hybridization solution completely. Diaphragm 5 x 5 min with 120 ml of buffer I (29 mM Na ₂ HPO _4/8 mM NaH ₂ PO ₄ / 34mM NaCl / 0.5% SDS, 0.2% block reagent) wash. Always remove the washing solution completely. Make sure that there are no large air bubbles below the membrane. Rocky: 12 ° / 10 for filling the solution, then slowly regulate down to 12 ° / 6 for washing.
• 4. Remove the washing solution completely. Membrane 10 min with 4 µl streptavidin-alkaline phosphatase conjugate (before addition 5 min at max. Centrifuge revolution, Boehringer Incubate in 100 ml buffer I. Rocky: 12 ° / 0.5.
• 5. Remove SvAP solution completely. Membrane 1 x 5 min with 120 ml of buffer I, and then 6 x 5 (/ 29 mM Na ₂ HPO _4/8 mM NaH ₂ PO ₄ 34 mM NaCl / 0.5% SDS) wash min with 120 ml of buffer II. Then equilibrate the membrane 4 × 5 min with 120 ml buffer III (20 mM Tris-HCl / 100 mM diethanolamine / pH: 9,751). Rocky: 12 ° / 10 for filling the solution, then slowly regulate down to 12 ° / 6 for washing / equilibrating.
• 6. In the meantime, 50 µl CDP-Star (Tropix) to 10 ml  Add buffer III (1: 200) and mix well. Then the Substrate solution in the middle of the "CDP-Star only" bowl give.
• 7. Membrane after the last equilibration step from the Take the bowl and let it drain briefly. After that the Membrane with the DNA side down on the substrate solution placed. By repeatedly picking up and dropping the Membrane ensures that the substrate solution evenly distributed under the membrane.
• 8. Let the membrane drain briefly and then in one piece Fix plastic and cover with cling film. 30 min. let it lie.
• 9. Place X-ray film and expose for 30 min. Perhaps Carry out shorter or longer (maximum 2 h) exposures.
• 10. After successful exposure, the membrane back into the Transfer the dish and wash with 120 ml preheated buffer IV (5th mM EDTA / 1% SDS, 85 ° C) strip 6 × 5 min. Rocky: 12 ° / 10 to fill the solution, then slowly to 12 ° / 4 to Regulate stripping down.
• 11. Membrane 1x with 120 ml buffer V (50 mM Tris-HCl / 150 mM Wash NaCl) and start again from step 1. Should the Membrane cannot be rehybridized immediately so it can either in buffer IV or between cling film be temporarily stored.

Claims (11)

1. A method for the non-radioactive detection of membrane-bound nucleic acids (NA), including nucleic acids which, for. B. "Single Nucleotide Polymorphisms"(SNP's) contain, DNA arrays (Cosmide, Yeast artificial chromosomes [YACs], Bacterial artificial chromosomes [BACs], cDNAs, PCR fragments, oligonucleotides), RNA arrays and any nucleic acid fragments that come from Gels (agarose or PAA) were transferred to membranes, including genomic DNA / plasmid DNA fragments (Southern) and mRNAs (Northern), which are characterized in that an NA obtained by methods known per se is bound to hybridizes a membrane at room temperature with a hybridization buffer and then detects the bound NA. 2. The method according to claim 1, characterized in that a membrane-bound DNA

• a) pre-incubated with a special hybridization buffer
• b) hybridized with a labeled probe in the same buffer,
• c) washes several times with a buffer I,
• d) incubated with streptavidin-alkaline phosphatase conjugate in buffer I,
• e) washed once with buffer I, then washed several times with buffer II, and equilibrated several times with buffer III,
• f) after equilibration, take the membrane and transfer it to a substrate solution composed of CDP-Star (Tropix) and buffer III,
• g) after a short drip, the membrane is fixed, covered and
• h) exposed,
• i) after exposure, the membrane is taken and stripped several times with a preheated buffer IV, then washed once with a buffer V and evaluated according to techniques known per se.

3. The method according to claim 1 or 2, characterized in that "single nucleotides Polymorphisms "(SNP's) detected. 4. The method according to claim 1 to 3, characterized in that the hybridization buffer Tris HCl, Tris-Base, NaCl, Triton-X-100, SDS and Block Reagent contains. 5. The method according to claim 1 to 4, characterized in that five times with buffer I, the PBS (pH 7.3), SDS and block reagent, is washed. 6. The method according to claim 1 to 5, characterized in that six times with buffer II, the PBS and containing SDS is washed. 7. The method according to claim 1 to 6, characterized in that the equilibration at pH 9.75 four times with buffer III, the Tris-HCl and diethanolamine contains, takes place. 8. The method according to claim 1 to 7, characterized in that buffer IV contains EDTA and SDS. 9. The method according to claim 1 to 8, characterized in that buffer V Tris-HCl and NaCl contains. 10. Test kit for the non-radioactive detection of membrane-bound DNA, characterized in that it consists of

• a) a hybridization buffer containing 10 × TNT, 20% SDS, block reagent,
• b) 10 × PBS, containing Na ₂ HPO ₄ , NaH ₂ PO ₄ , NaCl, pH: approx. 7.3,
• c) a buffer I containing block reagent, 10 × PBS, 20% SDS
• d) a buffer II containing 10 × PBS, 20% SDS,
• e) 10 × buffer III containing TrisHCl, diethanolamine at pH: 9.75,
• f) a buffer IV containing EDTA, 20% SDS
• g) a buffer V containing NaCl, TrisCl, pH 8.0
• h) 10 × TNT containing TrisHCl, TrisBase, NaCl, TritonX-100 pH: approx.8.0.

11. Test kit according to claim 10, characterized in that it consists of

• a) containing 5 l of hybridization buffer
  500 ml 10 x TNT
  1250 ml 20% SDS
  10 g block of reagent
  3250 ml H ₂ O (bidist)
• b) containing 5 l of 10 × PBS
  516.2 g Na ₂ HPO ₄
  132.6 g NaH ₂ PO ₄
  198.7 g NaCl
  H ₂ O ad 5 l
• c) containing 5 l of buffer I.
  10 g block reagent
  250 ml 10x PBS
  125 ml 20% SDS
  4625 ml H ₂ O (bidist),
• d) containing 5 l of buffer II
  250 ml 10x PBS
  125 ml 20% SDS
  4625 ml H ₂ O (bidist).
• e) containing 2 l of 10 × buffer III
  64.4 g TrisHCl
  1770 ml H ₂ O (bidist)
  210.4 g diethanolamine
• f) containing 5 l of buffer IV
  4700 ml H ₂ O (bidist)
  50 ml 0.5 M EDTA, pH 8.0
  250 ml 20% SDS
• g) containing 1 liter of buffer V.
  920 ml H ₂ O (bidist)
  30 ml 5 M NaCl
  50 ml 1 M TrisCl, pH 8,
• h) containing 1 l of 10 × TNT
  44.4 g TrisHCl
  25.6 g TrisBase
  73 g NaCl
  818 ml H ₂ O (bidist)
  1000 ml TritonX-100