DE3722958A1 - Method for the identification and/or isolation of specific molecules or populations of molecules - Google Patents

Abstract

The method is characterised in that specific properties and/or interactions are utilised. The method according to the invention is highly sensitive, it permits any molecule to be separated out and isolated and populations of molecules containing rare molecules to be worked up and is universally applicable; it permits in an advantageous manner for example the isolation, identification, separation out and fractionation of differentially expressed genes.

Description

The invention relates to a method for identification and / or isolation of specific molecules or molecule po populations.

The following references on the state of the art should be mentioned:
Alt FW et al: J. Biol. Chem. Vol. 253.5 (1978) 1357-1370: Selective Multiplication of Dihydrofolate Reductase Genes in Methotrexate-resistant Variants of Cultured Murine Cells
Aviv H. and Leder P .: Proc. Acad. Sci. Vol. 69, 6 (1972) 1408-1212: Purification of Biologically Active Globin Messenger RNA by Chromatography on Oligothymidylic acid-Cellulose
Berger SL and Birkemeyer CS: Biochemistry Vol. 23 (1979) 5143-5149: Inhibition of Intractable Nucleases with Ribonucleoside- Vanadyl Complexes: Isolation of Messenger Ribonucleic Acid from Resting Lymphocytes
Bobek L. et al: Proc. Natl. Acad. Sci. Vol. 83 (1986) 5544-5548: Characterization of a female-specific cDNA derived from a developmentally regulated mRNA in the human blood fluke Schistosoma mansoni
Brickell PM et al: Nature Vol. 306 (1983) 756-760: Activation of a Qa / Tla class I major histocompatibility antigen gene is a general fearure of oncogenesis in the mouse
Chirgwin JM et al: Biochemistry Vol. 18, 24 (1979) 5294-5299: Isolation of Biologically Active Ribonucleic Acid from Sources Enriched in Ribonuclease
Clancy MC et al: Proc. Nat. Acad. Sci. Vol. 80 (1983) 3000-3004: Isolation of genes expressed preferentially during sporula tion in the Yeast Saccharomyces cerevisiae
Davis MM et al: Proc. Nat. Acad. Sci. Vol. 81 (1984) 2194-2198: Cell-type-specific cDNA probes and the murine I region: The localization and orientation of A
Dworkin MB and Dawid IB: Dev. Biol. 76 (1980) 435-448: Construction of a Cloned Library of Expressed Embryonic Gene Sequences from Xenopus laevis
Dworkin MB and Dawid IB: Dev. Biol. 76 (1980) 449-464: Use of a Cloned Library for the Study of Abundant Poly (A) RNA during Xenopus laevis Development
Hedrick SM et al .: Nature Vol. 308 (1984) 149-153: Isolation of cDNA clones encoding T cell-specific membrane-associated proteins
Helman LJ et al .: Proc. Nat. Acad. Sci. Vol. 84 (1987) 2336-2339: Molecular markers of neuroendocrine development and evidence of environmental regulation
Hirschhorn RR et al .: Proc. Nat. Acad. Sci. Vol. 81 (1984) 6004-6008: Cell-cycle-specific cDNAs from mammalian cells temperature sensitive for growth
Kavathas P. et al .: Proc. Nat. Acad. Sci. Vol. 81 (1984) 7688-7692: Isolation of the gene encoding the human T-lymphocyte diffe rentiation antigen Leu-2 (T 8) by gene transfer and cDNA substraction
Lasky LA et al .: Proc. Nat. Acad. Sci. Vol. 77,9 (1980) 5317-5321: Messenger RNA prevalence in sea urchin embryos measured with cloned cDNAs
Lau LF and Nathans D .: EMBO Vol. 4, 12 (1985) 3145-3151: Identification of a set of genes expressed during the G0 / G1 transition of cultered mouse cells
Lee KL et al .: J. Biol. Chem. Vol. 30 (1985) 16433-16438: Molecular Cloning of cDNAs Cognate to Genes Sensitive to Hormonal Control in Rat Liver
Love JD and Minton KW: Analytical Biochemistry 150 (1985) 429-441 Screening of Library for Differentially Expressed Gene Using in Vitro Transcripts
Linzer DIH and Nathans D .: Proc. Nat. Acad. Sci. Vol. 80 (1983) 4271-4275: Growth-related changes in specific MRMAs of cultered mouse cells
Matrisisian LM et al .: Nucl. Ac. Res. Vol. 13, 3 (1985) 711-726: Epidermal growth factor or serum stimulation of rat fibro blasts induces an elevation in mRNA levels for lactate dehydrogenase and other glycolytic enzymes
Rothberg PG et al .: Mol. Cell. Biol. Vol. 4, 6 (1984) 1096-1103: Structure and Expression of the Oncogene c-myc in Fresh Tumor Material from Patients with Hematopoietic Malignancies
Rhyner TA, Borbely AA and Mallet J .: SENSITIVE HYBRIDIZATION TECHNIQUES AS POWERFUL TOOLS IN MOLEKULAR GENETICS TO IDENTIFY BRAIN-SPECIFIC GENE PRODUCTS in ROLE OF RNA DNA IN BRAIN FUNCTION edited by Giuditta A., Kaplan BB and Zomzely-Neurath C. (1986 ) 303-307 Martinus Nÿhoff Publishing Boston / Dordrecht / Lancaster
Rhyner TA et al .: J. of Neuroscience Res. 16 (1986) 167-181: An Efficient Approach for the Selective Isolation of Specific Transcripts From Complex Brain mRNA Populations
Roewekamp W. and Firtel RA: Dev. Biol. 79 (1980) 409-418: Isolation of Developmentally Regulated Genes from Dictyostelium Royer-Pokora B. et al .: Nature Vol. 322 (1986) 32-38: Cloning the gene for an inherited human disorder-chronic granulomatous disease-on the basis of its chromosomal location
Sargent TD and Dawid IB: Science Vol. 222 (1983) 135-139: Differential Gene Expression in the Gastrula of Xenopus laevis
Scott MRD, Westphal KH and Rigby PWJ: Cell Vol. 34 (1983) 557-567: Activation of Mouse Genes in Transformed Cells
St. John TP and Davis RW: Cell Vol. 16 (1979) 443-452: Isolation of Galactose-Inducible DNA Sequences from Saccharomyces cerevisiae by Differential Plaque Filter Hybridization
Timberlake WE: Dev. Biol. 78 (1980) 497-510: Developmental Gene Regulation in Aspergillus nidulans
Vannice JL, Taylor JM and Ringold GM: Proc. Nat. Acad. Sci. Vol. 81 (1984) 4241-4245: Glucocorticoid-mediated induction of α ₁-acid glycoprotein: Evidence for hormone-regulated RNA processing Williams JG and Lloyd MM: J. Mol. Biol. 129 (1979) 19- 35: Changes in the Abundance of Polyadenylated RNA During Slime Mold Development Measured Using Cloned Molecular Hybridization Probes
Zimmermann CR et al .: Cell Vol. 21 (1980) 709-715: Molecular Cloning and Selection of Genes Regulated in Aspergillus Development
Zurita M. et al .: Proc. Nat. Acad. Sci. Vol. 84 (1987) 2340-2344: Cloning and characterization of a female genital complex cDNA from the liver fluke Fasciola hepatica

Textbooks:
Molecular genetics, R. Knippers, Thieme Stuttgart / New York 1982
Molecular and Cell Biologist, P. v. Sengbusch, Springer Berlin / Heidelberg / New York 1979
Molecular Biology Of The Cell, Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson JD Garland New York / London 1983
Molecular Cell Biology, Darnell J., Lodish H., Baltimore D., Scientific American 1986

In the methods of the prior art, for example show the entire RNA molecules of one or two different their tissue or cell types or two different ones States of the same cell type in labeled cDNAs led and replica filters from genomic or cDNA banks (differential) screened. Another method described assumes two cDNAs that are made up of two different total or Poly A⁺ RNAs are made from. The so won cDNAs are each with the "opposite" RNA population tion hybridizes and then the double stranded cDNA / RNA molecules from the single stranded cDNA molecules separated. With these "state-specific" cDNAs are then screened for gene banks.

The methods and methods of the prior art point insufficient sensitivity, sensitivity and detection probability on. They only allow the Iso "frequent" or only randomly "rare" specific molecules. Hybridization in particular when screening the gene banks depending on the concentration and therefore too insensitive.

In contrast, the present invention has the object to provide a process that is highly sensitive which allows each molecule to be isolated and To process molecular populations with "rare" molecules, and which is universally applicable.

This task is carried out in a method of the type mentioned at the beginning Genus solved according to the invention by that you have specific properties and / or interactions exploits.

Special embodiments are characterized in that
that the molecules or molecule populations involved are deoxy-ribonucleic acids and / or ribonucleic acids and / or proteins and / or peptides and / or organic and / or inorganic and / or inorganic-organic substances or compounds or the like ,
that the molecules involved have a certain and / or any structure,
that the molecules involved are at least partially labeled,
that it is a non-radioactive and / or radioactive label with at least one marker and / or at least one isotope,
that an interaction between molecules of different substance classes and / or the same substance classes is exploited,
that certain (inherent) molecular properties are exploited,
that properties based on the molecular structure are used,
that the base sequence (s) are used by deoxyribonucleic acids and / or by ribonucleic acids and / or by analogues or the like,
that the interaction between (certain) base sequences of DNA and / or RNA with like and / or not like molecules is exploited.

Other special embodiments are characterized in that
that he

• a) from RNA population 1 (RNA ₁) and RNA population 1 (RNA₂) cDNA₁ and cDNA₂ produces
• b) cDNA₁ with RNA₂ and cDNA₂ with RNA₁ hybridizes and
• c) the hybridization mixture is separated or worked up,

that the separation takes place according to double-stranded and single-stranded molecules,
that the separation is carried out by means of hydroxylapatite column chromatography,
that the separated single-stranded cDNA is cloned by suitable measures,
that

• a) the ss cDNA is converted into the ds cDNA
• b) the ds cDNA is converted into ligatable form and
• c) this cDNA is ligated and transformed,

that the ds cDNA is converted into "blunt end" ds cDNA,
that suitable vectors may be used for ligation after dephosphory lation,
that one and / or more components of one and / or more process steps are present at least temporarily and / or at least partially in immobilized form,
that various combinations of procedural steps are used,
that individual process steps are carried out with the help of naturally occurring and / or synthetic and / or described agents, substances, catalysts, enzymes and the like,
that one and / or more of the properties of the interactions in the acted upon connections and / or methods and / or process steps are linked or combined,
that compositions and / or mixtures and / or various process step combinations are applied or applied.

The invention enables in an abruptly increased manner isolation and / or identification more specific Molecules or molecular populations.

The method according to the invention offers particular advantages isolation / identification differentially expressed Genes.

For example, the scope of the invention includes:
Multiple hybridizations with the same or different RNAs, hybridization of cDNA with poly A⁺ RNAs to Iso-regulation of poly A ^- - and regulatory RNAs, interactions with proteins, hybridization of DNAs with DNAs and RNAs with RNAs. Molecule populations are understood to be a mixture of molecules of different structure, sequence and, if necessary, length, each individual molecule being present at least once.

So 1 describes the Fig. Is a schematic of one of the possible methods and procedures according to the invention applications. Two different starting materials (e.g. stimulated and unstimulated, growing and resting cells, old and young cells, differentiated and non-differentiated cells or tissues or organs or the like) are marked with (+) and (-) for distinction ) using a suitable method (for example GTC method (guanidine thiocyanate (guanidine rhodanide) method)) RNA obtained. Then the RNA is either separated by chromatography on an oligo dT column or otherwise and then converted into cDNA with the appropriate enzyme (reverse transcriptase) (cDNA = complementary, copy DNA).

It is also possible and sometimes preferable to go straight out to produce total RNA cDNA.

This cDNA is labeled with "other" ("opposite" RNA) or another molecular population to be subtracted brought under appropriate conditions (hybridized). The resulting mixture of cDNA-RNA hybrids and single stranded cDNA is on a hydroxyapatite column separated and at least the ss cDNA obtained.

This single-stranded (ss) cDNA is converted into double-stranded (ds) cDNA transferred by suitable means such as polymerase or reverse transcriptase. The ends of this ds cDNA are converted into ligatable form ("end polishing"), for example as by means of T 4 DNA polymerase and / or S 1 nuclease and / or mung bean nuclease or the like. The so on prepared ds cDNA is converted into an appropriate vector (Plasmid, phage, cosmid, virus, shuttle vector or the same, who may be prepared in a suitable manner was ligated, such as dephosphorylation, linker addition, etc.) and in competent cells, specially prepared and / or mo  transformed E. coli strains or the like. The transformed cells are under appropriate and Se lesson conditions bred (possibly on plates) and the Colonies, plaques or other resulting cells (after Selection or by means of marked samples) and isolated.

Figs. 2 shows in schematic form the processes for the isolation of poly A ^- and regulatory RNAs. As in the scheme of FIG. 1, cDNA is produced from RNA (ss). After hybridization with "other" RNA and HAP column separation, the (ss) cDNA is hybridized with "other" or "same" poly A⁺ RNA. This hybridization can also take place simultaneously or immediately after the first hybridization.

This is followed by (again) separation on a hydroxyl apatite column.

The thus isolated (+) or (-) cDNAs corresponding poly A ^- and regulatory RNAs and the like, for further characterization as in scheme of Fig be processed first.

Within the scope of the present invention, one or more levels existing at this point which molecules and the like with reagents and / or To apply substances and / or methods and / or agents, such that one and / or more molecular species and / or molecules and / or molecule populations specifically in Interaction occur or modified or converted will. For example, DNA and / or RNA molecules and / or proteins and the like methylated, hydrolyzed, split, broken down, synthesized and the like. This exposure is possibly molecular, moles cool structure, sequence, nucleotide, primary structure, seconds specific structure, tertiary structure, quaternary structure specific, such as single-strand-specific binding proteins (RNA and / or DNA, ss and / or ds), RNA specific hydro lysis for single and / or double strands, DNAse (s) (single and / or double-strand-specific), methylases, modifying enzymes, intercalating agents, fragmenting agents, modifying agents and / or (partial) combinations of these agents and / or the like. Furthermore, it is within the scope of the present Invention non-specific at one or more levels and / or to effect specific syntheses, for example in the production of the first and / or second strand of DNA and / or in the production of a first and / or second  RNA strands and the like. This can be done through specific and / or non-specific "priming" and / or by Setting / selection of suitable reaction conditions will. This can be specific and / or non-specific (Sub) populations are produced and / or enriched. These molecular populations and / or mixtures can then for specific separation and / or implementation accordingly, for example as stated above acted upon and / or processed.

Fig. 3 illustrates in exemplary schematic form, the interaction between a molecular species such as a protein and / or DNA and / or RNA, and any other type of molecule, such as DNA and / or RNA, as well as subsequent steps. It can also Molekülpopula functions accordingly are used according to the invention.

Total DNA is broken down into fragments (restrictions zyme, chemically, mechanically, physically etc.) and with one wholly or partially of cells, tissue or the like purified protein is applied. After corresponding Incubations are the result of interactions DNA-protein complexes separated from free DNA. Out the DNA-protein complexes, the DNA is obtained and after Transfer into ligatable form, such as above executed or in any other suitable way in a ligation introduced with a suitable vector into competent cells transformed, plated and isolated in a suitable manner. One and / or more components of this sequence can also be used in immobilized form.

By combining the different process steps and / or sequences or parts thereof, for example way possible to identify certain cDNA "genes" and / or isolate, if necessary, genomic sequences identify and / or isolate. Proteins deliver (expression systems) and other interacting Isolate DNA and / or RNA sequences. You can at for example, antibodies (against proteins and / or peptides and / or DNA and / or RNA) are used.

"Differential foot printing" can also be done with a combination nation of steps according to the invention will.

A possible (further) processing method for cDNA obtained is described below. The sequence of reactions for the isolation and / or identification of DNA and / or RNA sequences which represent "open reading frames (ORFs) is described in the following in a schematic manner. For example, a λ phage DNA with a" frame Shift mutation "in a suitable gene (for example: the lac Z gene). One of the cDNA populations obtained above is ligated with this vector. In doing so, every 18th ORF statistically abolishes the mutation Host transformed and at a suitable temperature, for example on minimal medium, plates which contain lac tose as the only sugar, wherein only hybrid molecules which have restored the "frame shift mutation" can grow cells. Induction of the phages provides plaques for the isolation of corresponding clones.

To explain the basic biochemical operations, terms and Anglicisms is given to the state of the art Literature and literature cited there, as well as on ange referenced textbooks.

The following examples do not limit the present invention.  

Examples Nick Translation

Materials: Nick-Translations buffer 10 ×:

0.5 MTris-HCl pH 7.5 100 mM MgCl 10 mMDTT

DNA polymerase I E. coli
DNAse 10 mg / ml
Nucleotides: dATP, dTTP, dGTP, α 32 PDCTP
NT mix: 0.5 M each of dATP, dTTP, dGTP, EDGEL 250mM column

Total volume 20 µl

• - Dry 5 µl α ³²PdCTP in Speed Vac
• - Add 2 µl of NT buffer
• - Add 3 µl nucleotide mix
• - Add X µl DNA (plasmids 0.5 µg, genomic 1-2 µg)
• - Add X µl H₂O
• - Add 1 µl DNA polymerase I = 10 U
• - Add 1 µl DNAse 1: 100,000
• - Mix
• - Leave at 15 ° for 1.5-2 hours
• - Add 2.4 µl EDTA 250 mM
• - Apply to the biogel column
• - Take 400 µl fractions
• - Measure 1 µl radiation in the Eppendorf Tscherenkov

Nucleotide mix: 0.5 M each of dATP, dTTP, dGTP
DNAse 10 mg / ml diluted 1: 100,000 in H₂O

Repair of the DNA ends ('end-polishing')

Materials: T 4 DNA polymerase 1 U / µl

• - 100 µl 2nd strand synthesis reaction
• - 8 µl T 4 DNA polymerase = 8 U
• - Incubate at 15 ° <6 hours
• - Add 0.1 vol. LiCl 4 M.
• - 3 vol EtOH
• - Fall at -20 ° ON
• - centrifuge for 20 min. Eppendorf
• - Let the pellet dry
• - Take up in 40 µl H₂O

c-DNA synthesis 1st strand

Materials:

Tris-HCl pH 8.52 M MgCl₂0.1 M NaCl 0.5 M

Nucloids: dATP, dTTP, dGTP, dCTP α ³ SdCTP
Actinomycin D
RNAsin 40 u / µl
DTT
Random primer pd (N) ₆
Reverse transcriptase

HCl1 M NaOH 0.2 M NaAc 0.5 M SDS1%

Biogel column
Scintillation cocktail
PAA urea gel

Reaction volume 100 µl:

c-DNA synthesis 2nd strand

Materials: second strand buffer 10 ×:
Random primer pd (N) ₆
Nucleotides
T 4 DNA ligase
E. Coli polymerase I
ATP 10 mM

Reaction volume 100 µl:

• - 50 µl single strand of c-DNA
• - 2 µl primer = 200 ng (100 ng / µll)
• - 10 µl 10 × ss buffer
• - 18 ul H₂O
• - annael 2 hours at 56 °
• - cool briefly on ice
• - 2 µl ATP 10 mM
• - Add 2 µl dATP, dTTP, dGTP, dCTP 100 mM each
• - 6 µl ligase
• - 4 µl polymerase
• - Incubate at 15 ° ON

the reaction was straight to repair the ends used

Ligations:

Materials: Ligase Buffer 10 ×:

ATP 2 mM
T 4 DNA ligase

Reaction volume: 20-100 µl depending on the ends to be ligated and the amount of DNA
e.g. B. for 20 µl:

• - 2 µl ligase buffer 10 ×
• - 2 µl ATP 2 mM
• - x µl DNA approx. 0.1-0.5 µg
• - y µl H₂O
• - 1 µl T 4 DNA ligase = 10 U
• - Ligate at 15 ° for at least 18 hours

Reaction mixture can be used directly in the transformation be set  

Southern blot:

• - usual agarose gel with EtBr
• - take normal photos after the run
• - Gel in 0.4 M NaOH, 0.6 M NaCl 30 min. at RT under light Let shake
• - Also leave in 1.5 M NaCl, 0.5 M Tris-HCl pH 7.5 for 30 min
• - Prepare the filter:
  Mark side B.
  wet with bidest
  15 minutes. leave in 10 × SSC
• - Set up the blot as usual
• - blot for at least 16 hours
• - Remove the blot as usual
• - rinse briefly in 0.4 M NaOH
• - in 0.2 M Tris-HCl pH 7.5-2 x SSC
• - air drying
• - at 80 ° 30 min. to bake

Double strand sequencing:

• - 2 µl DNA supercoiled in TE (Mini Prep DNA possible ??)
• - add 20 µl 0.2 M NaOH, 0.2 M EDTA
• - 5 min. RT
• - add 2 µl 3 M NaAc pH 4.8 (solution C plasmid prep)
• - add 2 vol.EtOH 30 min. 20 °
• - [wash 2 times with 70% etOH]
• - spin Eppendorf 20 min. full speed
• - dry pellet speed vac
• - Dissolve pellet in 10 µl primer mix + 2.5 µl H₂O
• - anneal 60 min. 60 °
• - cool down to RT (approx. 30 min.)
• - add 3 µl mix to 2 µl "A", "C", "T", "G" solution
• - 20 min. 37 °
• - add 2 µl chase sol.
• - 20 min. 37 °
• - add 4 µl stop sol.
• - 2 min. 95 °
• - load 3 µl on gel

DNA-RNA hybridization in capillaries

Materials: 1st strand c-DNA
Excess RNA
50 µl micropipettes
Hybridization buffer:

NaPi pH 6.8120 mM NaCl820 mM EDTA pH 5.210 mM Poly U10 µl / ml

• - Total yield of a 1st strand c-DNA synthesis in Absorb 20 µl hybridization buffer
• - approx. 200 µg total RNA in 30 µl hybridization p. record, tape
• - Add the c-DNA solution to the RNA solution
• - 20 min. heat to 70 °
• - melt into the capillary
• - check whether the capillaries at both ends are completely melted under the binocular repeat melting if necessary
• - Hybridize at 60 ° for at least 100 hours

Then HAP column

Biogel column

Materials: Biogel buffer:
5 mM Tris-HCl pH 7.8
5 mM NaCl
Biogel A-5 m 200-400 mesh (BIO-RAD)
Pasteur pipette
Glass wool silanized (Serva)

• - Stuff a little glass wool into the pipette
• - Fill up Biogel
• - if possible without air bubbles
• - refill if Biogel has settled
• - Ultimately, the gel bed should constrict the Be a dropper
• - Equilibrate 2-3 ml with Biogel buffer
• - Let the buffer sink in completely
• - Apply the sample in the same volume of the fractions
• - take the appropriate number of fractions

Hydroxyapatite column (HAP)

Materials: Hydroxyapatite SC Serva Suspension 10-20% pure
covered column
Elution buffer Prepare 1 M NaPi unbuffered
[1 mol Na₂HPO₄ · 2 H₂O (MW 177.99) + 1 mol NaH₂PO₄ · H₂O (MW 137.99) / 2 l]
from it
PB 10 = 10 ml 1 M NaPi, pH 6.8 with H₃PO₄
PB 120 = 120 ml 1 M NaPi, pH 6.8 with NaOH
PB 400 = 400 ml 1 M NaPi, pH 6.8 with NaOH
all buffers without SDS
Scintillation cocktail

• - temper the columns to 20 °
• - Fill in the HAP
• - Have it set and refill up to a column bed approx. 4 cm high
• - Do not let surface dry. Give up PB 10
• - Equilibrate with 2-3 column volumes of PB 10
• - Dilute the hybridization sample with H₂O 1:10
• - let the entire 500 µl sink into the column
• - Add another 500 µl PB 10
• - Take 1 ml fractions each
• - add PB 120 after 10 fractions
• - take 10 fractions again
• - then 10 PB 400 fractions
• - 50 µl of all fractions each with 500 µl cocktail measure with the window open
• - the fractions containing the single strands in the Speed Vac
  Concentrate to 250 µl
• - The DNA can be used directly for the synthesis of the second strand be used

Sequencing gel

Materials: 2 glass plates 40 × 20 cm
Spacer 0.2 mm thick
Comb 0.2 mm
Tris-Borat 10 × running buffer:
89 mM Tris base
89 mM boric acid
2mM EDTA

Sample buffer: TBE 1 ×
90% formamide 0.2% xylene cyanol 0.2% bromophenol blue 0.4% orange G

Dimethyldichlorosilane
urea
Acrylamide
Bisacrylamide
Ammonium persulfate
N, N, N, N-tetramethylethylenediamine (TEMED)

• - Apply acrylamide stick:
  30 g acrylamide
  1 g bisacrylamide
  per 100 ml of water
• - Prepare 25% ammonium persulfate solution
• - Clean glass plates very well (EtOH)
• - Silanize the glass plate with "ears" on one side
• - Place a glass plate on 3 sides of the spacer
• - Place the silanized plate with the silanized side on the inside
• - hold together with clamps
• - Seal with agarose if necessary
• - Prepare gel in a feeding bottle:
  • - 22.5 g urea
  • - 20 ml of H₂O
  • - 10 ml acrylamide stick
  • 5 ml TBE 10 ×
• - Dissolve the gel while heating
• - Degas with water jet pump
• - Add 100 µl ammonium persulfate
• - Add 20 µl TEMED
• - mix briefly
• - Gel quickly with 50 ml pipette between the glass plates to water
• - Insert comb
• - Lay down horizontally and wait for the polymerization to occur
• - Carefully remove the lower spacer after polymerizing
• - Clamp the gel in the appliance
• - Fill up the running buffer 1 ×
• - Remove air bubbles from under the gel  
• - carefully pull out the comb
• - Allow the gel to run at 2000 V for 2-3 hours
• - Apply samples prepared by the sequencing reaction using a Hamilton syringe
  Rinse the appropriate gel bag with buffer before each sample application
• - Apply 2000 V.
• - Place X-ray film in the dark room after approx. 3 h
• - Develop film the next day

Genomic DNA Prep. from frozen liver

Materials: liquid N₂
Mortar with pestle
RNAse 10 mg / ml
Phenol saturated with TE + 0.1%

Hydroxyquinolines

TE
LiCl 4 MM
Isopropanol
Proteinase K 10 mg / ml
SDS 10%
Buffer: ½ TE + ½ EDTA 0.2 M and 0.3 M NaCl

• - Crush the liver in a mortar in liquid N₂ to dust with chilled pestle
• - Put the dust in a 20 ml buffer with a chilled spoon
• - fill in falcon tubes
• - Add 200 µl RNAse
• - Mix
• - Add 2 ml 10% SDS
• - 30 min. stir at RT
• - Add 2 ml Proteinase K.
• - with 4 ° shaking until solution has become clear (at least ON)
• - add proteinase if necessary
• - phenol extraction 2-3 hours each
• - Separate phases by centrifugation Sorvall SS 34 4 min 2500 rpm
• - Carefully remove the phenol phase (below)
• - Repeat 2-3 times
• - centrifuge SS 34 for 20 min. 10,000 rpm
• - Supernatant against TE dialyze first at RT for 1 hour, then
  ON in the refrigerator, a factor of 1000 should be reached
• - Add 0.1 vol. LiCl 4 M.
• - Add 0.6 vol. Isopropanol
• - shake at RT ON
• - Spin down SS 34 for 20 min. 4000 rpm 4 °
• - Add 500 µl TE and let ON dissolve at 4 °

Formaldehyde gel

Materials: Agarose type IV
Gene screen buffer 10 ×:
120 mM Tris-HCl
60 mM NaAc
3mM EDTA
pH 7.4
Formaldehyde 37%
Formamide
Load buffer
EtBr (10 mg / ml)

Preparation gel

Total amount 100 ml:

• - 1 g agarose type IV
• - 73 ml of H₂O
• - 10 ml Gene Screen Buffer 10 ×
• - dissolve while heating
• - Allow to cool slightly
• - Add 16.2 ml formaldehyde
• - Pour gel

Preparation sample

• - RNA 30 min. Centrifuge at 13 krpm
• - Carefully remove the excess
• - Dry Speed Vac
• - Record pellet in mix depending on the slot size
• - Shake at RT until pellet is dissolved
• - 10 min. heat to 70 °
• - Add load buffer according to slot size
• - Load gel
• - run at 20 V for 24 hours
• - 1 min. stain in 15 µl EtBr / l H₂O
• - take pictures

GTC RNA preparation

Materials: PBS
GTC:
Adjust 50 g guanidine thiocyanate 0.5 gn-lauryl sarcosine 2.5 ml 1 M NaCi 0.7 ml 14 M β- mercaptoethanol 0.33 ml 30% Antifoam pH 7 with NaOH
pour through pleated filter
filter through Millipore Millex GS 0.22 µm
pick up in the dark
CsCl pillow:
7.5 MCsCl 0.1 MEDTA pH 7.2 5 mM β- mercaptoethanol Depc H²O: autoclave 0.1% diethyl pyrocarbonate
LiCl 4 M
EtOH

• - Centrifuge cells at 4 °
• - Pick up the pellet in sterile PBS
• - centrifuge again (20 min. 2600 rpm)
• - wash again
• - Record pellet in GTC approx. 6 vol.
• - Vortex violently at the moment
• - in polyallomer tubes for SW 40 rotor 6 ml sterile CsCl Fill pillow
• - Give 6 ml each of the cell suspension
• - balance with GTC
• - Centrifuge for 20 hours at 31 krpm 20 °
• - Carefully remove the excess with the baked Pasteur pipette
  up to approx. 1 cm above the bottom of the tube
• - Drain the rest
• - Let the pellet dry
• - Add 450 µl sterile Depp-H₂O
• - Shake at 4 ° ON
• - transfer to baked Eppendorf
• - Add 0.1 vol. LiCl 4 M.
• - Add 2 vol ethanol
• - Record at -20 °

Mini prep

Materials: Phenol buffered with NaAc 0.3 M pH 5
Extraction buffer:
0.3 MNaAc pH 5 10 mM EDTA 0.2% SDS PBS
chloroform
LiCl 4 M

• - centrifuge depending on the cell type (e.g. 15 min 2000 rpm)
• - wash in 2 ml PBS
• - Remove the excess
• - Add 1 ml of 70 ° hot phenol
• - vortex
• - Add 200 µl extraction buffer
• - vortex
• - Add 1 ml chloroform
• - vortex
• - Centrifuge for 2 min 10 krpm
• - Transfer aqueous phase to new Eppendorf
• - extract again
• - Add 1 vol. 4 M LiCl
• - fall at 4 ° ON

Northern blot a) blotting

• - Normal formaldehyde gel
• - Decolor ½ hour in H₂O after photo
• - Prepare filters like Southern
• - Build a normal blot
• - Blot for at least 16 hours
• - Briefly wash the filter in 2 × SSC
• - air drying
• - Bake at 80 ° for 2 hours

b) hybridization

• - Prehybridize at 37 ° in Church buffer for 2-3 hours
• - 500,000-1,000,000 cpm / ml of sample 10 min. cook at 95 °
• - Hybridize at 37 ° ON in Church buffer
• - pour hot sample (spout)
• - 3 × 5 min. wash with warm washing solution at 37 °
• - Wash for 1 hour
• - wet exposure at -70 °
• - develop film after one night
• - Visit the signal

Production of competent E. coli cells

Competent cells are bacteria cells that have an increased receptivity for exogenous DNA. This is due to changes in the structure of the membranes, the exact mechanism is unknown. Is achieved by treating the cells with inorganic ions.

Required materials:
1 colony E. Coli HB 101 or 7118
LB full medium
100 mM CaCl₂ (4 °)

execution

1 day:

• - Inoculate 5 ml LB of a colony
• - incubate at 37 ° ON

2 day:

• - inoculate 200 ml LB with 1 ml of the ON culture
• - Let grow at 37 ° to OD 600 = 0.2
• - centrifuge at 4 °, Sorvall GSA 6000 rpm 10 min
  From here, carry out all steps in the cold room
• - pour off the supernatant
• - Take up the pellet in 100 ml CaCl₂
• - resuspend
• - Leave on ice for 20 min
• - centrifuge as above
• - pour off the supernatant
• - absorb in 5 ml CaCl₂
• - resuspend
• - divide into 400 µl aliquots
• - Freeze immediately in liquid N₂
• - Save at -70 °

Transformation of competent cells with plasmid DNA

Transformation is the uptake of foreign DNA by E. coli cells. With the help of imported Resistance genes can be selected from transformed cells.

Required materials:
Competent cells see DNA 1
Plasmid DNA
Agar plates with the appropriate antibiotic

execution

1 day:

• - Place 1 aliquot of competent cells directly in an ice bath
• - let thaw in ice about ½ hour
• - Add 1ng of plasmid DNA in no more than 5 µl volume
• - mix well
• - Leave on ice for 30 min
• - 2 min 37 °
• - Leave on ice for 2 min
• - Add 600 µl LB
• - Leave at 37 ° C for 60 min
• - 30 and 300 µl on 1 LB plate with antibiotic plate out
• - incubate at 37 ° ON (inverted)

2 day:

• - Count colonies per plate
• - Determine transformation efficiency as: Kol / µg DNA

Plasmid mini preparation

The mini-preparation of plasmids is used in relative a large number of different plasmids in a short time to extract and analyze. Thereby in Accepted that the DNA is quite dirty. The purity is sufficient, however, for. B. a restriction digest perform.

Required materials:
24 colonies containing plasmid
LB medium
Isopropanol
1% agarose gel
24 sterile large test tubes with caps

Solution A:
50 mM glucose
25 mM Tris-HCl pH 8.0
10mM EDTA pH 8.0

Solution B:
200 mM NaOH
1% SDS

Solution C:
3 M NaAc pH 4.8

TE:
10 mM Tris-HCl pH 7.5
1mM EDTA pH 7.5

autoclave all solutions except solution A

execution

1 day:

• - Inoculate 2 ml LB with the appropriate antibiotic with a colony
• - Incubate ON in a test tube at 37 °

2 day:

• - Transfer 1.5 ml of each culture to Eppendorf
• - centrifuge at maximum speed for 2 min
• - Decant the supernatant
• - Take up the pellet in 100 µl solution A.
• - resuspend well
• - Add 200 µl of solution B.
• - mix well
• - Leave on ice for 5 min
• - Add 170 µl of solution C.
• - mix well
• - Leave on ice for 15 minutes
• - Spin down for 10 min
• - Transfer 400 µl of supernatant to fresh Eppendorf
• - Add 400 µl isopropanol
• - mix well
• - Leave at -20 ° for 15 min
• - Centrifuge for 10 min
• - Remove the excess
• - Dry the pellet in Speed Vac for 5 min
• - Take up the pellet in 200 µl TE
• - Apply 10 µl to an agarose gel (see agarose gel)
• - Save at -20 °

Restriction enzyme digestion

Using digestion with restriction enzyme (s) one characterizes z. B. plasmids and their inserts. Man receives a characteristic restriction fragment car by placing the fragments obtained on a gel separates and usually by means of an intercalating Makes dye visible under UV light.

Required materials:

Appropriate enzyme buffer 10 times concentrated (10 ×) bidest water
DNA about 0.5 µg per digestion
Restriction enzyme approx. 2-5 U / µg DNA

execution

1 day:

General:

• - set the appropriate DNA concentration approx. 0.5 µg / 1-2 µl
• - pipette together in an Eppendorf tube: (final volume: 10 µl)
  1 µl enzyme buffer 10 ×
  1-2 µl DNA
  6-7 µl H₂O
  1 µl enzyme
  Add the enzyme last and get it out of the freezer as short as possible
• - Incubate at 37 ° for 60 min
• - Perform agarose gel electrophoresis
  with plasmid mini preparation:
• - 10 µl Mini-Prep DNA (without concentration determination
• - 1.5 µl enzyme buffer 10 ×
• - 1 µl RNAse (boiled 10 mg / ml)
• - 1 µl H₂O
• - 1.5 µl enzyme
• - Incubate at 37 ° for 90 min
• - agarose gel electrophoresis

Agarose gel electrophoresis

Electrophoresis in agarose gels is usually used to Separate DNA or RNA according to their size. Depending on The size of the fragments to be expected is the agarose concentration tration selected. The smaller the pieces, the higher the Concentration. Usual concentrations vary between 0.8 and 2% agarose. The separation takes place at a pH of 7.8 in an electrical field at 60-80 V. The DNA Fragments migrate to the anode.

Required materials:

Type IV agarose
Chamber for pouring the gel with a comb
Running chamber
Power supply
Tris acetate 10 × running buffer:
400 mM Tris base
50 MM NaAc
10mM EDTA
adjust to pH 7.8 with acetic acid

Sample buffer:
Tris acetate buffer 1 ×

0.25% bromophenol blue 0.25% xylene cyanol 60mMEDTA 50% glycerin

Ethidium bromide 10 mg / ml

execution

• - Weigh the required amount of agarose
• - for 100 ml 1% gel 1 g agarose all information for 100 ml gel
• - Add 10 ml 10 × buffer
• - fill up to 100 ml with Bidest
• - dissolve while heating
• - Add 5 µl EtBr
• - Insert the comb into the casting chamber
• - Pour dissolved gel into the chamber
• - freeze
• - Prepare 1 l running buffer (1 ×)
• - 50 µl EtBr added
• - Carefully remove the comb
• - Place the gel in the chamber filled with buffer
• - Cover gel 1-2 cm with buffer
• - Add 4 µl sample buffer to the sample
• - centrifuge briefly
• - Carefully load the sample into the pockets
• - Apply 60-80 V voltage
• - Direction of travel from - to +
• - Take Polaroid photo after 2-3 hours

Sanger sequencing

DNA sequencing enables us to do this Order of the four nucleotides in any DNA determine. There are different methods for this. The two most commonly used are chemical fission after Maxam and Gilbert, as well as the chain termination method Singer.

The chain termination method used here is based on the Incorporation of nucleotide analogs and the resulting Termination of the chain synthesized by the polymerase becomes. As nucleotide analogs are 2 ', 3'-dideoxynucleotides used. The marking is done with a ³⁵S- Nucleotides (in this case dCTP). Then the Separation of the fragments obtained in a polyacrylamide Gel. The resulting band pattern is shown by an x-ray film made visible.

Required materials:
F1 phages
LB medium
Colony with pEMBL plasmid or the like or M 13 phage
TM buffer 10 ×:
100 mM Tris-HCl pH 8.5
50 mM MgCl₂

Chase solution:
0.25 mM dNTP mix (dATP + dTTP + dGTP + dCTP)

Dideoxy / Deoxy mixture:
"C" 20 µM ddCTP, 37.5 µM dATP, dGTP, dTTP each
"A" 100 µM ddATP, 5.4 µM dATP. each 54 µM dGTP, dTTP
"G" 100 µM ddGTP, 5.4 µM dGTP, each 54 µM dATP, dTTP
"T" 450 µM ddTTp, 5.4 µM dTTp, each 54 µM dGTP, dATP
all mixtures in 10 mM Tris-HCl pH 8.0, 5 mM MgCl₂, 7.5 mM DTT
³⁵S dCTP (500 Ci / mM, 10 mCi / ml)
DNA polymerase I large fragment (Klenow fragment)
Stop solution: sample buffer + 10 mM EDTA
M 13 primer
small microtiter plates
TE buffer
PEG solution 20% + 14.6% NaCl
Phenol equilibrated with Tris-HCL 10 mM pH 9
chloroform
LiCl 4 M
Ethanol

execution a) Production of the template

1 day:

• - Inoculate 1 ml LB medium + antibiotic with one colony
• - Incubate at 37 ° ON

2 day:

• - Inoculate 3 ml LB medium + antibiotic with 100 µl ON culture
• - grow at 37 ° to OD 600 = 0.2 (= 2 × 10⁸ Bakt./ml) approx. 60 min
• - infected with F 1 phages with 20 pfu / bakt.
• - incubate at 37 ° for 5-6 hours
• - Transfer to two large Eppendorf
• - centrifuge at 14,000 rpm for 10 min
• - Transfer the supernatant to fresh Eppendorf
• - centrifuge as above
• - save an Eppendorf to the reserve at 4 °
• - in a second Eppendorf (1.1-1.2 ml) 150 µl 20% PEG, Add 14.6% NaCl
• - Leave at RT for 10 min
• - Centrifuge for 5 min
• - Centrifuge again and carefully remove the supernatant
• - Take up the pellet in 200 µl TE
• - Leave at RT for 60 min
• - Extract phenol (pH 9) 1 ×
• - Extract chloroform 1 ×
• - add 1/10 vol. LiCl 4 M + 2.5 vol. EtOH
• - Leave at -20 ° ON

3 day:

• - centrifuge for 10 min 14 000 rpm
• - Wash the pellet with 70% EtOH
• - Dry in Speed Vac for 10 min
• - Take up the pellet in 30 µl H₂O
• - Check 1 µl for agarose gel
• - Save at -20 °

b) Annealing the template

• - Place 7.5 µl template in Eppendorf
• - Add 5 µl M 13 primer mix (24 µl TM + 2.4 µl = 6 ng primer + 21.6 µl H₂O)
• - Let anneal at 60 ° for 60 min
• - Let cool to RT

c) sequencing reaction

• - prepare in a small microtiter plate: 4 × 2 µl "A" or "C" or "G" or "T" mix
• - make the following mixture:
  10 µl annealing reaction
  + 2.5 µl ³⁵S dCTP
  + 2 µl Klenow fragment (5 U / µl)
  mix well
• - Add 3 µl of this mixture to the microtiter plate
• - Incubate at 37 ° for 20 min
• - Add 2 µl Chase solution each
• - Leave at 37 ° for 20 minutes
• - Add 4 µl stop buffer each
• - Leave at 95 ° for 2 minutes
• - Load 2.5 µl of this onto the sequencing gel.

Claims (22)

1. A method for identifying and / or isolating specific molecules or molecular populations, characterized in that specific properties and / or interactions are exploited. 2. The method according to claim 1, characterized, that it is the molecules or molecule involved populations around deoxy-ribonucleic acids and / or ribo nucleic acids and / or Proteins and / or peptides and / or organic and / or inorganic and / or inorganic-organic substances or connections or the like. 3. The method according to claims 1 to 2, characterized, that the molecules involved have a certain and / or have any structure. 4. The method according to claims 1 to 3, characterized, that the molecules involved are at least partially are marked. 5. The method according to claims 1 to 4, characterized, that it is a non-radioactive and / or radioactive labeling with at least one marker and / or at least one isotope. 6. The method according to claims 1 to 5, characterized, that an interaction between different molecules Substance classes and / or the same substance classes is exploited.   7. The method according to claims 1 to 6, characterized, that certain (inherent) molecular properties are exploited will. 8. The method according to claims 1 to 7, characterized, that properties based on the molecular structure be exploited. 9. The method according to claims 1 to 8, characterized, that the base sequence (s) of deoxyribonucleic acids and / or of ribonucleic acids and / or of analogs or the like is used. 10. The method according to claims 1 to 9, characterized, that the interaction between (certain) Base sequences of DNA and / or RNA with similar ones and / or molecules of the same type are not used. 11. The method according to claims 1 to 10, characterized in that

• a) from RNA population 1 (RNA 1) and RNA population 2 (RNA 2) produces cDNA 1 and cDNA 2
• b) cDNA₁ hybridized with RNA₂ and cDNA₂ with RNA₁ and
• c) the hybridization mixture is separated or worked up.

12. The method according to claims 1 to 11, characterized, that the separation according to double-stranded and single-stranded molecules.   13. The method according to claims 1 to 12, characterized, that the separation by hydroxyapatite Column chromatography is carried out. 14. The method according to claims 1 to 13, characterized, that the separated single-stranded cDNA by appropriate measures are cloned. 15. The method according to claims 1 to 14, characterized in that

• a) the ss cDNA is converted into the ds cDNA
• b) the ds cDNA is converted into ligatable form and
• c) this cDNA is ligated and transformed.

16. The method according to claims 1 to 15, characterized, that the ds cDNA converted to "blunt end" ds cDNA becomes. 17. The method according to claims 1 to 16, characterized, that suitable vectors for ligation Dephosphorylation can be used. 18. The method according to claims 1 to 17, characterized, that one and / or more components of one and / or several process steps at least temporarily and / or at least partially in immobilized form is present. 19. The method according to claims 1 to 18, characterized, that different combinations of process steps be used.   20. The method according to claims 1 to 19, characterized, that individual process steps with the help of Naturally occurring and / or synthetic and / or be written agents, substances, catalysts, Enzymes and the like can be performed. 21. The method according to claims 1 to 20, characterized, that one and / or more of the properties of the Interactions in the loaded connections and / or methods and / or process steps are linked or combined. 22. The method according to claims 1 to 21, characterized, that compositions and / or mixtures and / or different Process step combinations acted upon or be applied.