DE3544459C2 - Process for synthesizing long chain deoxyribonucleic acid (DNA) - Google Patents

Description

The invention relates to a method for synthesizing long chain deoxyribonucleic acid (DNA), the information for the synthesis of specific proteins according to the preamble of claim 1. In particular The invention is based on a method for synthesizing long chain DNA in pure form and chemically, d. H. without the use of enzymes, through the so-called solid phase technique using blocks with 4 to 8 base sequences and directed using aminated CPG as a carrier.

It is already known since the synthesis of polypeptides through genetic engineering measures using synthetic Genes through stages (1) synthesis of structural gene; (2) recombination of the gene into the appropriate plasmid; (3) transformation of a suitable host organism through the formed Chimeric plasmid; and (4) recovering the desired polypeptide is possible by culturing the transformed substance.

In recent times, the development of DNA probes has drawn that Interest as a novel method of genetic engineering. This is an identification method for unknown DNA and RNA (ribonucleic acid), which is a transcribed, by hybridization obtained from known single-stranded DNA and RNA Product are, the ability of DNA and RNA, by selection a complementary substance as well as the relationship of Template and cast to form a double helix, exploited becomes. Because a sensitive and rapid identification through Application of the hybridization method is possible by Finding specific DNA and RNA in the gene level of blood and cells from patients and pathogenic bacteria using this method for the diagnosis of the exact name of a pathogen be applied. Hence DNA is when used as a DNA probe of great value as a diagnostic tool.  

Like the DNA mentioned above as a structural gene and as Source for use as a DNA probe is concerned with their nature became known that the longer the Base sequence is all the more important as information carrier and the wider the field of application as a DNA probe. However, it is also known that their synthesis is all the more so the more difficult the longer the base sequence is.

Hence the development of a technique for synthesizing of long chain DNA is extremely desirable in a simple manner.

Conventional methods for synthesizing DNA run like follows from: First, a comparatively short DNA Chemically synthesized fragment with 10 to 20 base residues, then they become fragments with the overall structure of double stranded DNA coupled to the information for the desired peptide synthesis, and eventually they will suitably coupled in aqueous solution using an enzyme called DNA ligase.

This method, however, is used before block condensation only comparatively short fragments with one (monomer), two (Dimer) or three (trimer) bases made and it is not possible to synthesize long-chain DNA with 80 residues or the like.

It is also essential with this method that DNA- To use ligase designated enzyme. So it's for synthesizing of double-stranded DNA as a gene required that all base sequences that make up the double-stranded DNA can be synthesized simultaneously. Hence this method in a method of synthesizing double-stranded DNA not particularly effective.

According to the invention, the studies are continued to the mentioned overcome technical difficulties. It could by using the procedure known as the Trieste Method  (under the so-called solid phase techniques), in which 1% Polystyrene used as a carrier and tetramers (with 4 bases) or pentamers (with 5 bases) repeated block condensation undergo the synthesis of DNA with about 46 bases can be achieved.

Even with this method, however, the number of bases was in the DNA obtained at most 50, and the difficulty of the Synthesis of DNA with long chains, such as with 80 to 150 residues, persisted.

The object of the invention is therefore to provide a method for synthesizing of deoxyribonucleic acid (DNA) with which (1) the synthesis of long-chain DNA with correspondingly extensive Genetic information and (2) synthesis under cheaper Conditions is possible.

According to the invention, this object is achieved by features specified in the claim solved.

The present invention will hereinafter be described in more detail explained.

Each block can be conventionally pre-condensed can be obtained, each base of a liquid phase synthesis is subjected.  

Animated CPG (glass with regulated pores; controlled pore Glass) (cf. Tetrahedron Letters, 24, 747-750, 1983) is invented used as a carrier in solid phase technology. To the amino group This substance binds to deoxythymidine is converted into 3'-succinate in the usual way. This is called Carrier used for the nucleoside. Any block you want is successively on this resin towards the 5'-terminal Group extended. As for the condensing agent, it can for example, mesitylenesulfonyl-3-nitrotriazolide (MSNT) is used will. The DNA obtained in this way is single-stranded, and the complementary strand DNA, which is used to generate the DNA Double helix required can easily be done in a similar way be preserved. Such a DNA double helix can also easily by using DNA polymerase using short Fragments (10 base pairs or the like) are obtained as a template, with the 3'-terminal region of the single-stranded DNA obtained are complementary. The fact that DNA polymers are used is one of the most beneficial merits of the present invention, since the condensation reaction without Use of DNA ligase can be performed at the conventional methods are widely used.

The double helix DNA obtained becomes the in a known manner Vector plasmid then coupled to bacteria like Escherichia coli, transformed, and the strain cultivated, to get the desired polypeptide. With the above Different genetic engineering measures can be used in stages have already been introduced.

According to the invention, it is possible to use long-chain DNA, such as at 80 to 150 residues, to synthesize, and consequently polypeptides with 15 to 30 amino acids by known genetic engineering Measures are synthesized. For example the following polypeptides are synthesized: This is the release growth hormone inhibiting somatostatin (14 Amino acids), the gastric acid secretion stimulant gastrin  (17 amino acids) that stimulates pancreatic secretion Duodenal hormone secretin (27 amino acids), which the Secretion of growth hormone, insulin and the rise in blood sugar stimulating glucagon (29 amino acids), morphine-like agents (β-endorphin, 31 amino acids) and Lime level increasing agents (calcitonin, 32 amino acids) u. the like

In addition, the long chain DNA according to the invention is not only used for DNA base sequences of structural genes, but also for the production of general DNA, including regulatory areas and specific sequences, as well for long-chain DNA probes to clarify their structures. Consequently, the invention can be successfully used for development diagnostic tools.

According to the invention, long chain DNA can be simple and large Quantities are synthesized. The long chain DNA after the Invention can be effective in the field of genetic engineering (1) as a gene information carrier for polypeptide synthesis and (2) used as a starting material for use in DNA probes will.

So far, the production of DNA was at best per approach 0.1 OD (1OD is about 50 µm equivalent). According to the invention however, it is now possible to use large quantities of 30 to 50 OD to manufacture per approach. As a result, an expansion of the Applicability in the field of long chain DNA as a gene and expected as a DNA probe.

The invention is explained in more detail below with the aid of examples, which concern the synthesis of endorphin, its physiological Effectiveness, such as the effect than on the central nervous system acting analgesic and endocrine hormone, known are.  

(1) Synthesis of each block containing the base sequences for represents the endorphin gene

The amino acid sequence of endorphins is known and the DNA Base sequence corresponding to this can be done using a Coding table can be chosen freely. You are below along with their relationship between each block that the DNA base sequence as used according to the invention are specified. The top, the middle and the bottom Columns (numbers in it are block numbers) denote each Block, the base sequence or the corresponding amino acid sequence. Incidentally, at both endpoints are the DNA base sequences restricted enzyme sites specified. These places are used to insert plasmit.

Among the blocks representing the above endorphin genes block 7 was synthesized in the manner explained below.

Further blocks representing the genes of the endorphin type (1-6 and 8-17) can be made in a similar manner. The respective yields are summarized below.

(2) Synthesis of endorphin genes

α-endorphin gene (deoxy-80-mer) with restricted enzyme sites was synthesized using the solid phase technique as follows:

• 1. Deoxythymidine-CPG resin is washed with CH₂Cl / MeOH.
• 2. It is detritylated with 2% BSA / CH₂Cl₂-MeOH (this process is repeated and carried out quickly until the discoloration disappears).
• 3. After replacement with pyridine it becomes azeotropic drying subjected. A pyridine solution of each block is added Dried azeotropically and MSNT and pyridine for implementation added. It is left to stand at room temperature and washed with pyridine.
• 4. 0.1 M dimethylaminopyridine / pyridine solution and acetic anhydride are added at room temperature left standing and washed with pyridine.

This process is repeated, i. H. performed a total of 13 times. The average yield of this implementation is 84%. Then the resin comes in at room temperature a 0.1 M solution of tetramethylguanidine pyridine doxime (see C. B. Reese et al., Tetrahedron Letters, 2727, 1978) freed from the protective groups in dioxane water, then with Pyridine water washed, the wash water in a vacuum concentrated, conc. Added ammonia water, and that Mixture warmed. The ammonia is evaporated, and one Part of the residue using the dimethoxytrityl group as a target for calculating the output of the final stage taken.  

The residue reaction solution was subjected to reverse phase chromatography (C₁₈ silica gel for Prep 500, manufactured by Waters), ion exchange chromatography (DEAE-Toyopal) and Phase reversal chromatography (C₁₈ silica gel, TSK gel 10-20 µm) (open) for obtaining pure α-endorphin gene subjected to restricted enzyme sites (= deoxy-80-mer).

The purity was brought about by HPCL (Nucleosil 300-7 C₁₈) and by electrophoresis, and the base sequences confirmed by the Maxam-Gilbert method. The results are shown in FIGS. 1 to 3.

In the same way, α- (Leu⁵) endorphin gene (with restrictive Enzyme sites) (deoxy-77-mer), γ- (Leu⁵) endorphin gene (with restrictive enzyme sites) (deoxy-77-mer) and γ-endorphin Gene (with restrictive enzyme sites) (Deoxy-80-mer).

(3) Synthesis of double-stranded DNA and their combination with vector plasmid

1 mole deoxy-80-mer and synthetic starter nucleotide (nucleotide primer), which with the 3'-terminal group of the former is complementary, mixed, heated to 65 ° C and cooled to room temperature to the deoxy-80-mer and that Anneal starter nucleotide. Then E. coli polymerase I (Klenow fragment) added in a conventional manner and during 30 minutes at 37 ° C, so that a double-stranded DNA was formed.

The DNA was obtained as a precipitate in ethanol, 30 min long at 37 ° C using T₄ polynucleotide kinase implemented, and both 5'-terminal groups double-stranded DNA phosphorylated.  

Then the vector plasmid pUC-8 DNA with a restrictive Enzyme Pst 1 cut the above solution from double-stranded DNA added overnight at 16 ° C with T₄ DNA ligase implemented, and the double-stranded D-80-mer DNA coupled to the vector plasmid.

(4) Cloning the plasmid containing endorphin gene

The plasmid prepared as described above was transformed in E. coli JM-103 strain in the usual way, then taking advantage of a defect in the pUC 8 as Selected existing β-galactosidase activity, and the plasmid molecules by cloning from the strain receive.

It was confirmed by the Maxam-Gilber method that Plasmid with the endorphin gene in the correct orientation and position as intended was inserted.

(5) Recovery of the endorphin

Transformed E. coli JM-103 strain was grown overnight in pre-cultured in an LB medium, planted in a 2YT medium and subjected to a shaking culture at 37 ° C.

IPTG was in logarithmic stages of the manufacturing stages (Initial, middle and final stages) added to a concentration of 0.5 mM and the synthesis of endorphin was induced. After the induction by IPTG that became extracted molten protein and analyzed by HPLC, an adequate amount of protein production was found was observed (1 to 5.0 × 10⁵ molecules per Cell) if the induction at the initial stage of logarithmic Manufacturing phase is exercised.  

With reference to natural α-endorphin and γ-endorphin with a methionine residue in the molecule they were with Trypsin treated in a conventional way. In reference to on α- (Leu⁵) -endorphin and γ- (Leu⁵) -endorphin with one Leucine residue instead of methionine, they were treated with BrCN. In any case, each of the desired endorphin proteins column chromatography according to the general Process of protein purification, whereupon each was separated from them and cleaned.

The fact that each of the endorphin molecules obtained has the desired amino acid sequence was determined by the Fact confirmed that they were identical to that already samples obtained by peptide synthesis as by testing determined by HPLC using a phase reversal carrier has been.

Fig. 1 is an autoradiogram showing the result of 20% polyacrylamide electrophoresis of deoxy-80-mer containing the α-endorphin gene and synthesized after being determined by the Maxam-Gilbert method;

Fig. 2 is an autoradiogram showing the result of 8% polyacrylamide electrophoresis of deoxy-80-mer containing the α-endorphin gene and synthesized after being determined by the Maxam-Gilbert method; and

Fig. 3 shows the result of high performance liquid chromatography (Nucleosil 300-7 C₁₈) of deoxy-80-mer, which contains α-endorphin gene and is synthesized after determination by the Maxam-Gilbert method; the ordinate and the abscissa show the absorption and the time, respectively; triethylamine acetate / acetonitrile was used as the solvent system and the flow rate was 1.0 ml / min.

Claims (1)

A process for synthesizing long-chain deoxyribonucleic acid (DNA) using the solid phase technique (Triester method) and aminated CPG as a carrier, characterized in that blocks with 4 to 8 base sequences are chemically coupled in pure form, the purification of the long-chain DNA by a) phase reversal chromatography ,
b) ion exchange chromatography and
c) Perform reverse phase chromatography in that order.