DD265427A1 - PROCESS FOR THE PRODUCTION OF SYNTHETIC DNA BLOBS - Google Patents

Abstract

The invention relates to a process for the preparation of synthetic DNA blocks of larger length. It aims to produce such blocks with higher effectiveness. The object associated with this object is achieved by alternatively providing an intermediate region which contains at least one strand of the recognition sequence for a restriction-cleavage site in both oligonucleotides to be provided per block in the first step of the process and alternately to this end of the oligonucleotides a poly-G or a poly-C tail is attached. After hybridization (including enzymatic refilling involving the Klenow enzyme) and cloning in a bacterial host-vector system, preferably an E. coli system, in the second step of the method, the double-stranded DNA vector, which is then present as a plasmid insert, is used. Rohduplex eliminates the auxiliary inserted GC section, and finally the thereby obtained liniearisierte plasmid DNA, optionally after use of the enzyme S1 nuclease, recircularized by a ligation step, wherein the DNA block is obtained in its desired design. In one embodiment, the expediency of the invention is demonstrated in the case of preparing an about 120 bp DNA block.

Description

-2 26B427

Field of application of the invention

The invention relates to a method for providing synthetic DNA blocks of greater length, which are increasingly required in genetic engineering laboratories.

The application field of the invention lies in the genteclinological research and development as well as downstream in those areas of the biotechnological industry that use gontechnisch modified microorganisms or cell lines for product recovery.

Characteristic of the known state of the art

As a major method for providing functional DNA sequences, genetic engineering laboratories have established gene synthesis from several double-stranded DNA blocks, which in turn are constructed from correspondingly complementary oligonucleotides. For the preparation of such synthetic DNA blocks, the now classic KHORANA method has been known since the end of the 1960s (NK GUPTA et al., Proc. Natl. Acad., Sci., USA, vol. 60, 1968, 1338-1344; KHORANA et al., J. Molec. Biol. Vol. Ti, 1972 , 209-217).

In this earliest synthesis method for D '. Blocks are short oligonucleotides, the length of which is normally 12 to 40 nucleotides, phosphorylated at its 5 'end by a polynucleotide kinase reaction and hybridized by a temperature regime (so-called "annealing".) The overlap region of complementary oligonucleotides comprises about 9 to 10 NuV leotide The high starting temperature of home annealing (about 90C) largely suppresses the generation of mishybridizations between non-complementary or only partially complementary oligonucleotides, resulting in partial DNA duplexing with so-called "nicks", since the oligonucleotides within of a strand are not yet linked. The closing of the nicks is carried out by enzymatic wave in a subsequent l.igationsreaktion the addition of T4 DNA Li (jnse. The resulting double-stranded DNA in KOHRANA process block after its purification, ι., Via polyacrylamide electrophoresis in Since only short oligonucleotides are used in this method, their use entails the disadvantage that a comparatively large number of starting oligonucleotides must be prepared for the synthesis of a functional DNA sequence.

After becoming available DNS synthase machine a modified version of Khorana method found its way into the laboratory technique used in the identical oligonucleotides having a length of 30 to 50 Nuklm ^l. The principle of this modification is to heteroarately hybridize two complementary oligonucleotides with a much larger overlap area. The double-stranded C \ S block is then wildly assembled from several short double-stranded sub-blocks, and these are in turn linked to each other with the aid of T4 · DNA ligase to the respective desired functional DNA sequence.

The general disadvantage of the KHORANA method, including the modified variant, is that the DNA block to be constructed, since it must be fully synthesized, has a high degree of oligonucleotide synthesis capacity. Since 1982, a method developed by ITAKURA for linking synthetic oligonucleotides has also been acknowledged (K. ITAKURA, Trends Biochem., Vol., 7, 1982, 422-445). This method also requires the availability of a synthesizer. In contrast to the KHORANA method, in which the entire sequence of a DNA double block is synthesized; In the ITAKURA procedure, this sequence is still partially synthesized, and the remaining gaps ("gaps") are then filled in enzymatically.

Dio iTAKUKA method has now been developed in 2 variants. In the first variant, several oligonucleotides are phosphorylated analogously to the KHORANA ancestor and subjected to annealing, in the course of which a partial DNA block (duplex) is formed with intervening single-stranded sections, by reaction with the Klenow enzyme, ie a subunit of DNA polymerase I, which has a templete-dependent 5'-3 'polymerase function, and with the addition of nucleotides, the gaps were then closed, the resulting complete DNA duplex is finally cut with Restrictusan to appropriate overhanging block -Ends to advertise in each selected cloning vectors to create.

In the second variant of the ITAKURA method only 2 sufficiently long oligonucleotides are used, whereby both oligonucleotides must have an overlap region at the 3 'end. After an annealing reaction in which the two oligonucleotides hybridize to one another at the 3 'end, the two single-stranded sections are filled in by the template-directed polymerase function of the Klenow enzyme. Re-annealing the resulting DNA duplex with restrictases is also required.

The disadvantage of both variants of the ITAKURA method is that Mißhybridisierungon arise in the overlap region of the oligonucleotides relatively many sequence errors.

Object of the invention

The invention pursues the goal of providing synthetic DNA blocks of greater length in a more effective manner.

Explanation of the essence of the invention

The invention has for its object to describe an improved, easy to handle procedure, with dom provided in a few enzymatic steps oligonucleotides under avoid the <on hybridization errors can be joined to synthetic blocks of greater length.

As a result, this basic problem is solved as follows:

On the synthesis machine, two sufficiently long oligonucleotides (oligonucleotides ie A and B, the latter for the opposite strand of the block) are initially provided for each double-stranded DNA block to be produced. This oligonucleotide A, B

carry in their nucleotide sequence (as viewed from the 5 'end) the desired design with regard to the particular gosamt-ONS to be generated; at the 3 'end, however, they have a provisional auxiliary region in the reading direction, which contains at least the recognition sequence for the cleavage site of a restriction enzyme X or of a pair of mutually compatible restrictionases X1, X2. At this end of both oligonucleotides is in process step I by enzymatic alternately a poly-G- 'zw. Poly-C tail attached or synthesized by the DNA Syntheseauiomaien.

The crude double-stranded DNA block obtained after hybridization as well as enzymatic filling is then cloned in a bacterial host vector system, and embedded in the recombinant vector DNA then becomes the auxiliary inserted GC section from the crude DNA block with the help dor Restrictase (s) or X1, X 2 removed (step II). According to this section of the overall procedure, the pieces of the DNA block to be produced are located at the ends of the linearized vector DNA. By final circularization of this recombinant Voktor DNA (ligation step), the present block pieces, if appropriate after use of the enzyme S1 nuclease, are then combined to form the synthetic DNA block in its final structure (method step III).

This basic feature is further developed in step I of the method to the effect that oligonucleotides A, B are each provided with an auxiliary region arn 3'endi which has the structure in the reading direction

- Erkennungssequon / for restrictase-Spältort X or X1 or X2 (component 1) and

- Extension piece containing two G- or C-nucleotido, (component 2) has.

It proves to be advantageous in this context, in the auxiliary region of the oligonucleotide A or B as component 1, to build up the recognition sequence for a restriction-site cleavage site which is unique in block design and in the cloning vector used, preferably for a uniculate cleavage site with overhanging ends. In particular, in this sub-step in Hilfsberreich the two oligonucleotides a unique cleavage site for one of the following group E

(Accl, BamHI Bell, BglII, ClaI, Sail, Xbal, Xhol) called rostriktases created.

Erfindungswesontlich in this context, in the oligonucleotides A and B, a corresponding unikalo cleavage site for each same or for 2 mutually compatible unique restriction enzyme (s) to create. The preferred group E residues also allow the generation of, for example, a unique cleavage site in the two oligonucleotides for the conclusion of mutually compatible restrictase pairs:

BamHI -BgIII, Bell -BgIII

 BamHI -Bell or Sail -Xhol.

The formation of a corresponding temporary restrictase cleavage site may be initiated at any mature AT, TA, GC or CG dinucleotide occurring at the 3 'end of the oligonucleotides to be delivered. For example, in the dinucleotide GC (by inserting d, tetranucleotide GATC, a cleavage site for the restrictase BamHI can be built up.) If, in the functional total DNA to be generated, naturally "a correspondingly unique restriction enzyme spp" ¹ is present, then the residues [to be delivered oligonucleotides A, B zweckmäßigerweiso so chosen that this Spaltstello comes to rest at the beginning of the TF.ndo each to be aligned auxiliary area.

In the synthesis of the extension piece in the auxiliary region of each oligonucleotide, the following relationship is still to be maintained according to the method: If the two nucleotido GGs are synthesized at the 3 'end of the Α strand, the boide nucleotides are synthesized at the 3' end of the B gene strand. Add CC and vice versa.

With the addition of the supernatant, this interference is conveniently eliminated in the present method, and, assuming that it is not necessary in the synthesis of long oligonucleotides, the desired single stranded membrane is eliminated. (length N) may be separated from its synthesis precursors (of lengths N-1 and N-2).

As will be noted in the Resume to Verfihrensschritt I thus, according to the method according to the invention, two single-stranded elements are supplied by the oligonucleotide, which - apart from the attached minimum so-called extension pieces - still have no overlap region.

In continuation of method step I, in the further embodiment of the invention, at the 3'end of the two oligonucleotides prepared in this way with the aid of the enzyme terminal transferase with the addition of dGTP or of dCTP to the already provided extension piece a poly-G- or poly- C-tail with a length of at least 8 to 10 nucleotides attached (tailing). In this way, in the present method, the overlap region for the subsequent hybridization of both oligonucleotides to a partial DNA duplex is created.

Thus, if oligonucleotide A has a partial initial step at its 3 'end via an extension -GG (and ·· oligonucleotide B thus has an extension -CC), then it becomes a member of torminal transferase with dGTP addition G tail (and oligonucleotide B for the opposite strand under ilCTP addition corresponding to a poly-C tail) attached. In general, by preliminary preliminary experiments, these are process conditions with regard to enzyme addition, Nu. Determine leotide concentration and reaction time, which ensure the addition of 8 to 10 nuklootide Schwonztoilos long.

After the reaction, the Transfernse boids so completed oligonucleotides are subjected in known manner to a temperature Rogime, which leads to ihrei Haybridisierung r .vischen poly-G and poly-C-end (process step II, annealing). For the resulting in this step partial DNA block (C'iplox) with long single-stranded sections at the 5'- and at the 3 'end a Hybridisigrungsbereich of greater stability is characteristic, because this area is according to the book exclusively from GC pairs, between on per For each nucleotide or base pair, in each case 3 hydrogen bonds have been formed.

Then the single-stranded sections of this partial DNA duplex are filled in enzymatically in a manner known per se (closure of the gaps) with the introduction of the Klenov enzyme, and a complete double-stranded crude DNA block is obtained Cut the block ends with one or two restrictase (s) by means of

Ligation immediately in each case a selected bacterial cloning vector, preferably in a polylinker-carrying cloning vector, is inserted. For the cutting at the duplex ends wc'den expediently used such Restriktasen, for the one hand inside the respective constructed doppelrrängigen block no further restriction sites exist and for the selected vector on the other hand a singular winding unit or - about i ι a Polylinker area - also has two unique cleavage sites, i. H. here (X or X1, X2) - foreign restrictases are used.

As bacterial coding hosts, E. coli vectors of the pBR family, such as pBR322, of the E. coli family, such as pUC18 or pUC19, are preferably used in this step of the overall process, and the cloning is carried out in E. coli recipients such as strain HB101, strain JM101 and strain TG1, respectively.

After cloning, the auxiliary inserted G-C segment is removed from the crude DNA block now contained in the recombinant Voktor DNA. For this purpose, in the further embodiment of the present method, within the recombinant vector, the block is cut open at its two temporary internal restriction sites (see method step I) with or with the corresponding unique group E restriction X or X1, X 2 and GC Area separated.

Subsequently, the overhanging ends of these restriction sites are digested using the enzyme S.1 nuclease. At the resulting blunt ends of the recombinant vector DNA is then in turn circularized (üationsation), at the same time the synthetic DNA block assembled to its final structure, ie to the desired in terms of the respective functional total DNA to be generated design becomes (ancestral step III).

If, in step I, the insertion of the auxiliary region into the oligonucleotides A, B could be based on a "naturally occurring" block design of a unique restriction cleavage site, then the S1-nuclease restriction is omitted in step III.

Advantages of the invention

A first advantage of the invention compared to the known technical solutions is seen in the fact that the synthesis performance for oligonucleotides to be provided is further reduced.

In addition, by cutting out the overlap area, i. H. of the G-C auxiliary section, eliminates an essential source for the generation of sequence errors in synthetic DNA blocks.

Furthermore, in the present method, as already mentioned in connection with the presentation of step I, each disturbance is eliminated, which otherwise results from the difficulty in long oligonucleotides: single-stranded-gel molecule, length N) of its synthesis precursors (of lengths N-1 and N-2).

embodiment

Operation and capability of the described method will hereinafter be related to the production of the DNA block

Xbal Hindill

5'-TCT AGA CTT ATG ATT AAA AGA AGC ITA TTA TTT TTA ACT GTT TTA · 3'-AGA TCT GAA TAC TAA TTT TCT TCG AAT AAT AAA AAT TGA CAA AAT

Clal

TTG TTA TTA TTC TCA TTT TCA TCG ATT ACT AAT GAG GTA AGT AAC AAT AAT AG AG T AAA AGT AGC TAA TGA TTA CTC CAT TCA

GCG TCG AC-3 'CGCAGCTG-5'

closer executed or occupied.

The two oligonucleotides required to make this DNA block had the following design: The auxiliary region was introduced at the site marked *, via a unique, unique BglII site. For cloning reasons, an additional EcoRI site was inserted in front of the Xbal site.

The provided synthesized oligonucleotides thus had the following appearance:

EcoRI Xbal Hindlll

A: B'-TTTTTGAAnCTCTAGACTTATGATTAAAAGAAGCTTATTATTTTTAACTGTTTT

BgIII AGATCTGG-3 '

Sail B: 5'-CTGGACGTCGACGCACTTACCTCATTAGTAATCGATGAAAATGAGAATAATAACa

BgIII AGATCTCC-3 '

The process further processing of this oligonucleotide took place according to the following procedure:

1. Attaching the poly-dG or poly-dC tail »

0.3 OD of oligonucleotide A was spiked with poly (dG), a corresponding amount of oligonucleotide B with poly (dC). The reaction was carried out in 25μΙ volume of crude in the following buffer

120mMNa'-cacodylate 0.1mM dithiotreitol 10 MCi Ia- ³² PIdGTP or [a- ³² P] dCTP SOOpg / ml bovine serum albumin 1mM CoCI ₂ The reaction was initiated by the addition of terminal transferase (22 U / pmol of 3 '). -End up). The reaction was carried out at 37 ⁰ C for 20min. Preliminary experiments had shown that under these conditions about 10-15 dG or dC residues are added per end.

The samples were extracted after reaction with phenol / chloroform (equal volume) and twice with ether followed by precipitation from Hiuizjfügon of Vu V 3M Na-aceiate, 10pg tRNA and 2 V ethanol.

2. Hybridization

The combined samples were heated to 90 ° C and then a slow (about 3h) cooling to 30 ⁰ C. After precipitation and Zsntrifugation the samples were dissolved in 10gl ii0mMTris HCl, pH 7.2 1OmMMgCI ₂ 0.1 mM DTE and then united.

3. Klenow Reactlon

The samples were then treated with 3 μl of 110 μl Klenow buffer (0, BM Tris-HCl, pH 7.2, 1, 1 M MgCl ₂ , 1 mM DTE) and 2.5 μl 250 ng / ml bovine serum albumin 2.5 μl 4mMdATP 2.5 mMMdTTP 2, 5MUmMdGTP 2.5MUmMdCTP 11.5MlHiO

filled in and mixed with 10 U Klenow enzyme. The reaction was carried out at 30 ° C for 30 min and abgostoppt by a lOminütiges Incubate at 7O ⁰ C. Subsequently, the filled-in synthetic crude DNA block was reprecipitated.

4. Nachspalton the ends with Restriktasen

The resulting DNA pellet was dissolved in 8 ml 5 × high buffer 28 M redistilled.

Dissolve 2mIcoRI (20U) -plSall (20U) and incubate for 2h at 37 ° C. The 5 χ high buffer consists of 50 mM NaCl 25 mM Tris-HCl, pH 7.6 5OmMMgO ₂ 5mMDTE

Thereafter, the reaction was extracted once with phenol and once with chloroform and reprecipitated. The pellet was taken up in 20 μM 10 mM Tris-HCl, pH7.6, 1 mM EDTA.

5. Ligation in pBR322

10μΙ of the crude synthetic DNA block was spiked with .mu.-cut vector pBR322 (EcoRI-S'ail split and purified by agarose electrophoresis) and ligated. The ligation was carried out in SOmM Tris-HCl, pH 7.6 10mM MgCl ₂ 5mMDTE

1 mM ATP 10UT4 ligase carried out overnight at * 4 'C. Subsequently, the reaction was stopped by heating at 70 ° C for 10 minutes.

β. Transformation in E. coll HB101 Dio Transformation of the ligation mixture was carried out by a standard method (CaCl ₂ , heat shock) in <ism E. coli strain HBI01. The transformants were first selected for ampicillin resistance (40My Ap / ml) and then tested for tetracycline sensitivity (12.5 mg death / ml). All tctracycline-selective colonies were further investigated. A'js η were the plasmids isolated: and tested for unique restriction sites that are not normally present in pBR322 (XbaI, BglII, CIaI).

The plasmid DNA of a clone, which provided the additional 3 sites, was sequenced, showing that the desired sequence of the above-mentioned DNA block was present without errors

7. Removal of the help area

2 μg of DNA from the above pBR322-Derival were in 5OmMNaCI 10mM Tris-HCl, pH 7.5 1OmMMgCl ₂

1 mM DTE

incubated with 10 U RgIM for 2 h at 37 ⁰ C and extracted after checking the cleavage once with phenol and once with chloroform. After precipitated ethanol precipitation, the pellet was poured into 50μl S 1 nuclease buffer:

0.2MNiCI 5OmM N trium acetate, pH 4.5

1mM2r.SO ₄

0.5% glycerol

dissolved and with oil) S 1-nuclease is incubated for 30 min at 37 ⁰ C. After the reaction was again stopped by a one-time phenol extraction and subsequent chloroform extraction. Thereafter, the DNA was reprecipitated. The DNA was then added in 20μΙ 50mM Tris-HCl, pH 7.6 10mMDTE

I mM ATP

dissolved and mixed with 10U T4 ligase. The ligation was again overnight at 14 ° C. The transformation was then again into the E. coli Stamrrv HB101.

Among the obtained trisformants, those could be found in each experimental batch whose plasmidic DNA no longer had a BglII cleavage site. By simple Xbal-Sall restriction cleavage, the DNA of this DNA could be used. DNA block can be isolated in the required Men / β.

Claims (10)

claims: 1. A process for the preparation of synthetic DNA blocks using each 2 oligonucleotides per block (oligonucleotides A, B, the latter for the opposite strand of the block), which in their * Nucleotide sequence that with regard to the respective functional total DNA to be generated wear desired design, characterized in that one - Provides oligonucleotides A, B, each with an auxiliary region at the 3 'end, which contains at least the recognition sequence for the cleavage site of a Restriktase X or einos pair of mutually compatible Restriktasen X1, X2, as well as to this end of the oligonucleotides enzymatically alternately a poly Attaching G- or polyc-C-tail (step I), - The obtained after hybridization and enzymatic filling double-stranded raw DNA block in a bacterial host vector system Moniert and now from the recombinant vector DNA the helically inserted GC section using the Rostriktase (s) X or X1, X 2 removed (step II) and Hereinafter the present block pieces, optionally after insertion of the enzyme S1 nuclease, joins by final circularization of the vector DNA to the synthetic DNA block in its final structure (step III). 2. The method according to claim 1, characterized dvdurch that one provides in step I oligonucleotides A, B with J9 a Hilfabereich which in the reading direction ¹ the structure Recognition sequence for restriction gas cleavage site X or X1 or X2 and - Extension piece containing two G or C nucleotides having. 3. The method according to claim 2, characterized daduro i, that builds in the auxiliary region of the oligonucleotide A and B, the recognition sequence for a unique in the block design restriction cleavage site. 4. The method according to claim 3, characterized in that one builds in the auxiliary region of the oligonucleotide A and B, the recognition sequence for a unique cleavage site of a restrictase with overhanging ends. 5. The method according to claim 4, characterized in that in the auxiliary region of the oligonucleotide A and B, the Erkennungssequer.z fü for a unique cleavage site of a restrictase of der'Gruppo (Acd, BomHI, Bell, BglII, CIuI, Sail, Xbal, Xhol ) (Group E Restrictases). 6. The method according to claim 1 and 2, characterized in that one provides Oligonukleotide A, B, whose auxiliary region is placed at the beginning of the recognition sequence for a naturally occurring in the block design restriction cleavage site X. 7. The method according to claim 1 to 6, characterized in that in step I with terminal transferase with the addition of dGTP or dCTP compliant to the respective extension piece to the oligonucleotide A and B, a poly-G or poly-C Tail of 8 to 10 nucleotides in length appends. 8. The method according to claim 1 to 7, characterized in that in step Il Preparing the complete double-stranded crude DNA block by cutting with one or with two X- or X1, X2-foreign residues for insertion into a bacterial cloning vector, A cloning vector used is an E. coli plasmid of the pBR family or a polylimer-carrying E. coli plafimide of the pUC-f amily, and The cloning is carried out in E. coli receptors such as strain HB101, strain JM101 or strain "Io1. 9. The method according to claim 8, characterized in that one uses in step II plasmid pBR3.?2 or plasmid pUC18 or plasmit pUC19 as a cloning vector. 10. The method according to claim 1,2 and 6, characterized in that in step III, the S1-nuclease reaction entfr'H, when, when inserting the Hilfsbereichcs in the oligonucleotides A, B on a naturally occurring in the block design restrictase Slit location can resort.