DK175230B1 - Method for producing double-stranded DNA as well as oligonucleotide - Google Patents

Abstract

It is possible by synthesis of a DNA single strand with a hairpin structure at one or both ends, filling in to give the double strand and opening or elimination of the hairpin structure(s) to obtain even very long double-stranded DNA fragments or genes. It is also possible to attach mutagenic primers to the single strand and thus to carry out site-specific mutagenesis. Furthermore, the preparation of highly labelled DNA sequences is possible in the filling-in reaction. <IMAGE>

Description

in DK 175230 B1

The invention relates to an efficient method for producing double-stranded DNA as well as an oligonucleotide (see Table I). In particular, the invention relates to an efficient method for producing DNA fragments and genes which, due to the simple and rational method, also allow the synthesis of very long DNA sequences.

The usual method for producing double-stranded DNA is to chemically prepare overlapping single-stranded oligonucleotides, and because of the natural base pairing, these are converted by DNA ligase to the double helical form (HG Khorana (1979) Science 203, 614-625). An alternative to the Khorana method is the so-called "fill-in" method, which utilizes the repair properties of the DNA polymerase. Hereby, two chemically produced single-stranded oligonucleotides having a short region of complementary nucleotide sequences in common are used (J. J. Rossi et al. (1982) J. Biol.

Chem. 257, 9226-9229). The first method has the disadvantage that one always has to chemically prepare the two strands of the double stranded DNA, thereby chemically producing and enzymatically linking at least three oligonucleotides, in general, however, a larger number of oligonucleotides. Although the synthetic unfolding is reduced by the "fill-in" method compared to the Khorana method, however, at least 25 oligonucleotides are required. Furthermore, it is a major problem that the two oligonucleotides must coincide with the desired defined hybrid structure. The longer the oligonucleotides used, the greater the risk of formation of false hybrids.

European Patent Publication No. 70,675 relates to a method for synthesizing dsc DNA encoding calcitonin, thereby spontaneously forming hairpin structures.

Thus, this method has limited utility as it is fortuitous whether hairpin structures are formed and whether a formed hairpin structure is suitable for producing the desired double-stranded DNA.

I DK 175230 B1 I

I I

In the process of the invention for preparation I

I of double-stranded DNA does not have these drawbacks. According to op- I

In the finding, a single strand of DNA is synthesized with a hairpin

In structure at one end or at both ends, the complementary I

In 5 nucleotides are filled in and the hairpin structure (s) Γ I

You open or split. The process of the invention I

Thus, you enable targeted production of the desired double

In belt-stranded DNA. IN

The figure illustrates a particular embodiment of I

In the invention:

I (1) denotes a single DNA strand with a hairpin structure I

At the 3 'end, whereby within the region of the paired I

In bases (indicated by vertical lines) lies an I

At the intersection of a restriction enzyme (indicated by I

In 15 a small arrow). IN

I (2) denotes after intersection with the restriction enzyme I

I obtained DNA strand which at the 3 'end has a short sequence of I

In paired bases (vertical dashes) and the one for restriction- I

In the zymet characteristic "supernatural" ending. IN

20 (3) denotes another single DNA strand with a hair I

In needle structure at the 3 'end, which at the 5' end has bases that are I

In complementary to the "above" end bases from (2). IN

In (4), the ligation product denotes (2) and (3). IN

I (5) denotes the completed doublet obtained from (4)

In 25 belt string. IN

In the invention, a number of embodiments which are suitable for use are provided

You are further elucidated below or defined in pa- I

In the tent requirements. Thus, in the following, individual I is stated

In embodiments in a form in which e.g. only you are

In a hairpin structure, this mode of expression should serve you only

In simplification, it must in no way rule out a variation

In or in combination with other embodiments. IN

In a preferred embodiment of the invention,

You first synthesize an oligonucleotide having at the 31-end an I

In 35 cards opposite repeat of the sequence. This leads to you

In this oligonucleotide, it falls backward at the 3 'end, and thus I

DK 175230 B1 3 assumes a defined secondary structure. Eg. the oligonucleotide (1) in the figure assumes the hairpin structure shown in Table I. The thus hybridized 3 'end now serves as a "primer" for the filling of the second DNA-5 strand. It has been found that these hybrids with a hairpin structure are exceptionally stable and are formed preferentially. The invention additionally allows synthesis of practically arbitrary length double-stranded DNA structures, thereby only synthesizing a single oligo or polynucleo-10 single strand. The length of the double strand is practically limited only by the synthesis possibilities of the single strand that is being further developed. For the synthesis of the single strand all known methods are considered, the advantageous phosphoric acid triester or phosphite method.

Long single-stranded polynucleotides may optionally be formed by linking (ligating) fragments. The oligo or polynucleotides can be derived from a coding or non-coding DNA strand and used in the 5 'hydroxy or phosphate form.

The filling into a DNA double strand takes place according to methods known per se. However, in a particular embodiment of the invention, mutagenic "primers" can also be used, whereby, based on a single synthesized oligonucleotide and the corresponding "primer", 25 variations of the DNA sequence can be produced. Thus, without cloning in single-stranded phages, it is necessary to obtain approx. 50% mutants. By this method, any mutations - base replacement, removal, or insertion - can be provided. An overview of the known methods can be found in M. Smith (1985), Annu. Rev. Genet. 19, 423-462.

Also "misincorporation" mutagenesis can be used (M.

Sing et al. (1986) Prot. Engin. 1, 75-76) and likewise "saturation" mutant agenesis (K. Derbyshire et al. (1986) Gene 46, 145-152).

Because the hairpin structure is not suitable for linking with another DNA fragment, e.g. a vector,

DK 175230 B1 I

4 I

this structure is either opened or decomposed by means of I

of a restriction enzyme. Suitable recognition sites are provided

in this case, by the synthesis of the single strand. Of course you

For example, those resulting from the removal of the hairpin structure may also occur

5 ends are modified in a manner known per se, e.g. can you

they are blunt-ended by decomposition or filling, or I

they can be modified using adapters or linkers. IN

Also in this connection, the method of I

the invention is an adaptation to the present one

10 requirements. Eg. you can also include recognition sites for you

class of IIS restriction enzymes, so-called "shifters" (W. I

Szybalski (19S5), Gene 40, 169-173). By cutting it you

double-stranded DNA with these enzymes is recognized

sites, so that fragments can be combined on I

15 anywhere. The incorporation of one or more recognisers

sites for a restriction enzyme before the opposite gene I

determination also permits a restriction analysis for determination I

of whether the desired structure has emerged. IN

In accordance with the invention, hair is arranged at both ends

20 needle structures, suitably incorporate different I

recognition sites for restriction enzymes, which I

can provide a gene fragment capable of in the desired direction

used in a vector construct. IN

The modification of the hairpin structure may also occur

25 upon opening by a single strand specific nuclease, I

such as "mung bean" nuclease or nuclease SI. IN

The length of the opposite repetition, thus leading I

to a double string, is variable and it comprises the advantage I

like 8 to 60, especially 12 to 32 bases, corresponding to 4 to 1

30, preferably 6 to 16 base pairings. The number of those I

unpaired bases in the "loop" region of the hairpin structure I

is also variable and preferably represents 0 (direct cova- I

lent bond) to 6. I

Constructs a hairpin structure with a favorable I

35 restriction enzyme cross-site (1) appears

thus, after cleavage of the hairpin structure with this I

Enzyme supernatant ends (2) which can be used for ligation with an additional oligonucleotide (3). Optionally, a residual short double strand residue obtained within the ligation region of the long polynucleotide (4) obtained above may be removed by heat denaturation if a mutagenesis is to be performed in this region.

A further advantageous embodiment of the invention consists in that a marking is performed upon filling in the double strand. In this way, many labeled DNA-10 fragments become very advantageously available. In this way, highly radiolabeled DNA fragments obtained are suitable after denaturation as hybridization or gene probes.

Thus, it is found that the method of the invention can be extremely versatile and allows for the synthesis of very long double-stranded DNA fragments with a minimum of reaction steps.

In the following examples, the invention is further illustrated. Percentages are based on the weight, unless otherwise stated.

20

Examples 1

Chemical synthesis of a single-stranded oligonucleotide

Example Oligonucleotide 1 (Table I) explains the synthesis of the DNA building blocks. For the solid phase synthesis, the 3 'end nucleotide is used, in this case thymidine (nucleotide no. 130) which is covalently linked to a carrier via the 3' hydroxy function. The carrier is functionalized CPG ("Controlled Pore Glass") with long chain aminoalkyl groups.

In the following synthesis steps, the base components are used as 5'-O-dimethoxytrityl nucleoside-3 '-phosphoric acid-6-cyano-ethyl-dialkylamine, whereby adenine is present as N®-benzoyl compound, N2-isobutyryl compound and thymine without protecting group.

I DK 175230 B1 I

I 6 I

In 25 mg of the polymeric carrier to which I is attached

In 0.2 μη / ol 5'-O-dimethoxytrityl-thymidine is treated in series.

In accordance with the following reagents:

In A) acetonitrile, I

I 5 B) 3% trichloroacetic acid in dichloromethane, I

I C) acetonitrile, I

I D) 5 μmol of the corresponding nucleoside 3'-O-phosphite, I

I and 25 μιηοΐ tetrazole in 0.15 ml of anhydrous acetonitrile, I

I E) acetonitrile, I

In 10 F) 20% acetic anhydride in tetrahydrofuran with 40% I

In lutidine and 10% dimethylaminopyridine, I

I G) acetonitrile, I

In H) 3% iodine in lutidine / water / tetrahydrofuran in volume I

In the ratio of 10: 1: 40. IN

I "Phosphite" is understood herein to mean 21-deoxyribose-3'-mono-I

In the phosphoric acid mono-S-cyanoethyl ester, whereby the third I

In valence is saturated with a diisopropylamino group. The yields I

I of the individual reaction steps can be determined each time spectro-I

I photometrically after the detritylation reaction B) by measuring I

In the absorption of the dimethoxytrityl cation at a wavelength of

At 496 nm. IN

After completion of synthesis, the cleavage of I is performed

In the dimethoxytrityl group by methods known to those skilled in the art, I

I with the reagents mentioned in A) to C). When treated with I

In ammonia, the oligonucleotide is cleaved from the carrier, and co

Early on, the β-cyanoethyl groups are eliminated. At a 16-hour I

In treating the oligomer with concentrated ammonia at 50 ° C

I quantitatively cleaved the amino protecting groups of the bases. IN

The crude product thus obtained is purified by poly-I

In acrylamide gel electrophoresis. IN

The solution is heated to 95 ° C for 5 minutes. After cooling to 37 ° C, add 2 μΐ EcORI (40 units) or 2 μΐ Sau96I and incubate for 12 hours at 37 ° C. Then the digestion mixture is transferred to an analytical 10% polyacrylamide / 7M 5 urea gel and separated by electrophoresis.

The resulting oligonucleotide runs against a label such as a 130-mer (oligonucleotide 1, uncut), 103-mer (oligo nucleotide 1-EcoRI cut) and a 96-mer (oligonucleotide 1 - Sau96I cut).

10 3. Formation of a DNA double strand and provision of overhanging ends: preparation of DNA sequence I.

0.5 nmol of the oligonucleotide 1 is taken up in 23 μΐ water and 5 μΐ 10 x Nick-Translations buffer (0.5 M Tris-HCl, pH 7.5; 0.1 M magnesium chloride, 0.1 M DTT, 500 / g per ml of BSA) and heated for 5 minutes to 95 ° C. After the solution is cooled to room temperature, 20 μΐ dNTP solution (containing 2.5 mM dATP, dTTP, dCTP and dGTP each) and 2 μΐ DNA polymerase (Klenow fragment, 10 units) are added and incubated one hour at room temperature. After 2, 5, 10, 20 and 60 minutes, 1.5 μΐ samples can be taken electrophoretically to follow the reaction process using a 10% native polyacrylamide gel. The mark used is pBR322 cut with Mspl. To provide the overhanging ends, 25 μΐ of the DNA polymerase reaction solution is heated to 95 ° C for 5 minutes, then tempered to 37 ° C and diluted with 15 μΐ of 5 x Eco RI buffer. After addition of 12.5 μΐ 0.2 M NaCl, 37.5 μΐ water, 5 μΐ Eco RI and 5 μΐ HindIII (each 100 units of enzyme), incubate for 15 30 hours at 37 ° C. The mixture is purified by electrophoresis on a 10% polyacrylamide gel (without urea). The head bands running toward the pBR322 tag as a 99 base pair fragment are cut and eluted after homogenization with 0.2 M triethylammonium bicarbonate buffer. By desalinating via a 35 "sephadex G50" column, the purified DNA fragment corresponding to the DNA sequence I (Table II) is obtained.

I DK 175230 B1 I

I I

In 4. Preparation of a mutant DNA fragment (DNA sequence II). IN

In the oligonucleotide 1's for cysteine coding triplet TGT I

I (nucleotide # 58-60) can be converted by the mutagenic I

In primer I

I 5 5 'CTTGTCA (3TAGAATAGTTAC 3' I

I to one for Ser encoding triplet TCT. IN

In this, 1 nmol of primer is first converted by reaction I

I with kinase to the corresponding 5'-phosphate. 0.22 OD25q of I

In the 10 primer, dissolve in 15 μΐ HEPES buffer and dissolve the solution

You are heated to 95 ° C for 5 minutes, then cooled on ice. IN

After the addition of 9 μΐ 2.5 mM ATP (pH 7), 2 μΐ 0.1 M DTT, I

In 2 μΐ water and 1 μΐ T4 polynucleotide kinase (10 units) in- I

You are cubed for 2 hours at 37 ° C, then desalted via I

In 15 a "Sephadex G 50" column. IN

To form the double strand, 0.2 L is hybridized

In nmol of oligonucletotide 1 with 1 nmol of kinase-treated primer I

I as described in Example 3 and subjected to DNA polymer I

In race reaction. Before cutting with restriction enzymes I

In 20 EcoRI and HindiII, DNA ligase links the I

In newly formed DNA strand with the mutagenic primer. To this you are added

In 1 µl 10 times ligase buffer (660 mM Tris-HCl, pH 7.5; 50 mM I

In magnesium chloride, 50 mM DTT and 10 mM ATP) as well as 1 μΐ T4 DNA-I

I ligase to the polymerase reaction and incubate for 1.5 L

25 hours at 37 ° C. The additional reprocessing is carried out as it is

I is described in Example 3. Following the gel electrophoretic I

In purification, the DNA fragment (as described in Example I) is cloned

5). Since the mutagenesis destroys a Rsal incision site, you can

In which restriction analysis with Rsal distinguishes between I

In the 30 mutants (Ser coding) and the wild type plasmid (Cys coding). IN

I From the mutated hybrid plasmid is obtained by EcoRI-HindIII form I

In addition, DNA fragment corresponding to DNA sequence II (Table I

I III). IN

I 35 I

5. Construction of vectors containing the synthetic DNA fragments as well as their transformation and amplification.

The commercially available vector pIC18 is opened in known manner with the restriction enzymes Eco RI and HindIII according to the manufacturer's instructions. The digestion mixture is separated in a known manner on a 1% agarose gel by electrophoresis and the fragments are made visible by staining with ethidium bromide. The vector bands (about 2.6 kB) are cut out of the agarose gel and separated from the agarose by electroelution, phenol extraction and ethanol precipitation.

10 µmol of the EcoRI-HindiII-opened vector is ligated to 100 µmol of the synthetic DNA fragment of DNA sequence I or the DNA fragment of Example 4 containing overhanging ends corresponding to the restriction enzymes EcoRI and HindIII, in 20 μΐ ligase buffer in the presence of T4 DNA ligase overnight at room temperature.

A hybrid plasmid is obtained with which E. coli is transformed. To this end, the strain E. coli K12 is made competent by treatment with calcium chloride, and to this is added the solution of the hybrid plasmid. By the addition of isopropyl-1S-D-thiogalacto-pyranoside (IPTG), 5-bromo-4-chloro-3-indolyl - & - D-galacto-pyranoside (Xgal) and ampicillin, after dispersing on plates, colonies containing the insert into the vector are detectable. After amplification, the cells are dissected and the hybrid vectors isolated by known methods. From these, the gene fragments corresponding to DNA sequence I or DNA sequence II can be isolated by digestion with the initially used enzymes EcoRI and HindIII. The nucleotide sequences are verified by the common known methods of DNA sequencing.

6. Synthesis of a Salm calcitonin Glv gene (33).

DNA sequence III's oligonucleotide is prepared as described in Example 1. 0.5 nmol of this oligonucleotide is then hybridized as described in Example 3, and by DNA polymerase (Klenow fragment) DNA double strand

I DK 175230 B1 I

I 10 I

In gen. After the denaturation of the enzyme by heat treatment I

You increase the NaCl concentration to 100 mM and cut I

I with the restriction enzymes Sphl and EcoRI (each 100 units) I

In, the gene is provided with the above ends. IN

In the purification and cloning of the calcitonin gene, you can

You are performed in a manner known per se (eg, as suggested in I

In German Patent Application No. P 3,632,037.4). IN

DK 175230 B1 11

Table I: oligonucleotide 1 1 10 20 30 40 50 5 'AGTAAGCTTCACGAGTATTTTCCGGAAATGCAGATTCTGGCTGTTAGTGG 51 60 70 80 90 100 TAACTATTGTACTGACAAGAAACCTGCTGCTATCAATTGGATTGAGGGCC 101G

"Hairpins" structure: Sau96I EcoRI

1 in 5 'AGTAAGCTT ....................... GAGGGCCGTGAATTCGCT- ^

:::::::::::::::: T

:::::::::::::::: T

3 "TCCCGGCACTTAAGCGT ^" "Base Pairing In Cutting Point

I DK 175230 B1 I

I 12 I

In Table II: DNA sequence I I

I LEO HIS GLO TYR PHE PRO GLO WITH GLN ILE LEO I

ag CTT CAC GAG TAT TTT CCG GAA ATG CAG ATT CTG 'I

A GTG CTC ATA AAA GGC CTT TAC GTC TAA GAC I

IN ALA VAL SER GLY ASN TYR CYS THR ASP LIGHT LIGHT PRO “I

GCT GTT AGT GGT AAC TAT TGT ACT GAC AAG AAA CCT I

CGA CAA TCA CCA TTG ATA ACA TGA CTG TTC TTT GGA

I ALA ALA ILE ASN TRP ILE GLO GLY ARG

GCT GCT ATC AAT TGG ATT GAG GGC CGT G

CGA CGA TAG TTA ACC TAA CTC CCG GCA CTT AA

In Table III: DNA sequence II

I LEO HIS GLO TYR PHE PRO GLU WITH GLN ILE LEO

AG CTT CAC GAG TAT TTT CCG GAA ATG CAG ATT CTG

A GTG CTC ΑΤΑ AAA GGC CTT TAC GTC TAA GAC

IN ALA VAL SER GLY ASN TYR SER THR ASP LIGHT LIGHT PRO

GCT GTT AGT GGT AAC TAT TCT ACT GAC AAG AAA CCT

CGA CAA TCA CCA TTG ATA AGA TGA CTG TTC TTT GGA

I ALA ALA ILE ASN TRP ILE GLO GLY ARG

GCT GCT ATC AAT TGG ATT GAG GGC CGT G

CGA CGA TAG TTA ACC TAA CTC CCG GCA CTT AA

DK 175230 B1 13

Table IV: DNA sequence III

0 1 5 10 * With Cys Ser Asn Leu Ser Thr Cys Val Leu Gly List

5 'TTTGCATGC ATG TGC TCT AAC CTG TCG ACT TGC GTT CTT GGT AAG

1 10 20 30 40 _ 15 20 25

Leu Ser Gin Glu Leu H.is Lys Leu Gin Thr Tyr Pro Arg Thr Asn

CTT TCT C AG GAA CTT CAT AAA CTG CAG ACC TAT CCG CGC ACT AAT

50 60 70 80 90 30

Thr Gly Ser Gly Thr P :: o Gly Stp Stp ACC GGC TCT GGT ACC COT GGT TAA TAG AATTCGCTTTTGCGAATTCTATT 3 '100 110 120 130 140

Claims (7)

A method for producing double-stranded II DNA, characterized in that a single single-stranded DNA DNA with a hairpin structure is synthesized at one end II or at both ends, the complementary nucleotides are filled in, II and the hairpin structure (- the structures) are opened or split. IN 2. A method according to claim 1, characterized in that one or more recognition site (s) of a restriction enzyme (I) 10 is applied by the hairpin structure by which the hairpin structure can be cleaved. IN The method of claim 1, characterized in that the hairpin structure is opened by a single strand specific nuclease. IN Method according to one or more of the preceding claims, characterized in that the single-stranded goat DNA is hybridized with a mutagenic "primer" and site-specific mutagenesis is performed. IN Process according to one or more of the preceding claims, characterized in that labeled nucleotides are used in the filling of the double strand. IN Method according to one or more of the preceding claims, characterized in that starting from I I oligonucleotide 1 (Table I). IN 7. Oligonucleotide 1 (Table I). IN