FI64187C - FOERFARANDE FOER RENING AV EN PRODUKT BESTAOENDE HUVUDSAKLIGENAV CDNA-FRAGMENT MED SAMMA LAENGD VILKA HAR EN SPECIFIK N UKEOTIDSQUENSE OCH MINST ETT RESTRIKTIONSSTAELLE OCH FOER A NAYS AV PRODUKTENS RENHET - Google Patents

Description

r, advertisement publication, ΠοΠ JMTq LBJ (11) UTLÄGGNINGSSKRIFT 64187 q (45) n, t, ... · i! ‘Ί ιλ 1933 ^ ^ ^ (51) Kv.ik3 / Int.ci. 'C 12 N 15/00 FINLAND - FINLAND (2.1) PtMnttlhakemut - Pat * ntan * öknlng T81676 (22) Hikemljpilvi - Ansöknlngtdag 26.05 * 78 (23) AlkupUvi— Glltighetsdag 26.05. (8 (41) Become Public - 2 years off .03 79

National Board of Patents and Registration (44) Date of issue and hearing | - 30.06.83

Patent and Registration Authorities Anaökan utlagd och utl.skrlften publlcerad (32) (33) (31) Claim Requested — Begird prlorltet 23- 09. 77 USA (US) 836218 (71) The Regents of the University of California, 2200 University Avenue, Berkeley, California 9 ^ 720, USA (72) Howard Michael Goodman, San Francisco, California,

Peter Horst Seeburg, San Francisco, California, USA (US),

John Shine, Curtin, ACT, Australia-Australien (AU) (71 *) Oy Kolster Ab (5M Method for purifying and analyzing the purity of a product consisting mainly of e-DNA fragments of the same length having at least one restriction site and having a specific nucleotide sequence - Förfarande för rening av en The present invention relates to a method for the purification of product cNAs of at least one restriction site of the same product having at least one restriction site and having the same nucleotide sequence. for analysis of purity, wherein the fragments encode human fetal choroidal somatomammotropin or human growth hormone or parts thereof and are suitable for recombination with a DNA transfer vector and transfer to a microorganism, and wherein said purified The product of avista cDNA fragments is obtained by treating a population of cDNAs heterogeneous in length and nucleotide sequence in which at least a portion of the cENA molecules contain at least two restriction sites, first with one or two suitable restriction enzymes and then fractionating 2,6487 cDNAs of the same length. using electrophoresis and separating a fraction of cDNA fragments having the desired specific nucleotide sequence.

Living organisms are able to synthesize a wide variety of proteins and peptides. Several proteins and peptides are useful in medicine, agriculture or industry. Some proteins are enzymes that catalyze complex chemical reactions. Others act as hormones that affect the growth and development of an organism or the function of tissues in a medically significant way. Certain binding proteins may be of commercial importance in isolating and purifying substances or removing impurities. Both proteins and peptides consist of linear amino acid chains, and the latter term is used for short single-chain sequences and the former for long-chain multi-chains. The principles contained in this invention apply equally well to proteins as to peptides.

Proteins and peptides are generally high molecular weight substances, each with a specific amino acid sequence. With the exception of small peptides, chemical synthesis of proteins and peptides is often impractical, expensive, time consuming, and even impossible. In order for a desired protein to have practical significance, it must most often first be isolated from the organism that produces it. Often the desired protein is present in only a small concentration. The source of the desired protein is usually so limited that not enough protein is available. Thus, a number of agricultural, industrial, and medical applications are known for certain proteins that have not been able to be further developed due to the inadequacy of the desired protein or peptide.

In Ann. Rev. Biochem. 44 (1975) 45 (Gefter, Malcolm, L.) describes the principle of DNA replication, i.e. the synthesis of DNA in a cell, and mentions the necessary enzymes and proteins. In Ann. Rev. Biochem. 44 (1975) 273 (Nathans, D. and Smith, H.D.) describe restriction endonucleases and their use; mm. the classification of these enzymes, cleavage sites and methods of analysis, as well as the use of the enzymes in chromosome studies, gene isolation and DNA cloning are mentioned. In Ann. Rev. Biochem. 46 - (1977) 415 (Sinsheimer, R: L.) Describes recombinant DNA technology; the publication concerns e.g.

3,641,87 plasmids, DNA modification, isolation of cloned DNA, etc.

These publications do not describe a method according to the invention for purifying a nucleotide fragment, first isolating the mRNA, then preparing a fragment of the cDNA which is substantially pure, i. it does not contain contaminating cDNA sequences. In Ann. Rev. Biochem. 47 (1978) 607 (Wu, R.) describes a method for analyzing a DNA sequence; according to this publication, DNA is isolated and purified by gradient centrifugation, and restriction endonucleases are used to cleave the DNA into analyteable fragments.

The publication also briefly mentions DNA cloning; according to one method, starting from relatively pure mRNA, and obtaining an easily clonable cDNA, according to another method, the cDNA is prepared from a mixture of mRNAs. These publications also do not disclose the isolation and purification of specific cDNA from a mixture of mRNAs in which only small amounts of the desired mRNA are present, for example only 2%.

Science 196 (1977) 1313-1319 (Ullrich A * et al.) Describes a method for purifying cDNA. However, only the dominant cDNA could be purified by this known method, whereas cDNA, which is sparse, could not be purified and in a heterogeneous population. Surprisingly, with the new method, such a minority cDNA can be selectively purified to obtain, for the first time, a pure DNA fragment encoding human growth hormone and human chorionic somatic mammotropin.

Recently developed methods (see, for example, FI application No. 781 675) make it possible to use fast-growing and abundant microorganisms for the synthesis of commercially useful proteins and peptides, regardless of their source in nature. These methods can be used to confer on a suitable microorganism the genetic ability to synthesize a protein or peptide that is normally produced by another organism. The methods utilize the principle that prevails between the genetic material of all living organisms, usually DNA, and proteins synthesized by the organisms. According to this principle, the amino acid sequence of a protein is determined by the nucleotide sequence of the DNA. For each of the 20 amino acids most commonly found in proteins, there are one or more specific groups of trinucleotide sequences. The link between a particular trinucleotide sequence and its corresponding amino acid 641 87 4 forms the genetic code. It is believed that in all living organisms, the genetic code is either the same or similar. Thus, the amino acid sequence of each protein or peptide is determined from the corresponding nucleotide sequence in a known manner. In addition, in principle, any living organism can interpret this nucleotide sequence.

The method of rendering a microorganism capable of synthesizing a new protein comprises three main steps: (1) purifying and isolating a particular gene or nucleotide sequence containing the genetic information of the amino acid sequence of the desired protein, (2) recombining the isolated nucleotide sequence with a suitable transfer vector; the bacteriophage or plasmid DNA and (3) transferring the vector to a suitable microorganism and selecting a strain containing the desired genetic information from the microorganisms thus obtained.

When the method outlined above is to be developed commercially, the fundamental difficulties relate to the first step, namely the isolation and purification of the desired genetic information. In all living cells, DNA exists as very large molecular nucleotide chains. A cell may contain more than 10,000 structural genes encoding more than 10,000 amino acid sequences of a specific protein, and the sequence of each gene may contain many hundred nucleotides. All existing sequences consist essentially of four different nucleotide bases. These are adenine (A), guanine (G), cytosine (C) and thyrine (T). The sequences of the structural genes of certain proteins are thus very similar in chemical structure and physical properties. One such sequence cannot be distinguished by other physical or chemical methods from other sequences present in the isolated DNA.

The nucleotide sequence information of DNA is converted to the amino acid sequence of the protein by messenger RNA. In the first step of this process, called transcription, the DNA segment whose nucleotide sequence specifies the protein to be produced is copied into the RNA. RNA is a polynucleotide similar to DNA except that deoxyribose is replaced by ribose and thymine is replaced by uracil. The nucleotide bases of the RNA are capable of settling into similar base pairs known to be between complementary strands of DNA. A and U (T) are complementary and G and C are complementary. The RNA transcript of the DNA nucleotide sequence is complementary to the copied sequence. This RNA is called messenger RNA (mRNA) because it acts as an intermediary between the genetic mechanism of the cell and the mechanism that synthesizes proteins. In general, only mRNA sequences corresponding to the proteins currently being synthesized are present in a cell at a given time. Thus, a specialized cell whose function is primarily to synthesize a single protein contains an RNA that is primarily responsible for that protein. The nucleotide sequence encoding a particular protein can be isolated and purified in certain cases by isolating that mRNA from cells specialized in the production of that protein.

The disadvantage of the above method is that it can only be applied to those rather rare cells that specialize mainly in the production of a single protein. Most commercially interesting proteins are not synthesized in such a specialized manner. The desired protein may be one of about one hundred different proteins produced by the cells or organism of the tissue at a given time. However, the method of isolating mRNA is useful because the RNA species present in the cell usually represent only a fraction of the total number of DNA sequences, and on this basis, initial purification can be performed. In order for such purification to be useful, a method is needed by which sequences present in small concentrations, such as a few percent, can be isolated in very pure form.

The method of this invention can isolate and purify nucleotide sequences as low as 2% in a heterogeneous set of mRNA sequences. The method can further be combined with known mRNA fractionation methods to isolate and purify sequences that are even lower in the RNA population originally shown.

The method according to the invention is characterized in that a) the 5 'phosphate end groups of cDNA fragments of the same length having the desired specific nucleotide sequence are removed by phosphatase at a time of, for example, pH 7.5 and a temperature of about 60 ° C. b) cleaving the cDNA fragments treated according to a) from a site within the desired nucleotide sequence with a specific restriction enzyme not used in the previous step of the treatment, for example Hha I, Hpa II or

Using restriction enzyme endonuclease Pvu II at a pH of about 7.6, incubating at about 37 ° C for about 2 hours, c) fractionating the resulting subfragments by length, for example using gel electrophoresis, d) recovering fractions comprising two subfragments of a specific nucleotide sequence. , e) enzymatically combining the subfragments of d) with covalently linked DNA ligase in the presence of ATP, e.g. at a pH of about 7.6 at a temperature of about 15 ° C for about 2 hours, incubating in f) e) The product containing the cDNA containing the specific DNA sequence obtained according to f) is fractionated according to length, for example by gel electrophoresis, and g) the cDNA obtained according to f), free from up to 90% of the contaminating cDNA sequences, is taken from the cDNA having the desired specific nucleotide sequence. product consisting of fragments.

7 64187

The following table shows the symbols and abbreviations used.

Table 2 DNA deoxyribonucleic acid RNA ribonucleic acid cDNA complementary DNA (enzymatically synthesized from mRNA sequence)

mRNA messenger RNA

dATP deoxyadenosine triphosphate dGTP deoxyguanosine triphosphate dCTP deoxycytidine triphosphate dTTP thymidine triphosphate dT ^ 2_2g deoxythymidylate (12-18 bases) uricosinase Sicomammotinin

Tris 2-amino-2-hydroxyethyl-1,3-propanediol EDTA ethylenediaminetetraacetic acid ATP adenosine triphosphate

Hpa H-endonuclease i, res trict ioendonuclease that cleaves C CCG in the home fence (arrow indicates cleavage site)

Search for the III endonuclease diffraction endonuclease that cleaves at GG ^ CC

Hha I endonuclease, a restriction endonuclease that cleaves at

GCG ^ C

Hsu I-endonuclease, a restriction endonuclease that cleaves at

N AGCTT

Pvu II endonuclease, a restriction endonuclease that cleaves at

CA ^ GCTG

Because DNA replication and transcription are characterized by a known base pair system, isolating an mRNA that contains a nucleotide sequence encoding the amino acid sequence of a particular protein, the same sequence, or a gene is equivalent to isolating it from the DNA itself. If the mRNA is transcribed into complementary DNA (cDNA), a DNA sequence can be reconstructed that can be transferred to the genetic material of another organism by appropriate methods. The aforementioned complementary forms of a particular sequence are thus functionally equivalent.

The nucleotide units of DNA and RNA are joined together by phosphodiester bonds at the 5 'position of the second nucleotide sugar and at the 3' position of the adjacent nucleotide sugar. When such bonds are reconstituted, a linear polynucleotide is obtained that is polar so that one end can be separated from the other, the 3 'end may have a free hydroxyl group, or the hydroxyl group may be substituted with a phosphate or some more complex moiety. The situation is the same at the 5 'end. In eukaryotic organisms, i.e., those with a distinct nucleus and mitotic mechanism, the synthesis of a functional mRNA usually involves the incorporation of a polyadenylic acid at the 3 'end of the mRNA. For this reason, mRNA can be separated from other types of ENA isolated from a eukaryotic organism by column chromatography on cellulose to which polytymidylic acid has been added, cf. Aviv, H. and Leder, P., Proc. Natl. Acad.

Sci. U.S. 69, 1408 (1972). Also useful are other chromatographic methods that utilize the base pair affinity of poly A and the chromatographic packing material when the packing material contains oligo-dT, poly-U, or some combination of poly-T and poly-U.

Reverse transcriptase catalyzes the synthesis of DNA complementary to the RNA template strand in the presence of an RNA template, a primer that can be any complementary 3'-hydroxyl-containing oligo- or polynucleotide, and four deoxynucleoside triphosphates (dATP, dGTP), dGTP, dGTP. The reaction is initiated by non-covalent attachment of the oligo-deoxynucleotide primer near the 3 'end of the mRNA and then stepwise coupling of appropriate deoxynucleotides to define the mRNA nucleotide sequence at the 3' end of the base pair. The resulting molecule can be described as a hairpin structure in which, alongside the original RNA, there is complementary DNA joined by hydrogen bridges and partially folded over itself at one end. The DNA and RNA strands are not covalently linked. Reverse transcriptase is also capable of catalyzing a similar reaction using a single-stranded DNA hairpin in which the ends are joined by single-stranded DNA, cf. Aviv, H. and Leder, P., Proc. Natl. Acad. Sci. U.S. 69, 1408 (1978) and 9,641 87

Efstratiadis, A., Kafatos, F.C., Maxam, A.M. and Maniatis, T., Cell 7, 279 (1976).

Restriction endonucleases are enzymes capable of hydrolyzing phosphodiester bonds in DNA and thus cleaving the DNA strand. If the DNA is a closed ring, it becomes linear under the influence of this enzyme. The main feature of a restriction enzyme is that its cleavage effect is limited to a site with a particular nucleotide sequence. Such a sequence is called a restriction endonuclease recognition site. Restriction endonucleases have been isolated from several sources and identified by the nucleotide sequence of their restriction sites. Upon cleavage of double-stranded DNA, some restriction endonucleases cleave the phosphodiester bond at the same site in each strand to form a blunt end. Others, in turn, catalyze the cleavage of bonds separated by a few nucleotides to form free single-stranded regions at the cleavage site. Such single-stranded ends are self-complementary and thus cohesive and can be used to reattach truncated DNA. Because such an enzyme cleaves any DNA from only the same recognition site, the same cohesive ends are created, and this allows heterogeneous DNA sequences to be ligated together as long as they have first been treated with the same restriction enzyme. Restriction endonucleases are named according to the bacterial strain source; the first letter of the surname and the first two letters of the species name are used, and if more than one endo-nuclease has been obtained from the same strain, a further Roman numeral is added. For example, Hae III means one of three Haemophilus aegyptius restriction endonucleases. The name may contain other letters in addition to this, if a special isolate or serotype also needs to be reported (see Roberts, R.J., Crit. Rev. Biochem. 4, 123 (1976)).

It has been found that restriction sites for a particular enzyme are quite sparse and unevenly distributed. It must be investigated experimentally whether a particular restriction site exists in any segment. However, it has been possible to isolate a large and growing number of restriction endonucleases with different restriction site specificities from various sources, and thus it can be assumed that, for example, a particular thousand nucleotide sequence contains one or more restriction sites.

64187 This invention relates to a novel method for purifying a cDNA having a desired nucleotide sequence that is complementary to a particular mP.NA. The method uses a restriction endonuclease to cleave a cDNA transcribed from a heterogeneous mRNA mixture. The method does not require precise purification of the RNA, but instead utilizes transcription of the RNA into cDNA, specific division of this cDNA by one or two restriction endonucleases into fragments, and isolation of these cDNA fragments by length. Using restriction endonucleases, heterogeneous size is avoided and DNA fragments of the same length are obtained from any cDNA containing at least two restriction sites. From an initially heterogeneous population of cDNA transcripts, fragments of the same size with the desired sequence are obtained. The fragments can be several hundred nucleotides in length and in some cases may contain the entire structural gene of the desired protein. The length of the fragment depends on the number of nucleotides between the restriction sites, which is usually different at different sites in the DNA. When the separation is performed on the basis of length, homogeneous populations of fragments with the desired sequence are obtained. The fragments are obtained as homogeneous in size and very pure in nucleotide sequence. The separation and analysis methods of this invention make it possible to separate fragments from the corresponding xnRNA species in an amount of at least 2% by weight of the RNAa to be transcribed. If the mRNA is purified prior to transcription by known RNA fractionation methods, a lower limit of less than 2% of the total mRNA isolated is obtained.

Purification of specific sequences purified by the method outlined above is continued by allowing a second restriction endonuclease to cleave the desired sequence from one of the sites. This cleavage results in two subfragments of the desired structure that can be separated by length. The subfragments are separated from unbroken and specifically broken impurity sequences whose original size has been virtually sauna. The method is based on the rarity and random location of restriction endonuclease recognition sites, as it is therefore highly unlikely that an impurity fragment of the same length as the desired sequence will initially be cleaved into fragments of the same length as the desired alpha fragment. Once the impurities have been removed, the subfragments of the desired sequence can be joined together by known methods to reconstruct the original sequence. Joining of the two subfragments to each other in reverse to the original sequence must be prevented. In the context of the present invention, a method is described by which the subfragments are joined together in the correct order.

The method described above can be modified and combined with appropriate labeling methods to provide accurate information on the purity and amount of sequences isolated. Combined methods have made it possible to prepare a known nucleotide sequence with a purity of more than 99%.

The cDNA isolated and purified by the method described can be recombined with a suitable transfer vector and transferred to a suitable host microorganism.

The method of this invention uses polyadenylated, impure or partially purified messenger RNA as a starting material, which may have heterogeneous sequence content and molecular size. It is important to select a suitable tissue containing the desired mRNAs as the starting material for the method. The choice is often made on the basis that only certain specialized tissues of the differentiated organism produce the protein of interest. This is the case, for example, with peptide hormones such as growth hormone and human fetal choroidal somatomammotropin. In other cases, it can be noted that many cell types or microbial species can serve as sources of the desired raRNA.

Once the RNA has been separated from the cell, the desired mRNA sequences can be enriched using conventional RNA fractionation techniques for pre-purification. All methods in which RNA is not degraded are useful. Highly suitable methods include preparative sedimentation in a sucrose gradient and gel electrophoresis.

mRNA should be isolated from source cells under conditions where it does not degrade. In particular, ribonuclease enzymes must be inhibited because they hydrolytically degrade the nucleotide sequence of RNA. If one bond is hydrolyzed, that sequence is broken and the fragment containing the original 5 'end of the sequence is lost. FI patent application No. 781675 describes a suitable method for inhibiting ribonuclease during extraction. The method uses 4-molar guanidinium thiocyanate and 1-molar mercaptoethanol in the cell disruption step. Degradation of the RNA to be isolated by ribonuclease can be further reduced by using a low temperature and a pH close to 5.

Before the method of the invention can be applied, the mRNA must be purified from impurity proteins, DNA, polysaccharides and lipids. Prior art methods are used for this purification. The RNA thus obtained contains both mRNA and other RNA.

The preferred method for isolating eukaryotic mRNA is column chromatography with oligo-dT cellulose or some other oligonucleotide-substituted filler, as this can take advantage of the hydrogen bond specificity due to the polyad at the 3 'end of the eukaryotic mRNA.

DNA is then generated that is complementary to the isolated heterogeneous mRNA sequences. Reverse transcriptase is selected as the enzyme for this reaction, although in principle any enzyme capable of making a complementary copy of DNA for an mRNA template can be used. The reaction can be performed under known conditions using mRNA as a template and a mixture of four deoxynucleoside triphosphates (dATP, dGTP, dCTP and dTTP) as DNA strand precursors. It is preferred that one deoxynucleoside triphosphate is labeled with a radioisotope, 32 for example Λ-P, as it can monitor the progress of the reaction, act as a marker when the product is recovered after, for example, chromatographic or electrophoretic separation and allow quantitative estimates of recovery, cf. . Efstra-tiadis, A. et. ai., supra.

CDNA transcripts prepared by reverse transcriptase are slightly heterogeneous at the 5 'end and 3' end, as the start and end points for the mRNA template may vary.

The variability at the 5 'end is thought to be due to the ability of the oligo-dT primer used to initiate the synthesis to bind 641 87 13 at several different sites in the polyadenylated region of the mRNA. cDNA synthesis begins at an unspecified site in the poly-A region, and a variable distance from the poly-A region is transcribed depending on the original junction of the oligo-dT primer. This ambiguity can be avoided by using a primer containing one or two nucleotides of the RNA sequence itself, as this will form a primer with a preferred and defined junction for initiating the transcription reaction.

The ambiguity of the 3 'end of the cDNA transcript is due to several factors influencing the reverse transcriptase reaction and the possible partial degradation of the RNA template. Isolation of cDNA transcripts of maximum length can be greatly facilitated if the reverse transcriptase reaction conditions are chosen so that the equilibrium is on the side of synthesis of full-length strands with less synthesis of short strands of DNA. Preferred reaction conditions for avian myeloblastosis virus reverse transcriptase are set forth in the Examples. Reaction temperature, salt concentration, amount of enzyme, ratio of primer to therapist concentrations, and reaction time are parameters that can be altered to effect the generation of as many similar DNA transcripts as possible.

The temperature and salt concentration are selected so that specific base pair formation between the oligo-dT primer and the polyadenylated portion of the RNA template occurs. Under properly selected conditions, the primer is capable of binding to the polyadenylated portion of the RNA template and substantially avoiding non-specific priming reactions due to binding of the primer to other sites in the template, such as short A-containing sequences. The effects of temperature and salt are interdependent. High temperatures and low salt concentrations reduce the persistence of the specific base pair interaction. The reaction time is kept as short as possible to avoid non-specific starts and to minimize decomposition. The reaction time and temperature are interdependent, so that a low temperature requires a longer reaction time. At 42 ° C, a reaction time of 1 to 10 minutes is suitable. The primer should be present in a 50- to 500-fold molar excess over the RNA template and the enzyme should be present in the same excess over the RNA template. Enzyme 14 64187 and an excess of primer promote the initiation of the reaction and the growth of the cDNA strand so that long-chain cDNA transcripts are efficiently formed within a short reaction time.

It is often possible to perform the remainder of the purification method of this invention using mDNA transcribed and single-stranded cDNA sequences. In the event that the desired restriction enzyme cleaves only double-stranded DNA, double-stranded DNA can be synthesized using cDNA prepared as described above as a template and DNA polymerase such as reverse transcriptase and single-stranded DNA hydrolyzing nuclease as enzymes. The preparation of double-stranded DNA in this way has been described in the literature, cf. e.g., Ullrich, A., Shine, J., Chirwin, J., Pictet, R., Tischer, E., Rutter, W.J. and Godman, H.H., Science 196, 131 3 (1977).

Heterogeneous cDNA transcribed from heterogeneous mRNA sequences is then treated with one or two restriction endonucleases. The choice of endonuclease depends primarily on which restriction sites of the enzyme are contained in the cDNA sequence to be isolated. The method depends on the existence of two such points.

One enzyme is sufficient if the sites are identical. The desired sequence is cleaved at both sites and a population of molecules or fragments of homogeneous length with the desired sequence and homogeneous length is formed, at least at the site of the desired cDNA sequence. If the restriction sites differ, two enzymes are required to produce the desired homogeneous fragments.

The restriction enzyme (or enzymes) required to produce all or part of the nucleotide sequence fragment encoding the desired protein must be selected experimentally. If the amino acid sequence of the desired protein is known, it is possible to compare the nucleotide sequence of restriction endonuclease-cut nucleotide fragments of homogeneous length with the amino acid sequence encoded by it, since the known genetic code is common to all life forms. The complete amino acid sequence of the desired protein is not necessary, as a relatively accurate identification can be made based on the partial sequence. If the amino acid sequence of the desired protein is not known, homogenous polynucleotides of restriction endonuclease can be used as samples to synthesize and identify the protein by a suitable in vitro method. Alternatively, the mRNA can be purified by affinity chromatography. Other methods suitable for this purpose may be known to those skilled in the art.

The number of restriction enzymes useful depends on whether single-stranded or double-stranded cDNA is used. Preferred enzymes are those that act on the single-stranded DNA, which is the direct reaction product of mRNA reverse transcription. Currently, only a few restriction endonucleases are known to act on single-stranded DNA, such as Hae III and Hha I.

CDNA for restriction endonuclease treatment

can be radiolabeled so that it can be identified after subsequent separation steps. It is preferred to include a radioactive label, such as O-P, in one of the four deoxynucleoside triphosphate precursors. The highest activity is obtained if the concentration of the radioactive precursor is high compared to the concentration of the non-radioactive part. However, the total concentration of each deoxynucleoside triphosphate should be greater than 30 μ mol / l in order to achieve the longest possible cDNA in the reverse transcriptase catalyzed reaction, cf. Efstratiadis, A., Maniatis, T., Kafatos, F.C.,

Jeffrey, A. and Vournakis, J. N., Cell 4, 367 (1975). For nucleotide sequencing of the cDNA, it is a good idea to label the 5 'ends with P using a polynucleotide kinase catalyzed reaction, cf. Maxam, A.M. and Gilbert, W., Proc. Natl. Acad, Sci. U.S. 74, 560 (1977).

Fragments generated by a restriction enzyme or a combination of two restriction enzymes can be distinguished from each other and from heterodisperse sequences without a recognition site by their length. This can be done by electrophoretic methods and sedimentation methods using an ultracentrifuge. Gel electrophoresis must be given priority as it provides the best resolution based on the length of the polynucleotide. In addition, the method makes it easy to recover the separated substances quantitatively. Preferred gel electrophoresis methods are described in Dingman, C.W. and Peacock, A.C., Biochemistry 7, 659 (1968) and Maniatis, T., Jeffrey, A., and van de Sande, H. Biochemistry 14, 3787 (1975).

16

6418 V

Prior to restriction endonuclease treatment, cDNA transcripts from most sources are heterodisperse in length. When an appropriately selected restriction endonuclease or restriction endonuclease pair is allowed to act on polynucleotide chains containing the desired sequence, the chain is cleaved at the corresponding restriction sites to give polynucleotide idifragments of equal length. In gel electrophoresis, these are observed as distinct lines. Depending on the presence or absence of restriction sites, separate lines may also be obtained from other sequences, however, the lengths of the sequences contained in them are likely to differ from the length of the desired sequence. Thus, the effect of restriction endonuclease results in gel electrophoresis showing one or more distinct lines and leaving the remainder of the cDMA heterodisperse. When most of the polynucleotide sequences present are the desired cDNA, electrophoresis results in a single cDNA line.

Although it is unlikely that restriction enzymes will cleave two different sequences to produce fragments of substantially the same length, it is desirable that the purity of fragments of a certain length be ensured by some method. Sequence analysis of the electrophoresis line can detect impurities of 10% or more in the band material. Thus, a method has been developed which can express impurities even smaller than mentioned by e, and the method is based on the same factors as the separation method described above. The method requires that the desired nucleotide sequence fragment contain a restriction endonuclease restriction site that has not been used in the first separation. When the polynucleotide material eluted from the electrophoresis line is treated with a restriction endonuclease capable of cleaving the desired sequence internally, two subfragments are obtained which are very likely to be of different lengths. In electrophoresis, these subfragments form two distinct lines at their respective positions, the sum of the lengths of the fragments contained being equal to the length of the polynucleotide before cleavage. Impurities in the original line that are not affected by the restriction enzyme can be shown to migrate to the original site. Impurities with one or more restriction sites degrade into two or more subfractions. Since the location of the restriction sites is considered random, it is very unlikely that the impurities will result in subfragments of the same size as the desired sequence. The amount of radiolabeled polynucleotide contained in any line can be quantified by radioactivity measurement or any other suitable method. A quantitative measure of fragments of a desired sequence is obtained by comparing the amounts of material in the bands of the sub-fragment pathways of the desired sequence to the total amount of material.

After the separation described above, the desired sequence can be reconstructed. A DNA ligase that catalyzes the annealing of the ends of DNA fragments is suitable for this purpose. Subfragments of the desired sequence can be eluted separately from the gel electrophoresis lines and pooled under appropriate conditions in the presence of DNA ligase, cf. Sgaramella, A. Van de Sande, J.H. and Khorana, H.G., Proc. Natl. Acad. Sci. U.S. 67, 1468 (1970). If the sequences to be joined are not blunt-ended, E. coli ligase can be used, cf. Modrich, P. and Lehman, I.R., J. Biol. Chem. 245, 3626 (1970).

Reconstruction of the original sequence from the subfragments generated by restriction endonuclease treatment is greatly improved if the formation of false sequences is prevented. A detrimental outcome is avoided if a homogeneous length cDNA fragment containing the desired sequence is treated prior to restriction endonuclease treatment with a reagent capable of removing 5 'terminal phosphate groups from the cDNA. Alkaline phosphatase is a preferred enzyme for this purpose. DNA ligase is only able to catalyze the association of truncated fragments with each other if a 5 'terminal phosphate group is present.

Thus, ends without a 5 'end-phosphate cannot be joined. DNA subfragments can only be ligated to ends that contain 5 'phosphate generated by cleavage of separated DNA fragments by restriction endonuclease. This method is described in detail in FI patent application No. 781675.

Under the conditions used, most of the cDNA transcripts are derived from the mRNA region containing the 5 'end of the mRNA template, using as a primer in this template the fragment obtained by restriction endonuclease-catalyzed cleavage. In this way, both fragments containing only a portion of the nucleotide sequence encoding the desired protein and the entire nucleotide sequence can be obtained by the method described above.

18 64187

In the cloning of human genes, the purification method is very important, as the prevailing regulations state that the human gene must be purified very carefully before recombination with DNA and its transfer into the bacterium, cf. U.S. Federal Register, Vol. 41, No. 131, 7 July 1967, p. 27902-27943. The method of this invention has succeeded in producing sufficiently pure human genes that contain a major portion of the structure encoding human fetal choroidal somatomammotropin or human growth hormone. Human genetic material isolated and purified by the method described above can be recombined into plasmids or other transfer vectors.

The purification and analysis methods described above have been able to isolate most of the nucleotide sequence of the human fetal choroidal somatomammotropin structural gene with a purity of greater than 99%. The structural gene for human growth hormone has been isolated relatively pure. Novel plasmids have been synthesized that contain the isolated sequence of human fetal choroidal somatomammotropin or human growth hormone. Novel microorganisms have been prepared whose genetic structure includes an isolated sequence of human fetal vascular membrane somatomammotropin or human growth hormone.

The accompanying drawings and drawings are intended to illustrate the examples contained in this invention.

Figure 1 shows an autoradiogram of a series of gel electrophoresis 32 performed on P-labeled cDNA, described in detail in Example 1.

Figure 2 is a schematic representation of the nucleotide sequence code of human fetal choroidal somatomammotropin and shows the relative locations of the various restriction sites, as described in detail in Example 1.

32

Figure 3 is an autoradiogram of gel electrophoresis performed with P-labeled cDNA (see Example 2).

32

Figures 4 and 5 are autoradiograms of gel electrophoresis performed with P-labeled cDNA (see Example 3).

Example 1 A general method of separating a particular cDNA sequence is described herein, for example, by separating the sequence portion of the somatomammotropin coding region of the human fetal choroid from a placental tissue extract.

Extraction of 19,641 87 mRNA from placenta.

Full-time human placenta obtained by caesarean section was rapidly cooled in liquid nitrogen and stored at -60 ° C. To extract total RNA, 40 g of chilled placental tissue was minced and dissolved at 140 ° C in 140 ml of freshly prepared solution # containing 7 M guanidinium HCl (Cox, RA, Methods in Enzymology 12, 120 (1968), 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 1% sarcosyl (Ciba-Geigy Corp., Greensboro, NC). 5 g of CsCl, and the resulting dark brown solution was heated for 5 minutes at 65 ° C, rapidly cooled with ice and layered in nitrocellulose tubes (2.54 x 8.89 cm) based on 5 ml of a solution containing 5.7 mol / ml. 1 CsCl, 10 mmol / l Tris-HCl (pH 7.5) and 1 mmol / l EDTA, centrifuged for 16 hours at 15 ° C at 27,000 rpm on a SW27 rotor (Beckman Instruments Corp., Fullerton, CA) see after Clisin, V., Crkvenjakov, R.ja Ryus, C., Biochem, 13, 2633 (1974), centrifugation, dried tubes are decanted, tubes, and the bottom half cm.. an RNA-containing layer was cut into particles off the razor blade. The substance was dissolved in 20 ml of a solution of 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 5% sarcosyl and 5% phenol in a sterile Erlenmeyer flask. NaCl was then added to the solution to a concentration of 0.1 mol / L, and then a mixture containing 50% phenol and 50% chloroform was shaken vigorously with 40 ml. RNA was precipitated from the aqueous phase with ethanol in the presence of sodium acetate (0.2 mol / L, pH 5.5). The RNA product was washed with 95% ethanol, dried and dissolved in sterile water. Usually, about 30 mg of RNA was obtained from 40 g of placental tissue, and again about 300 mg of polyadenylated RNA when chromatographed twice with oligo-dT cellulose (see Aviv and Leder, supra).

cDNA synteesl ♦

Analytical reactions were performed in 5 μl of a solution containing 50 mol / l Tris-HCl (pH 8.3), 0.1 mmol / l EDTA, 7 mmol / l MgCl 2, 20 mmol / l KCl, 10 mmol / l β-mercaptoethanol, 40 μmol / l dCTP (50,000 32 computing units / min / pmol P), dGTP, dATP and dTTP (500 pmol / l each), 100 μg / ml polyadenylated RNA, 20 μg / ml oligo-dT ^ 21g (supplied by Collaborative Research, Waltham, Mass.), And 100 units / ml avian myeloblastosis virus reverse transcriptase. This enzyme is supplied by D.J. Beard, Life Science Incorporated, St. Petersburg, Florida, which manufactures the enzyme National Institutes of 20,641 87

Health with the following method: Kacian, D.L. and Spiegelman, S., Methods in Enzymology 29, L.

Grossman and K. Moldaye, eds., Academic Press, N.Y. (1974), p. 150.

o

Reactions were initiated by the addition of enzyme at 0 ° C and synthesis lasted 6 minutes at 42 ° C. Under these conditions, about 106 counter units / 32 min P were incorporated into the TCA-precipitated material, and each pg of RNA yielded about 50 ng of cDNA. In order to obtain sufficient cDNA for sequence analysis, the reaction volume was increased to 100 pg and the dCTP concentration to 250 pmol / L (specific activity 500 32 counter units / min / pmol P). Under these conditions, about 200,000 1 / min of P-labeled cDTPs were incorporated into cDNA 32. Restriktioendonukleaasikäslttely.

For the restriction endonuclease treatment, the analytical reactions were stopped by adding 20 μΐ of ice-cold water, boiling for 2 minutes, rapidly cooling with ice, and adding magnesium chloride to a concentration of 7 mmol / l. Samples (5 μΐ, approximately 2 x 10 ^ 1 / min) were treated with an excess of restriction endonuclease Hae III (Hha I or a mixture of Hae III and Hha 1 can also be used, see Figure 1) for 1 hour at 37 ° C: in. Search III was prepared by the following method: Middleton, J.H., Edgell, M.H. and Hutchinson, C. A. Ill., J. Virol. 10, 42 (1972). The enzymes Hha I and Hpa II were supplied by New England Bio-Labs, Berverly, Mass. The latter also supplies the enzyme Hae III. Based on the experimental results, more enzyme was used than is required for complete processing of restriction-sensitive DNA under identical reaction conditions. Reactions were stopped by the addition of 5 μΐ of a solution containing 20 mmol / l EDTA, 20% sucrose and 0.05% bromophenolizine and heating for 1 minute at 100 ° C, followed by analysis by polyacrylamide gel electrophoresis. The products were separated on a polyacrylamide composite plate gel (4.5% to 10%) in tris-borate-EDTA buffer (2.5 hours, 150 V) (see Dingman, C. W. and Peacock, A. C., supra) and visualized autoradiographically on a dry gel.

641 87 21

Figure 1 shows the results of gel electrophoresis and autoradiography of 32 P-labeled cDNAs prepared as described above. The sample spots passed electrophoretically from their starting point first through 4.5% acrylamide and then 10% acrylamide. The line on the left side of the image shows the boundary between the two gel areas. Lane A represents the electrophoretic mobility of the entire cDNA transcript. Lane B shows the mobility of cDNA treated with Hha I-ent enzyme. Lane C shows the mobility of cDNA treated with Hae III. Lane D shows the movement of cDNA treated with both Hha I and Hae III. Lane E shows the electrophoretic movement of the material with the strongest line in lane C. Lane F shows the mobility of the material isolated from the strongest line in lane C after first treatment with Hha I. Single-stranded phage M13 DNA with a 32 'P label at the 5' end and digested with Hae III (Houriuchi, K. and Zinder, ND, Proc. Natl. Acad. Sci.) Was used as the size standard. USA 72, 2555 (1975) and its mobility is shown in lane G. The average lengths of the nucleotide sequences of these DNA fragments are numbered to the right.

From the result of lane A, it can be concluded that the cDNA transcript of the full-time placental mRNA is heterodisperse. Treatment with Hha I, lane B, or Hae III, lane C, results in the accumulation of polynucleotides of a certain length. The emergence of such distinct lines proves that at least one sequence with two Hha I and Hae III recognition sites is present in multiple copies in a heterogeneous cDNA transcription population. Hha I treatment yields a fragment of about 470 nucleotides in length, and Hae III treatment yields a fragment of about 550 nucleotides in length. Treatment with both enzymes yields three fragments of 90 nucleotides (A), 460 nucleotides (B), and about 10 nucleotides (C) in length. Due to its small size, fragment C migrates away from the gel under the conditions of Figure 1. The line at the 10% and 4.5% gel boundaries represents a heterogeneous material that is too large to migrate to the 10% gel and therefore accumulates at the boundary. Judging by the band D vi ivoi.sl a, the fragment i t Λ and H oJcvan I ähl Γ »would appear at the same time as the cPNA-rnol el-. This johf-o decision was confirmed e iuoimal i a geo) A larger Maol Π fragment, i.e. moving in lane E in a visible way, and treating it with the enzyme Hhal. This treatment yielded two fragments with the same mobility as those obtained by treating either the cDNA simultaneously with HaeIII and Hhalr, as can be seen by comparing lanes D and F. The total cDNA in the degradation product, lane D, is the autoradiographic density of the total line the measure of dioactivity is greater for fragment A than for fragment B, although size could suggest otherwise. Based on this finding, it can be estimated that fragment A is transcribed closer to the 3 'end of the mRNA than fragment B.

Figure 2 is a schematic representation of the cDNA molecule and the HaellI and Hhal restriction sites are shown in relation to each other. DNA fragments A and B derived from the same cDNA molecule were classified according to their relative intensities obtained from the autoradiogram of lane D in Figure 1. The existence of fragment C was inferred from the different electrophoretic mobility of the line in lanes B and D of Figure 1. The size of DNA fragment A is well known by nucleotide sequencing, cf. Maxam, A. and Gilbert, W. supra. The size of DNA fragment B was determined by comparison to the M13 DNA size markers in lane G of Figure 1.

Nucleotide sequences for DNA fragment A and a portion of the 5 'end of fragment B were determined by the method of Maxam and Gilbert, cf. above. Because the amino acid sequence of human fetal choroidal somatomammotropin is known, the nucleotide sequences of the two fragments could be compared to the amino acid sequence based on a known genetic code. By comparison, it could be concluded that the sequences actually encoded portions of human fetal choroidal somatomammotropin, and this also confirmed the fragment sequence shown in Figure 2.

Example 2

The following reconstruction experiment demonstrates how the method of the present invention can purify a desired nucleotide sequence that is initially only a small amount in the entire nucleotide sequence population. RNA mixtures of known composition containing purified rat globin RNA and human 23 6 4 1 87 polyadenylated placental RNA were used for reverse transcription. 3 2 μm as templates in the presence of β-dCTP (final specific activity 10 μg counting units / min / pmol. The cDNA products were digested with HaellI endonuclease and the fragments were separated on polyacrylamide recombinant gel plates (4.5% to 10%). CDNA fragments). visualized by performing autoradiography on dried gels.

The experimental results are shown in Figure 3. Gel electrophoresis was performed essentially as described in Example 1. Lanes A and H are run with size tags prepared by digesting phage M13 DNA with HaellI endonuclease and labeling the 5 'ends of the fragments thus obtained with P. The average lengths of the nucleotide sequences of these DNA fragments are indicated by numbers on the left. Lanes B-G have electrophoretic patterns obtained by initiating the above reactions with mixtures containing globin RNA and placental RNA in the ratios shown in the following table.

Globin RNA Placental RNA

Lane ng ng B 300 0 C 60 240 D 30 270 E 15 285 F 7.5 292.5 G 0 300

It can be seen that the 320 nucleotide HaellI fragment is derived from globin cDNA. The cDNA transcript of the globin can still be detected, although only 2-5% of the globin RNA is present in the total RNA. (If there is so little desired RNA quality in the isolated RNA mixture that this method of analysis is not suitable, pre-purification can be performed by any known RNA purification method to a concentration of about 2-5% of the desired RNA quality).

Example 3 This example includes purification of a nucleotide sequence fragment containing about 550 base pairs encoding a portion of human fetal choroidal somatomammotropin and a method for determining the purity of the isolated sequence. The purity of the purified fragment was over 99%.

64187 24

Purification of human fetal choroidal somatomammotropin complementary DNA

The polyadenylated placental RNA isolated as described in Example 1 was enriched for human fetal choroidal somatomammotropin mRNA by centrifugation at 25,000 rpm for 16 hours in a Beckman Instruments ultracentrifuge SW27 rotor at a sucrose gradient (5-20% w / v). .). The 11S to 14S region of the gradient was recovered and 100 pg of this RNA was used to synthesize double-stranded cDNA (see Ullrich, A. et al., Supra). The synthesis of the second strand was stopped by extracting the reaction mixture with a volume of ethanol at -70 ° C. The CDNA was digested in 50 μl of a solution containing 6 mmol / l tris-HCl (pH 7.5), 6 mmol / l MgCl 2 and 6 mmol / l N-mercaptoethanol with 2 units of Hae III enzyme at 37 ° C. During 2 hours and thereafter, 0.1 units of bacterial alkaline phosphatase (BAPF type, Worthington Biochem. Corp., Freehold, NJ, unit as defined by the manufacturer) was added and allowed to act for 10 minutes at 60 ° C. The reaction mixture was then extracted with an equal volume of saturated chloroform of phenol. The DNA was precipitated with two volumes of ethanol at -70 ° C and dissolved in 20 μl of a solution containing 10 mmol / l Tris-HCl (pH 8) and 1 mmol / l EDTA, followed by electrophoresis on a polyacrylamide gel (6% w / v). ). Figure 4 (F) is an electrophoretic pattern of the above reaction mixture showing a single line corresponding to a nucleotide sequence of about 550 base pairs. When a 550 pair fragment was excised from the gel and eluted electrophoretically, the result shown in Figure 4 (E) was obtained.

The material corresponding to 550 base pairs shown in Figure 4 (E) was treated for 2 hours at 37 ° C with 4 units of Hha I endonuclease in 50 of the same buffer used in the Hae III endonuclease treatment. It was then extracted with ethanol-chloroform, precipitated with ethanol and the decomposition products were separated electrophoretically on a polyacrylamide gel (6% w / v). The result is seen in Figure 4 (D).

Both fractions were eluted electrophoretically and pooled by incubation for 2 hours at 15 ° C in 20 μl of a mixture of 66 romol / l Tris-HCl (pH 7.6), 6 mmol / l MgCl 2 15 mmol / l 64187 dithiothreitol, 1 mmol / l ATP and 20 pg / ml T4 DNA ligase. The reaction mixture was then diluted to 200 silicon with 0.1 M sodium chloride solution, extracted with one volume of phenol-chloroform mixture, and the DNA was precipitated with 2 volumes of ethanol. The product was suspended in 20 μl of a solution containing 10 mmol / l Tris-HCl (pH 8) and 1 mM EDTA and separated electrophoretically on a polyacrylamide gel (6% w / v). The result is shown in Figure 4 (C). It can be seen from the electrophoresis pattern of Figure 4 (C) that a 550 nucleotide fragment was reconstructed by ligase treatment. Alkaline-phosphatase pretreatment confirmed that the Hha I fragments were correctly aligned to the original 550 nucleotide segment. The additional lines shown in Figure 4 (C) are due to dimer formation between the Hha I fragment pathways, which cannot be prevented by alkaline phosphatase treatment.

The reconstructed 550 nucleotide fragment was excised from the gel and eluted electrophoretically. The electrophoresis pattern of the eluted material is shown in Figure 4 (B). Figure 4 (A) is a size 32 electrophoresis pattern obtained from the Hae III cleavage product of P-labeled double-stranded M13 DNA. Electrophoretic analyzes were performed on a polyacrylic strip gel (6% w / v for 2 hours at 100 V in a solution containing 50 mmol / l tris-borate (pH 8) and 1 mmol / l EDTA. After electrophoresis, the gel was dried and autoradiograms were prepared by imaging Kodak NS2T-X-ray film.

Purity of the reconstructed 550 nucleotide fragment of human fetal choroidal somatomammotropin cDNA. Isolated reconstituted Hae III fragments of human fetal choroidal somatomammotropin cDNA were labeled at the 5 'end with 32 P using a polynucleotide kinase obtained from bacteriophage-T4 infected E. coli by the following method: Panet, A. et al., Biochem 12, 5045 (1973). P-L Biochemical, Milwaukee, Wisconsin, also supplies polynucleotide kinase. The fragment was then treated for 2 hours at 37 ° C with either Hha I or Hpa 11 in 50 μl of a solution of 6 mmol / l Tris-HCl (pH 7.6), 6 mmol / l MgCl 2 and 6 mmol / 1/5-mercaptoethanol. The reaction mixture was then extracted with a volume of phenol-chloroform, the DNA was precipitated with 2 volumes of ethanol at -70 ° C, suspended in 26 μl of a solution of 10 mM Tris-HCl (pH 8) and 1 mM EDTA, electrophoresed and the gel was imaged on X-ray film as described above so that the labeled fragments were visualized.

The results are shown in Figure 5. Figures 5 (B) and 5 (E) show parallel runs performed on a 550 nucleotide fragment prior to restriction enzyme treatment. Figure 5 (C) shows Hha I cleavage and Figure 5 (D) shows Hpa II cleavage.

The purity of the 550 nucleotide fragment was measured by scanning the autoradiogram of restriction enzyme cleavage products and interpreting the radioactivity distribution of the fragment lines quantitatively. Measurements show that purification of the reconstituted Hae III fragment of human fetal choroidal somatomammotropin cDNA achieved greater than 99% purity.

Example 4 In this example, DNA whose nucleotide sequence contains most of the human growth hormone code is purified. Human growth hormone DNA was purified in the same manner as human fetal choroidal somatomammotropin DNA (see Example 3).

Five benign human pituitary tumors, postoperatively rapidly cooled in liquid nitrogen and weighing 0.4 to 1.5 g each, were thawed and homogenized at 4 ° C in a solution containing 4 mol / l guanidinium thiocyanate and 1 mol / l mercaptoethanol. , and having a pH of 5.0. The homogenate was layered in 1.2 ml of a solution containing 5.7 mol / l CsCl and 100 mmol / l EDTA and centrifuged at 15 ° C for 18 hours at 37,000 rpm in a SW50.1 rotor of a Beckman ultracentrifuge ( Beckman Instrument Company, Fullerton, California). RNA migrated to the bottom of the tube. Further purification was performed by the method described in Examples 1 and 3 using an oligo-dT column and sucrose gradient sedimentation. Approximately 10% of the RNA thus separated encoded growth hormone, judging from the incorporation of a radioactive amino acid precursor into wheat germ-derived anti-growth hormone precipitating material in a cell-free translation system (see Roberts, BE and Patterson, BM, Proc. Natl. Acad. Sci. Sci. 2330 (1973)). Single-stranded and 641 87 27 double-stranded DNA were synthesized as described in Example 3. The human growth hormone cDNA was then treated with restriction endonuclease Hae III and alkaline phosphatase as described in Example 3 and then fractionated by gel electrophoresis. A clear line was found at the site corresponding to a length of 550 nucleotides, which was separated for further purification.

Further purification used the methods described above, in which DNA was split into subfragments, which were purified separately and then combined, except that the restriction endonuclease Pvu II was used in conjunction with human growth hormone to obtain two subfragments; one was about 490 nucleotides in length and the other was about 60 nucleotides. All restriction enzymes used herein are supplied by New England Biolabs, Beverly, Massachusetts. The recombinant product, approximately 550 bp in length, was greater than 99% pure, as determined by subdivision into four different endonuclease systems.

Claims (1)

A method for purifying and analyzing the purity of a product consisting essentially of cDNA fragments of at least one restriction site having a specific nucleotide sequence and containing at least one restriction site, the fragments encoding human fetal choroidal transplantation and human growth hormone microorganism, wherein said product of the cDNA fragments to be purified is obtained by treating a population of cDNAs heterogeneous in length and nucleotide sequence in which at least some of the cDNAs contain at least two restriction sites, first with one or two suitable restriction enzymes and then fractionating for example, using gel electrophoresis and separating a fraction of cDNA fragments having the desired specific zero-tide sequence, t characterized in that a) the 5 'phosphate end groups of cDNA fragments of the same length having the desired specific nucleotide sequence are removed by incubation with alkaline phosphatase, for example at pH 7.5 and about 60 ° C for about 10 minutes, b) cleaving the cDNA fragments treated according to a) from a site within the desired nucleotide sequence with a specific restriction enzyme not used in the previous step of treatment, for example using Hha I, Hpa II or PvuII restriction endonuclease at pH about 7.6, incubating at about 30 ° C for about 2 hours, c) fractionating the obtained subfragments by length using, for example, gel electrophoresis, 64187 29 d) recovering fractions comprising two subfragments of a specific nucleotide sequence, e) enzymatically combining the subfragments of d) with by covalent bonding with DNA ligase in the presence of ATP, e.g. at a pH of about 7.6 at a temperature of about 15 ° C for about 2 hours (f) the product obtained according to (f) and containing the cDNA containing mainly the specific DNA sequence is fractionated according to length, for example by gel electrophoresis, and (g) the cDNA obtained according to (f), which is more than 99% contaminating, is taken from recovering a product consisting of cDNA fragments having the desired specific nucleotide sequence free of sequences. 641 87 30 For the preparation of a product of interest, the cDNA fragment containing the same nucleotide sequence, the specific nucleotide sequence and the restriction of the product, and for the analysis of the product, the color fragments of the codon for the uranium chorionic tomacomuron or for the purpose of recombination with a recombinant medium and a DNA transfer vector and for the transfer of a microorganism and colors of these products, the cDNA fragment of which is isolated from the genome name of the nucleic acid its heterogeneous cDNA population, i vane ätminstone en del av cDNA molecule innehäller ätminstone tvä restrictiona, med et ellia tvä warmpliga restriction enzyme och genome from därefter fractionation of lika cDNA fragments, t.ex. The genome is selected from the group consisting of gel electrophores and fractions of a cDNA fragment having its own specific nucleotide sequence, and a) a 51-phosphate group of the cDNA fragment having the same specific nucleotide sequence, avlägsnas medelst alkalisk fosfatas t.ex. the genome is incubated at a pH of about 7.5 and at a temperature of about 60 ° C for about 10 minutes, b) but en) a) the cDNA fragments have a specific nucleotide sequence and a specific restriction enzyme, vilket icke har använts i behandlingens tidigare skede, t.ex. with Hha I, Hpa II or Pvu II restriction endonucleases at a pH of about 7.6, genomic incubation at about 37 ° C under about 2 timmars, c) deferred fragments fractioning preferably from t.ex. (d) the enzymatic medium of covalently binding to the nucleotide sequence of the DNA-ligase. genomic incubation at a pH of about 7.6 and a temperature of about 15 ° C under about 2 hours,