DD143794A1 - PROCESS FOR ISOLATING LONG DOUBLE-STRANDED CDNS MOLECULES FROM A HETEROGENIC POPULATION - Google Patents

Abstract

The invention has the goal of a simple and fast method of isolation for a long time To develop double-stranded cDNA molecules that represent complete genes. According to the invention become relatively long homopolymeric ingrants of an average length of 30 to 60 Nucleotides are polymerized by terminal transferase to the Doppeistrang-cDNA molecules. Subsequently, this material is transformed in vitro with a linear plasmid vector following transferase reaction homopolymers, complementary prion rings with an average length of 8 to 12 Nucleotides possessed by the ends together and in pretreated accordingly Recipient cells, such as CaCl2-treated E. coli bacteria.

Description

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Method for isolating long dsDNA cDNA molecules from a heterogeneous population

A field of application of the invention

The invention relates to a biological in vivo selection method for long double-stranded cDHS molecules from a heterogeneous population.

Areas of application of the invention are medicine, plant and animal production as well as the pharmaceutical industry.

Characteristics of the Known Technological Solutions The previously known methods of isolating long double-stranded cDNA molecules representing complete genes are based on prefractionation by size by gel electrophoresis or density gradients (Villa Komaroff et al., Proc. IJatl. Acad Sei, USA, 75 (1978), pp. 3727-3731) · This prefractionation is laborious and results in losses of material used.

Objective of the invention

The invention aims to provide a simple and rapid method of isolating long double-stranded cDUS molecules representing complete genes.

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Explanation of the essence of the invention

The method of the invention is based on double-stranded cDNA molecules of heterogeneous length, which are obtained enzymatically from the unicron transcription of mRUS, poly (A) -RIiS or with rA-chained single-stranded RUS. According to the invention, relatively long homopolymeric intrinsic lengths of an average length of 30-60, preferably 40, nucleotides are polymerized to the double-stranded cDUS molecules. The polymerization takes place by means of terminal transferase. Due to the kinetics of this' enzymatic reaction ', shorter homopolymeric ends, preferably longer, are preferably synthesized within a double-stranded cDNA population of heterogeneous length at the 3' ends of the long duplex cDUS molecules, preferably longer double stranded cDUS molecules ,

The plasmid pBR322 is used as a vector. The plasmid DNA is cleaved by the restriction endonuclease Psti only at one point. To the resulting linear plasmid of defined length v / ground only short complementary homopoly-. mers of average length of 8-12, preferably 10, huidotides synthesized by terminal transferase (short-tailed vector). Vector and double-stranded cDEFS molecules are then connected together in a ring by hybridization of the complementary homopolymeric inguinal endings and transferred into correspondingly pretreated recipient cells, Z ₀ B. CaCl ₂ -contacted E. coli bacteria.

The new process leads to a high yield of long double-stranded cDHS molecules. The effectiveness of the method can be demonstrated by comparison with a parallel experiment, but using vector molecules to which long homopolymeric ends of scintigraphic length of 65 nucleotides were synthesized (long-hatched vector). Surprisingly, it has been found that the cloning according to the invention with the short-tailed vector is considerably more successful, characterized by the absolute number

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of the tetracycline-resistant transformants, by altering the ampicillin resistance of the vector, by the absolute number of strongly hybridizing clones in the üHUUSUSällT-HOüiiESS method (Grunstein and Hcgness, Proc. Hatl. Acad. Sei, USA, 22 0975). Pp. 3961-3965) and by the restriction analysis for the length distribution of the integrated ds-cDITS, which is obtained from the strongly hybridizing clones.

The new method is simple to implement and widely applicable, with the specific conditions of the terminal transferase reaction being slightly varied depending on the material used (ds-cDHS from the reverse transcription of mHlTS, poly (A) "- HES or with rA-tailed single-stranded EITS) In order to obtain the different lengths of homopolymeric ends necessary for the process on the double, stranded cD'KS and the vector to be selected, the process is explained in more detail below with reference to an embodiment, whereby the invention is not limited.

example

Production of ds-cDiS

Fractionated poly (A) - RHS (9S) from Brockmann bodies of carp are transfected with AHV reverse transcriptase into complementary WS (cDHS) in a 2o ο /u.1 batch containing 3o / ug / ml HHS, 8o / ug / ml oligo (d'I) ₁₂ -1s * 5 ° ¹ ^ ³ T ^ is / HCl pH 8.2, 5o mil KCl, 5 xaM ISgA-C ₀ , 1o mM DTT, o, 5 η & ΐΐ of each dlTTP ( G, G, A, T), 4OO / UCi / ml ^ H-dCTP and 11 units / ml of AI £ V reverse transcriptase, in 1 hr at 37 ° G in o, 72 / ug cDlTS (535,000 cpm ) rewritten. In the alkaline sucrose gradient (5 - 2o »linear sucrose gradient, ο, 2H ITaOH, 1o mEl EDTA, 2o ° C _s 4o ₅ ooo U / rain, Beclnnan SV / 4-o rotor), this material gives a uniform 9S spike , The gradient is divided into 26 fractions, fractions 9 to 16 are cut. This cDHS thus obtained serves as starting material for the double strand synthesis with the KIIQiTQW fragment of Eecoli DITS polymerase I (Boehringer) in a batch with 1.6 / ug / ml cDES 30 mM Sris / HGl pH 7 _S 5 , 4 mil 13gCl _? , o _? 5 ml of β-mercaptoethanol,

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o, 5h of each dITTP (G, C, A, T), 7o with KCl and 62.5 units of enzyme / ml. After 75 nin. Incubation at 18-2 ° C de-skews the mixture, desalting and preparatively treating the ds-cDNA with S 1 -nuclease to remove DIIS single-stranded areas from the material. This material is analytically characterized in the alkaline sucrose gradient. "The average length is only about 3oo bp (base pairs) based on the gradient profile. only half the length of the 9S starting material. The entire ds-cDNA material is prepared preparatively in the neutral saccharose gradient (5-2% linear sucrose gradient, 0.1 M NaCl, 1% with Tris / HCl pH 7.5, 20 ° C., Beckman SW4o rotor, 25.ooo IT / min, 2o hours "). The broad-spike material, with 26 fractions cut from Nos. 12 to 18, serves as the starting material for the synthesis of long homopolymeric single-stranded sequences to the ds-cDNA by terminal transferase (BOLLIM enzyme).

Synthesis of homopolymeric 3'-ends on double-stranded DNA. An assay with o, 57 / ug / ml of ds-cDNA, this concentration corresponds to 5 Bl. 3 'ends based on an average length of the 3'OO bp, 1 mM CoCl ds-cDNA ₂ , o, 2 M HEPES buffer pH 7.1, 5cyuM ⁵ H-d0TP (22 Ci / ml Tol) and 154 Snzyme units / ml is incubated for 5 min at 37 ° C. The incubation time, which is necessary for the synthesis of 3 ° to 6o nucleotides, is determined immediately beforehand analytically. In the preparative approach, the incorporation of "Ή-dCTP corresponds to an average length of 41 nucleotides per 3 'end of the DNA.

For cloning, the vector used is the plasmid pBR322 (Bolivar et al., Gene, 2 (1977), pp. 95-113). After linearization of the vector with the help of the restriction endonuclease PstI, which cleaves the plasmid only once in the ampicillin gene (the gene codes for a β-lactamase which renders ampicillin ineffective and thus causes plasmid resistance of the plasmid-carrying bacterium), is synthesized at its 3 'ends dG_ with terminal transferase. By

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Approach contains 5 ηΜ 3 'ends of the plasmid, ο, if? M HEPES buffer pH 7.1, 1 τΜ CoCl ₂ and 1 _{2 2} enzyme units / ml. For the synthesis of the short-tailed vector (with dG.) 2o / μCi Ίϊ-dGTP (8 Ci / mHol) is employed. "For the tin comparison, synthesis of a long-tailed vector (with 5oo / Ul.i - ^ H-dGTP (8 Ci / ml) The corresponding incubation times at 37 ° C. are determined analytically in each case immediately before and were approx

Hybridization of Ends and Transformation Following synthesis of the homopolymeric ends on ds-cDliS and vector DNA, 2 ng of ds-cDITS and 53 ng of plasmid DrTS (either pBE322-dG ^ ₀ or, for comparison, pBR322-aGg, -) are obtained ? Incubated conditions that allow a juxtaposition of the homopolymeric complementary Einrangenden. (15 ° / μl mixture containing 1o ml, Tris / HCl pH 7 _S 5, 1oo mil KaCl, ο, 2 mil EDTA and the indicated DKS lengths and first 3 Hin ~ at 64 ⁰ C, then 2 StcU at is slowly cooled 42 to 44 ⁰ C and finally incubated over a period of 3 hours at 25 ° C) "This raschung can be directly used to transform CaCIp treated Ε.οοϋ γ1776 * -. bacteria, said by Norgard et al. (Korgard et al., Gene, J5 (1977)> S 279 ~ 292).

Table 1 shows the characteristic data of the obtained transformants (clones). From the table it can be seen that the effectiveness of the molecular cloning with the erfindvaigsgemäßen, briefly ge schwänzten vector 2o-fold, with the comparatively long-tailed vector, however, only 3-fold above the respective control value (vector without ds-cDIiS) is. Among the found Clones are due to the integration of the ds-cDITS in the ampicillin resistance gene in accordance with the invention method 60J0, the comparatively performed method, however, only 16> & egg-sensitive clones «In the back-hybridization with the 93-Fraiction of poly (A) ^ - FdTS, originally also used as starting material for

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the cDITS synthesis served a similar picture; in the process according to the invention, 6%, but only comparatively 1% strongly hybridizing clones are found in the comparatively performed method. Finally, among the 38 are strongly hybridizing. Clones from the process according to the invention contain 6 clones with insert lengths of 6OObp, 15 with lengths of 300 to 600 bp and only 5 with lengths around 300 bp, with 26 clones only being investigated.

Table 1

Characterization and Comparison of the Efficiency of the Molecular Cloning of ds-cDHS-dC ,,. * In the method according to the invention with short-tailed vector and comparatively

carried out with long-tailed vector

Invention according to the process Proceed in parallel pBR322-dG _1o pBR322-dG ₆₅ Tc ^r transformants Ap ^r Ap ^(r > Ap ^s η % η % Of which with 2 ² JUp ₀ Iy (A) ^ - RHS strongly hybridizing clones lengths of integrated ds ~ cDITS in 26 of 38 strongly hybridizing clones 600 bp 3oo - 600 bp 300 bp 6o2 I00 72 12 171 28 359 60 138 I00 83 44 75 4o 3o 16 Controlled: Dc ^{r '} Iraris formants with linearized vector without ds-cDIT3 38 6 6 15 5 2 1 not examined 5o 5 65 35

Remarks: Tc tetrasyklin resistant

Ap ^r ampicillin resistant

Ap ^ low ampicillin resistant

Ap ^s ampisillin sensitive

bp base pairs -

Claims (5)

invention claim A method for isolating long dsDNA cDHS molecules from a heterogeneous population enzymatically derived from reverse transcription of mRNA, poly (A) ⁺ RUS, or rA-tailed single-stranded RI, characterized by the use of long polymers Single-stranded cDUS molecules polymerize, then this material with a linear plasmid vector having homopolymeric, single-stranded intrastranding sites with an average length of 8 to 12 nucleotides after the transferase reaction, via the ends abutted and transferred to recipient cells. 2. The method according to item 1, characterized in that polymerized long polymer Einstrangenden with an average length of 40 nucleotides to the double-stranded cDUS molecules. 3. The method according to item 1, characterized in that polymerizing the long polymeric Einstrangenden by means of terminal transferase to the double-stranded cDHS molecules « 4. Method according to item 1, characterized in that a linear plasmid vector is used which has an average length of 10 nucleotides « 5. The method according to item 1, characterized in that are used as recipient cells with CaCl ₂ treated Eocoli bacteria