Sub-cloning of cofilin and GFP cDNA

3.7.2.1. Cofilin and GFP cDNA amplification

The polymerase chain reaction (PCR) constitutes an enzymatic in vitro amplification of specific cDNA segments. Amplification occurs in automated, temperature-controlled cycles of denaturation, annealing and elongation in a thermal cycler. Initially, double-stranded template DNA is separated into its complementary single strands by heating (denaturation). At a lower temperature two oligonucleotides primers, flanking the DNA region to be amplified, hybridize to their respective complementary sequences on opposite strands (annealing) and serve as primers for DNA synthesis in a 5'→3' direction (elongation). Primer extension is catalyzed at a slightly increased temperature by a thermostable DNA polymerase that add deoxyribonucleotide triphosphates (dNTPs) to the recessed 3'-hydroxyl end of extending strands, thereby generating new double-stranded DNA across the primer-flanked region. The products of each reaction cycle are then denatured to permit a new amplification cycle. Theoretically, for n cycles a 2ⁿ-fold amplification of a specific DNA sequence is obtained.

Primer for amplification of cofilin cDNA was commercially synthesized (MWG Biotech, Ebersberg, Germany). It was designed corresponding to the DNA segment to be amplified, provided with restriction sites for endonuclease digestion. Pairs of primers were designed to have equivalent melting temperatures (Tm), calculated according to the formula

Tm[°C] = (A+T) ×2 + (G+C) ×4.

Where A, T, G, and C are the 2’-deoxyribonucleosides; adenosine (A), thymidine (T), guanosine (G) and cytidine (C) with in the primer sequence.

Following cofilin primers containing respective sites for restriction endonuclease (underlined) were designed to amplify cofilin cDNA from plasmid pEGFP-C1-cofilin:

Sense primer (Nco I) 5'-ATTATTCCATGGATGGCCTCCGGTGTGGC-3' Antisense primer (BamH I) 5'-TTATTGGATCCTCACAAAGGCTTGCCC-3'

Whereas to amplify GFP cDNA from same plasmid (pEGFP-C1-cofilin), following GFP primers were used:

Sense primer (BamH I) 5'-ATTAGGATCCCCGGTCGCCACCATG-3'

Antisense primer (Bgl II) 5'-AGAATTCGAAGCTTGAGCTCGAGATCTGAGTCC-3' The annealing temperature for each PCR was typically estimated experimentally. For preparative DNA amplification as part of cloning strategies, High fidelity DNA polymerase was used in preference to Taq DNA polymerase, due to its 3'→5' proofreading exonuclease activity which minimizes the risk of nucleotide miss-incorporation during elongation.

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PCR reaction composition for DNA amplification was as follows:

Template DNA 20-50 ng (Plasmid pEGFP-C1-cofilin) Sense primer (forward) 100 pmoles

Antisense primer (reverse) 100 pmoles

dNTPs 200 µM each

PCR reaction buffer 1x

DMSO 5-10% (Optional, to increase yield, specificity, consistency) DNA polymerase 1-2.5 units

Final reaction volume 100 µl (preparative PCR), 20-50 µl (analytical PCR)

Thermal cycle parameters for PCR were as follows:

Lid temperature 105°C

Initial denaturation 94°C, 5 minutes Denaturation 94°C, 30 seconds

Annealing 50-55°C (primer pair and template specific), 1minute Elongation 72°C, 1 minute/kb

Cycles 30

Final elongation 72°C, 10 minutes

The PCR amplified products were analyzed by agarose gel electrophoresis. Cofilin cDNA was further processed for cloning into the pQE-60 vector (Qiagen, Hiden, Germany). The resultant pQE-60-cofilin plasmid was used as backbone to ligate the GFP cDNA between the C-terminus of cofilin and the His-tag to get the pQE-60-cofilin-GFP plasmid.

3.7.2.2. Agarose gel electrophoresis for isolating cDNA Buffers and solutions

TBE buffer (0.5x): 45 mM Tris-base, 45 mM boric acid, 1 mM EDTA (pH 8.0) TE buffer: 10 mM Tris-base and 1 mM EDTA (pH 8.0)

DNA loading buffer (6x): 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol, 30%

(v/v) glycerol, and 50 mM EDTA

Agarose gel electrophoresis was used separate and purify DNA fragments from PCR reaction mixtures. This method uses the migration of negatively charged DNA towards the anode in an electric field. The fragments migrate through the gel matrix at rates inversely proportional to the logarithm (log10) of the number of base pairs. The agarose gel was prepared by melting 1-2%

(w/v) agarose in 0.5x TBE electrophoresis buffer, adding 0.5 µg/ml ethidium bromide to the melted agarose and casting in a tray of desired size. A plastic comb of desired thickness and well number was inserted before the solidification of agarose gel. After solidification, the gel was placed in an electrophoresis tank and submerged in 0.5x TBE buffer. The DNA samples were mixed with DNA loading buffer and loaded into the gel wells. In adjacent to the samples, 5 µl of a DNA standard (SmartLadder^TM Eurogentec GmbH, Germany) was loaded. Horizontal

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electrophoresis was carried out at approximately 100 V. DNA bands were visualized within agarose gel by illumination of DNA-intercalated fluorescent dye ethidium bromide under UV light. The length of DNA fragments were determined by comparing their mobility with that of DNA standards. The stained gel was photographed under UV light.

3.7.2.3. DNA recovery from agarose gel

For preparative purposes DNA fragments of interest were cut out from stained agarose gels with a sharp scalpel under UV illumination. The gel slices were solubilized and DNA was purified by using the QIAquick Gel extraction kit (Qiagen) according to the manufacturer’s instructions.

Agarose gel slices were weighed (1 volume) and dissolved in 3 volumes of solubilization buffer QG by incubating at 50°C for 10 minutes. A volume of isopropanol was added and mixed. The whole solution was applied to a silica-gel embedded in QIAquick spin column and centrifuged at 13,000 rpm for 1 minute in order to bind DNA. The column was washed once by adding 0.75 ml wash buffer PE and centrifuging at 13,000 rpm for 1 minute. An additional 1 minute centrifugation was done to remove residual buffer. DNA was eluted by adding 30-50 µl water or TE to the column followed by incubation for 2 minutes at room temperature and then centrifugation at 13,000 rpm for 1 minute. All the buffers were provided in the QIAquick Gel extraction kit.

3.7.2.4. Restriction endonuclease digestion of DNA

For sub-cloning cofilin into pQE-60 vector, 1-5 µg PCR amplified cofilin cDNA and pQE-60 vector were digested with 1-20 U of restriction enzymes (BamHI and NcoI) in 10-50 µl of reaction buffer. For construction of pQE-60-cofilin-GFP, the resultant pQE-60-cofilin plasmid and GFP cDNA were digested with BglII and BamHI restriction endonucleases. Complete digestion was confirmed by agarose gel electrophoresis. For analytical purposes, 0.2-1 µg DNA were digested with 1-5 U of enzyme in a volume of 10-20 µl of reaction buffer. In both cases digests were incubated for 3 hours at 37°C. Reaction buffers were supplied by the manufacturer.

Enzymes were heat-inactivated as recommended by the supplier or removed by purifying the digested DNA using QIAquick PCR purification kit.

3.7.2.5. Dephosphorylation of linearized plasmid DNA by CIP

In order to prevent self-ligation of vector ends, linearized plasmid DNA was treated with calf intestine alkaline phosphatase (CIP). CIP catalyzes the hydrolysis of phosphate to generate 5'-hydroxyl ends. T4 DNA ligase requires 5'-phosphate residues to catalyze new phosphodiester bonds. After hydrolysis of vector’s 5'-phosphate ends, ligation is only possible between vector and inserts ends, but not between vector-ends themselves. Dephosphorylation was carried out directly following plasmid linearization. CIP was added to the digested mixture at a concentration of 1 U per pmole of linearized vector DNA and incubated at 37°C for 45 minutes.

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3.7.2.6. Purification of the digested DNA

To inactivate and remove the proteins e.g. restriction enzymes, digested DNA were purified using the QIAquick PCR purification kit (Qiagen) and according to the manufacturer’s instructions with slight modification. Buffers were provided in the kit and all centrifugation steps were carried out at 13,000 rpm at 25°C using a tabletop micro-centrifuge. To the DNA solutions sought purification, 5 volume of buffer PB and one volume of isopropanol were added. The mixed solution was then applied to QIAquick spin column (Qiagen) and centrifuged for 1 minute in order to bind DNA. The column was washed with 0.75 ml buffer PE and centrifuged for 1 minute. Flow through was discarded and an additional centrifugation was done to remove the residual ethanol present in buffer PE. The column was then placed into a clean 1.5 ml eppendorf tube and the purified DNA was eluted with 30-50 µl of buffer TE or water as described for QIAquick Gel extraction kit (section 3.7.2.3).

3.7.2.7. Ligation of DNA fragments

DNA fragments of bearing either sticky or blunt ends can be ligated in vitro with bacteriophage T4 DNA ligase. This enzyme catalyzes the formation of new phosphodiester bonds between a 5'-phosphate residue of one and a 3'-hydroxyl residue of another double-stranded DNA fragment generated by restriction endonucleases.

Ligation was carried out using Rapid DNA ligation kit (Roche) according to the manufacturer’s instructions with slight modification. All the buffers were provided in the kit. A 2-5 molar excess of PCR amplified cofilin cDNA relative to the linearized and dephosphorylated pQE-60 vector DNA were taken and mixed. The vector and insert DNA mixture was diluted to a final volume of 10 µl using dilution buffer. To the diluted mixture, 10µl of ligation buffer and 5 units of T4 DNA ligase were added and mixed thoroughly. The ligation reaction mixture was then incubated at 20°C for 10 minutes subsequently used for transformation. An aliquot of 5 µl was used for transformation of 200 µl DH5α competent cells. Positive bacterial colonies were selected, screened by restriction digestion or PCR, and confirmed by DNA sequencing.

3.7.2.8. Quantification of DNA solution

The concentration of nucleic acid solutions was determined by spectrophotometry. The UV absorption was measured at a wavelength of 260 nm (OD260) using a quartz cuvette of 1 cm width. For double-stranded DNA an OD260 =1.0 corresponds to approximately 50 µg DNA/ml. In addition the OD260 was measured to estimate the purity of the nucleic acid sample. A ratio OD260/OD280 of significantly less than 1.8-2.0 indicates protein contamination.

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3.7.2.9. DNA sequencing

The sequence of specific target regions in recombinant plasmid DNA was determined by a commercial sequencing service (Agowa GmbH Berlin, Germany). The sequence data were verified on the basis of the corresponding fluorescence.