3. Materials and methods
3.4. Recombinant DNA processing and manipulation
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.
Primers for amplification of LIMK1 and LIMK2 cDNA were commercially synthesized (MWG Biotech, Ebersberg, Germany). They were 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 formula1 Tm [°C] = (A+T)x2 + (G+C)x4. The annealing temperature for each PCR was typically estimated by 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 misincorporation during elongation. PCR reaction composition for DNA amplification was as follows:
Template DNA 20-50ng (Plasmid PUC-SRα-LIMK2 or cDNA of LIMK1)
Sense primer (forward) 100 pmoles Antisense primer (reverse) 100 pmoles
dNTPs 200μM each
PCR reaction buffer 1x
DMSO 5-10% (Optional, to increase yield, specificity, consistency)
DNA polymerase 1-2.5 units
Final reaction volume 100μl (preparative PCR), 20-50μl (analytical PCR)
The PCR amplified products of LIMK1 and LIMK2 gene were analyzed by agarose gel electrophoresis, they were further proceed for cloning into the EGFP-C1 vector.
1 A, T, G, C: number of the 2’-deoxyribonucleosides adenosine (A), thymidine (T)guanosine (G) and cytidine (C)
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
3.4.2.
Buffers and solutions
TBE buffer (5X) Tris-base 54 g
(Working solution 0.5X) boric acid 27.5 g
EDTA 20 ml of 0.5 M, pH 8.0 Final volume 1 Liter
TE 10mM Tris (pH 7.6), ImM EDTA (pH8.0)
DNA loading buffer (6X) 0.25% (w/v) bromophenol blue
0.25% (w/v) xylene cyanol
30% (v/v) glycerol
50 mM EDTA
Ethidium bromide 1% (w/v) in water
DNA standard SmartLadderTM Eurogentec GmbH, Germany
3.4.3.
Restriction endonuclease digestion of DNA
Type II restriction endonucleases were used to digest double-stranded DNA for analytical or preparative purposes. These restriction enzyme bind to short, specific nucleotides sequence and cleave the DNA within this region by hydrolyzing a phosphodiester bond in each strand. The recognition sequences (palindromes) possess twofold rotational symmetry. Staggered cleavage generates complementary, cohesive 5' and 3' ends (sticky ends). Cleavage in the axis of symmetry yields blunt ends.
For preparative purposes, 1-5 µg PCR amplified DNA and EGFP-C1 vector were digested with 1-20 U of restriction enzymes (see section 3.2.7.1) in a volume of 10-50 µl of reaction buffer. 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 3h at 37°C. Reaction buffers were supplied by the manufacturer. Enzymes were heat-inactivated as recommended by the supplier or removed by purification of the digested DNA by column chromatography.
Materials and methods 40
3.4.4.
Purification of the digested DNA
To inactivate and remove the proteins e.g. restriction enzymes, digested DNA was purified by using the QIAquick PCR purification kit (Qiagen) 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 RT in a tabletop microcentrifuge. The 5 volume of the buffer PB and one volume of isopropanol were added to the DNA solution. The sample was then applied to QIAquick spin column (Qiagen) and centrifuged for 1 min in order to bind DNA to the column. For washing of DNA on the column, 0.75 ml of buffer PE was added to the column and spun down for 1 min. The flow-through was removed and the column was centrifuged for an additional 1 min to remove the residual ethanol. The column was then placed into a clean 1.5 ml tube and the DNA was eluted by the addition of 30-50 µl of buffer EB (10 mM Tris/HCl, pH 8.5) or dH2O
to the column followed by centrifuging for 1 min. If needed the DNA could be concentrated by precipitating with ethanol.
3.4.5.
Ethanol precipitation of DNA
In the presence of relatively high concentrations of monovalent cations, ethanol induces a structural transition in nucleic acid molecules, which causes them to aggregate and precipitate from solution. Ethanol precipitation was used to concentrate and/or desalt DNA solutions and to remove any residual impurity.
5 M NaCl solution was added to the DNA sample to a final concentration of 250 mM. Three volumes of ethanol (-20°C) were added to the solution and stored on ice for 30 min and then centrifuged at 13,000 rpm for 15 min at 4°C. The pellet was washed with one volume of ice-cold 70 % ethanol, recentrifuged for 5 min and dried at room temperature on desk. The DNA was dissolved in water or TE (pH 8) for 20 min at 37°C at an appropriate concentration.
3.4.6.
Dephosphorylation of linearized plasmid DNA by CIP
In order to prevent self-ligation of vector ends in cloning strategies linearized plasmid DNA was treated with calf intestine alkaline phosphatase (CIP). CIP catalyzes the hydrolysis of 5'- phosphate residues to 5'-hydroxyl ends. Since T4 DNA ligase requires 5'-phosphate residues to catalyze new phosphodiester bonds, ligation is only possible between vector ends and inserts, but not between vector ends themselves. Dephosphorylation was carried out directly following plasmid linearization. CIP was added to the digestion mixture at a concentration of 1 U per pmole linearized vector DNA. After 45 min incubation at 37°C, CIP and the restriction enzymes were removed as described previously.
3.4.7.
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 with the kit. The insert DNA (LIMK1 or LIMK2 PCR amplified DNA) was employed at a 2-5 molar excess relative to the linearized and dephosphorylated EGFP-C1 vector DNA. The vector DNA and insert mixture were diluted in dilution buffer to a final volume of 10μl. 10μl of ligation buffer was added in diluted DNA mixture. 5unit of T4 DNA ligase was added and mix thoroughly. The ligation reaction mixture was incubated 5-10 minutes at 15-25°C. Ligation reaction mixture (5μl) was used directly for transformation of the 200μl competent cells.
3.4.8.
Miniprep: small-scale preparation of plasmid DNA
Plasmid DNA was purified from bacteria cultures by alkaline lysis of the bacterial cells by using QIAprep Spin miniprep kit according to the manufacture instructions. In brief, a small culture (10ml) of bacteria was grown in order to amplify the plasmid of interest in vivo. The bacteria
were harvested by centrifugation at 13,000 rpm for 1 min at room temperature. This pellet was resuspended in 250µl buffer P1 (50 mM Tris·Cl pH 8.0, 10 mM EDTA, 100 µg/ml RNase A). Lysis buffer P2 (250µl; 0.2 N NaOH, 1 % SDS) was added in resuspended pellet followed by gently mixing and incubated for 5 minutes at room temperature. It causes denaturation of the DNA by NaOH and of the bacterial proteins by SDS. The alkaline mixture was neutralized by 350µl of buffer N3 (containing guanidine hydrochloride, 3M potassium acetate, pH 5.5) causing reannealing of plasmid DNA and precipitation of SDS. The white precipitate, containing proteins, chromosomal DNA, SDS and cell debris was removed by centrifugation for 10 minutes at 13,000 rpm while the plasmid DNA was left in solution. The supernatant was passed through the spin column by centrifugation at 13,000 rpm for 1 minute. After washing of the column with 0.75ml buffer PE, the column was additionally centrifuged for 1 minute to remove residual buffer. The DNA was eluted by adding 50µl water or TE and centrifugation of the column at 13,000 rpm for 1 min.
3.4.9.
Endofree Maxiprep: Large scale preparation of plasmid DNA
Endotoxins or Lipopolysaccharides (LPS) are cell membrane components of Gram-negative bacteria (e.g., E.coli). During lysis of bacterial cells for plasmid preparations, endotoxin
Materials and methods 42
structures lead to co-purification of endotoxins with the plasmid DNA. Endotoxins strongly influence transfection of DNA into primary cells like endothelial cells and increased endotoxins levels leads to sharply reduced transfection efficiencies. To solve this problem, Endofree Plasmid maxi kit was used for large-scale purification.
Bacterial culture (100 ml) was centrifuged at 4,000 rpm for 15 min at 4°C. The pellet was resuspended in 10 ml of P1 (50mM Tris.Cl, pH 8.0; 10mM EDTA; 100μg/ml RNase A). The resuspended pellet was lysed with 10 ml P2 (200mM NaOH, 1% SDS) by incubating for 5 min at room temperature. The lysis was stopped by adding 10 ml ice-cold P3 (3M potassium acetate, pH 5.5) and the white precipitated mixture was poured into the barrel of QIAfilter cartridge and incubated. After 10 minutes, the plunger was inserted into the QIAfilter cartridge and the cell lysate was filtered into a 50ml tube. 2.5ml of buffer ER (endotoxin removal buffer) was added to the filtered lysate and incubated on ice for 30 minutes. The ER buffer treated lysate was loaded on an equilibrated anion exchange column that was subsequently washed twice with 30 ml of buffer QC (1.0M NaCl, 50mM MOPS, pH7.0; 15% isopropanol). The bound DNA was eluted with elution buffer QN (1.6M NaCl, 50mM MOPS, pH7.0; 15% isopropanol) by gravity flow. The eluted DNA was precipitated by adding 0.7 volumes isopropanol at room temperature and subsequent centrifugation at ≥15,000xg for 30 min at 4°C. The pellet was washed with 5 ml 70 % ethanol, re-centrifuged for 10 min, dried under vacuum and re-dissolved in 1ml of endothelial electroporation buffer or TE.
3.4.10.
Quantification of DNA and RNA solutions
The concentration of nucleic acid solutions was determined by spectrophotometry. The ultraviolet (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.
For RNA an OD260 = 1.0 corresponds to approximately 40 µg RNA/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.
3.4.11.
Agarose gel electrophoresis
Agarose gel electrophoresis was used for analytical and preparative purposes. The method is based on 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. DNA bands were visualized within an agarose gel by staining with the intercalating fluorescent dye ethidium bromide and subsequent illumination under UV light. The length of a DNA fragment is determined by comparison of its mobility to that of DNA standards. For casting gel, 1-2 % (w/v) agarose was melted in 0.5xTBE electrophoresis buffer and supplemented with 0.5 µg/ml ethidium bromide and casted in a tray of desired size. The gel was
placed in an electrophoresis tank and submerged in 0.5xTBE buffer. The DNA samples were mixed with DNA loading buffer and loaded into the gel wells. In addition 5 µl of a DNA standard was loaded in parallel with the samples. Horizontal electrophoresis was carried out at approximately 100 V. The stained gel was photographed under UV light.
3.4.12.DNA recovery from agarose gel
For preparative purposes DNA fragments of interest were cut out from stained agarose gels with a razor blade 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 each slice in 3 volumes of solubilization buffer QG by incubation for 10 min at 50°C. 1 volume isopropanol was added and the solution was applied to a silica-gel QIAquick spin column. The column was centrifuged at 13,000 rpm for 1 min, washed with washing buffer PE and centrifuged again. The extra centrifugation was done to remove residual buffer. DNA was eluted by adding 30-50µl water or TE on the column and centrifugation of the column at 13,000 rpm for 1 min. All the buffers were provided by the manufacturer.
3.4.13.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 electropherogram and subjected to computer analysis.