Enzymatic reactions

5.1.3.1 Polymerase chain reaction (PCR)

PCR is an enzymatic reaction to amplify the specific DNA fragments in vitro. PCR reaction mixture includes double-stranded DNA template, a pair of primers specific for the beginning sequences of each DNA strand, dNTPs, a DNA polymerase and the appropriate buffer. Each cycle consists of DNA denaturation, binding of the primers to the single-stranded template DNA (annealing) and synthesizing new DNA (elongation), so that the desired double-stranded DNA will be amplified in large quantity at the end of PCR. The most commonly used polymerase is Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus.

The optimal elongation temperature for Taq-polymerase is 72°C, and with an amplification

rate of 1kb/min. All Taq-PCR used in the study were based on the following standard programs:

Standard PCR reaction for Taq-polymerase:

DNA template ca. 100 ng

5’ primer (10 pmol/μl) 2 μl 3’ primer (10 pmol/μl) 2 μl

10 x PCR buffer 2 μl

MgCl2 (25 mM) 2 μl

dNTP mix (2 mM each) 2 μl

Taq polymerase 0.5 μl

H2O ad to 20 μl

Standard PCR program for Taq-polymerase:

1^st step 95 ºC 5 min initial denaturation

2^nd step: 25-35 cycles

95 ºC 30 s denaturation 55 ºC variable annealing 72 ºC variable elongation

3^rd step 72 ºC 5 min final elongation

4 ºC hold the final products of PCRs

The use of Phusion (Biozyme) and KOD Hot Start (Novagen) DNA polymerase ensures high accuracy in amplification and high processivity because of the combination of a Pyrococcus similar DNA-polymerase with a double-stranded DNA binding domain. The amount of components in a PCR reactions and the PCR-program for these two DNA-polymerases are included in the manufacturer’s instruction.

5.1.3.2 A-tailing of PCR fragments and ligation into pGEM^®-T Easy vector

PCR product amplified by phusion and KOD Hot Start polymerase are blunt-end. In order to be used in the pGEM^®-T Easy Vector System from Promega, it must be subjected to the A-tailing reaction. This was achieved by addition of an adenosine at the 3’-end of the DNA-fragments using Taq DNA polymerase. 10 µl reaction mixture were usually composed of 5 µl purified PCR fragment, 2 µl 1 mM ATP, 1µl 10 x Taq DNA Polymerase Reaction Buffer, 1 µl 25 mM MgCl2 and 1 µl 5 U Taq DNA polymerase, mixed and incubated at 70°C for 30 min.

For the subsequent ligation, the components of 3 µl A-Tailing product, 5 µl 2 x Rapid Ligation Buffer, 1 µl pGEM^®-T Easy Vector and 1 µl T4 DNA ligase (the last three reagents are from the pGEM^®-T Easy Vector System I) were mixed and kept at room temperature for 1-2 hours. The ligation product was then transformed in competent E. coli cells (5.2.1.2). The detection of positive transformants was achieved by the blue-white selection (5.1.3.3).

5.1.3.3 Blue-white selection

LacZ gene encoding β-galactosidase enzyme is in the pGEM^®-T Easy vector. This enzyme breaks down a series of synthetic materials besides the natural substrate lactose, such as here used X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactoside). X-gal is hydrolyzed to the colorless galactose and the blue precipitate of 4-chloro-3-bromo-indigo. The colony appears blue, if it is capable of producing the β-galactosidase enzyme. However, the colony appears white if the expression of the β-galactosidase was destroyed by the insertion of foreign DNA.

In order to prove successful transformation of a cloned pGEM^®-T Easy Vector, the transformation mixture was plated out on the antibiotic selective plates together with 20 µl 100 mM IPTG (isopropyl-β-D-thiogalactopyranoside) and 35 µl X-Gal (50 mg/1 ml of DMF) and incubated overnight at 37°C. IPTG is a synthetic analogue of lactose and induces the synthesis of the β-galactosidase by inactivating the lacZ repressor. White Colonies were picked and incubated in 1 ml of selective medium at 37°C and subjected to a mini preparation (5.1.1.1) on the next day.

5.1.3.4 Restriction endonuclease digestion of DNA

Restriction endonucleases are widely used for analytical digestion of plasmids and for the construction of recombinant DNA molecules. It should be noted that the amount of enzyme is at most 10% of the total volume, in order to avoid negative effects of glycerol. 3-5 µg DNA were usually applied for the ligation reaction. After 1-2 h incubation at the appropriate temperature, the digestion was tested by agarose-gel electrophoresis (5.1.4.2).

5.1.3.5 Generation of blunt ends

Blunting of a cohesive end from a restriction digestion were used to inactive an enzyme site or prepare for the blunt-end ligation. T4 DNA polymerase was majorly used to removal of 3’-overhang due to its strong 3’→5’ exonuclease activity, while the Klenow-fragment, the large

fragment of DNA polymerase I, blunts DNA by filling-in 5’-overhangs. Both enzymes for DNA blunting were applied according to the protocol from manufacturer.

5.1.3.6 Dephosphorylation of 5’-OH group in vectors

If the vector was digested by a single enzyme, it was dephosphorylated before ligation in order to prevent a re-ligation with itself. For this purpose, 1 µl alkaline phosphatase (Fast AP from Fermentas) was added to the 20 µl digestion and incubated for 30 min at 37°C. Since Fast AP is active in all kinds of buffers for digestion, the specific buffer is not required.

5.1.3.7 Ligation of DNA fragments

The ligation of two DNA fragments is carried out in a reaction with total volume of 20 µl, where the concentration (in molar) of insert was 3-5 fold excess to that of the vector. The Amount of T4 DNA ligase and the T4 DNA ligase buffer were applied according to the manufacturer's instructions. The reaction takes place at room temperature: 20 min for "sticky end" ligation and 30 min for a "blunt end" ligation. The ligase did not need to be purified or deactivated for transformation into E. coli competent cells.

5.1.3.8 RT-PCR

The RT-PCR includes reverse transcription reaction (RT) of mRNA into cDNA and a following PCR reaction using the obtained cDNA as template. RT-PCR was used to detect RNA expression levels and also to amplify the coding sequence of the target gene. Before RT reaction, DNaseI (RNase-free, Fermentas) was added to RNA sample at 37°C for 30 min for removal of the DNA contamination. After addition of 25 mM EDTA, DNaseI was inactivated by heating at 65°C for 10 min. After denaturation of the template RNA at 70°C for 5 min, the Revert Aid™ M-MuLV Reverse Transcriptase catalyzed the RT reaction to generate total cDNA library by incubation at 42ºC for 60 min in the presence of oligo dT primer, dNTPs, RNase inhibitor and reaction buffer. The reaction was stopped by heating at 70ºC for 10 min.

2 µl of the reaction mixture was used in a PCR reaction with primers specific for a targeting DNA fragment (5.1.3.1).