Chapter 2 Materials and methods
2.2 Creation of ADAMTS13 missense mutations
2.2.1 Sequencing ADAMTS13 cDNA
ADAMTS13 missense mutations and polymorphisms were introduced into a pcDNA
3.1/V5-His TOPO ® vector (Life technologies) containing the complete ADAMTS13 cDNA sequence (kindly provided by Dr. F. Scheiflinger, Baxter Bioscience). ADAMTS13 was therefore expressed with a C-terminal V5 epitope and polyhistidine tag (6xHis tag). The entire ADAMTS13 cDNA within the vector was first sequenced to ensure there were no unexpected deletions or mutations within its sequence. The primers used are shown in the appendix (Table S 2).
Sequencing was carried out using the principle of dye-terminator DNA sequencing, originally described by Sanger (Sanger et al, 1977). This was set up with the big dye terminator v3.1 cycle sequencing kit (Life technologies) using 3.2μM of primer and 1µg of plasmid DNA. The cycle sequencing reaction was set up as follows:
96⁰C 10 seconds
}
x 25 cycles 50⁰C 5 seconds60⁰C 4 minutes
Polymerase chain reaction (PCR) products were purified using sephadex and loaded onto an automated ABI Prism 310 genetic analyser (Life technologies).
2.2.2 Site directed mutagenesis
Missense mutations and polymorphisms were introduced by site directed mutagenesis. The primers used for site directed mutagenesis are shown in the appendix (Table S 3 and Table S 4). The mutagenesis reaction was set up using the QuickChange II XL site directed mutagenesis kit (Agilent technologies) using 10- 50ng of DNA in the absence or presence of 5% dimethyl sulfoxide (DMSO).
63 The PCR conditions used were as follows:
95⁰C 1 minute x1 cycle 95⁰C 50 seconds
}
x18 cycles 60⁰C 50 seconds 68⁰C 5 minutes 68⁰C 7 minutes x1 cycle2.2.3 DpnI digestion and agarose gel electrophoresis
The restriction enzyme DpnI (1μl) was added to the reaction mix for 1 hour at 37⁰C to digest unmutated vector. The DpnI digested DNA was run on a 1.5% agarose gel prepared in TBE (Tris-Borate-EDTA (Ethylenediaminetetraacetic acid), 89mM Tris, 89mM boric acid, 2mM EDTA, pH 8.3). To 100ml of prepared agarose 1μl of ethidium bromide was added and for loading 5μl of loading buffer (0.25% bromophenol blue, 10% glycerol in TBE) was added to 5μl of water and 1μl of DpnI digested DNA. This was run at 120V for ~30 minutes.
2.2.4 Transformation
Transformation was performed after detection of a band on a 1.5% agarose gel, after DpnI digestion. Transformation was carried out either using TOP10 one shot chemically competent Escherichia Coli (E. coli) (Life technologies), XL10-Gold ultracompetent E. coli (Agilent technologies) or Sure 2 supercompetent E. coli (Agilent technologies). Briefly cells were incubated with 1-2µl of DpnI digested DNA (after addition of beta mercaptoethanol to the XL10-Gold and Sure 2 supercompetent cells) for 30 minutes on ice. The cells were then heated at 42⁰C for 30 seconds followed by incubation on ice for 2 minutes. S.O.C medium (Life technologies) was added to Top10 cells and NZY+ broth (prepared according to the manufacturer’s instructions) was added to the XL10-Gold ultracompetent cells and Sure 2 supercompetent cells (as recommended by the manufacturers). E. coli were then incubated at 37⁰C for 1 hour with shaking (250rpm). After this E. coli were plated onto LB (Luria-Bertani) agar (Anachem Ltd) containing 50μg/ml ampicillin and left overnight at 37⁰C (~16 hours). Plates were stored at 4⁰C until further analysis.
64 2.2.5 Mini preparation
For mini preparation a single E. coli colony was picked using a 1μl sterile inoculation loop (Greiner Bio-One Ltd) and added to 5ml of LB media (Anachem Ltd) containing 50μg/ml ampicillin. The cultures were left to grow overnight at 37⁰C with shaking at 250rpm for approximately 16 hours.
The next day cultures underwent mini preparation using a commercial kit, according to the manufacturer’s instructions (Qiagen). Before mini preparation glycerol stocks of the E. coli were prepared by adding 190μl of 80% glycerol (MP Biomedicals) to 810μl of culture.
In brief the cultures were centrifuged at 15,700g for 3 minutes and the supernatant removed. The pelleted E. coli were resuspended in 250μl of resuspension buffer containing RNase. After, 250μl of alkaline lysis buffer was added, followed by neutralisation buffer (350μl) 5 minutes later. Samples were spun at 15,700g for 10 minutes to precipitate and remove proteins, cell debris and chromosomal DNA from the cell lysate samples.
The cell supernatant was then passed through a silica membrane column, to which the plasmid DNA bound. The column was washed with PE buffer containing ethanol, to wash away impurities, followed by elution with 30μl of water.
The extracted plasmid DNA was quantified using a nano-drop spectrophotometer (ThermoFischer Scientific) and the purity assessed by running on a 1% agarose gel (Chapter 2.2.3).
2.2.6 Sequencing of construct to confirm presence of mutation
After site directed mutagenesis ADAMTS13 was sequenced to confirm the presence of the nucleotide change introduced. Sequencing was either carried out using the ABI Prism 310 genetic analyser (Life technologies) as described (Chapter 2.2.1) or using a Ceq™ 8000 genetic analysis system (Beckman Coulter). Reactions were set up using GenomeLab DTCS Quick start kit (Beckman Coulter).
65 The cycle sequencing PCR was set up as follows:
96⁰C 30 seconds
}
x 30 cycles 50⁰C 30 seconds60⁰C 4 minutes
PCR products were purified as recommended by the manufacturer. The primers used to sequence ADAMTS13 cDNA to confirm the presence of these nucleotide variations are shown in the appendix (Table S 2).
2.2.7 Large scale plasmid preparation
Once it was confirmed that the desired nucleotide change had been introduced into the plasmid without disrupting the surrounding ADAMTS13 sequence, maxi- preparation was carried out in order to produce large quantities of plasmid DNA for subsequent experiments. As with mini preparation (Chapter 2.2.5) this was performed using a commercial kit (Qiagen).
Briefly E. coli were resuspended in buffer P1, followed by addition of buffer P2 to enable cell lysis. This was neutralised by the addition of buffer P3. The lysates were cleared by centrifugation and then loaded onto an anion-exchange tip where plasmid DNA selectively bound. RNA, proteins and other impurities were removed by a medium salt wash and pure plasmid DNA was eluted using a high salt solution. The DNA was concentrated and desalted by isopropanol precipitation and collected by centrifugation.
The quantity of plasmid DNA extracted was measured using a nano-drop (ThermoFischer Scientific) and the quality was assessed on a 1% agarose gel (Chapter 2.2.3).
66