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at the University of Sydney Nepean Clinical School.
MATERIALS
Table 2-8 List of materials used for IGH gene clonality assay
METHOD
2.6.2.1. Isolation of DNA from peripheral blood
DNA was isolated from peripheral blood using the Wizard Genomic DNA purification kit (Promega), according to the manufacturer’s protocol. Briefly, blood samples were collected in lithium heparin vacutainer tubes. Whole blood (300 µL) was added to a sterile 1.5 mL micro- centrifuge tube containing Cell Lysis Solution (900 µL) and mixed by inverting the tube 5-6 times. The tube was left at room temperature for 10 min to lyse the red blood cells. The sample was centrifuged at 13,000 g for 30 s at room temperature and the supernatant was carefully removed. The white cell pellet was re-suspended and Nuclei Lysis Solution (300 µL) was added to the tube and pipetted up and down gently 5 times to lyse the white blood cells
MATERIALS COMPANY
1 Wizard Genomic DNA purification kit Promega, Cat No. A1120 2 Isopropanol Sigma-Aldrich, Cat. No. I-9516 3 Pure, ethyl alcohol Sigma-Aldrich, Cat. No. E7023 4 Water, molecular biology Sigma-Aldrich, Cat. No. W4502 5 Thermo Scientific Nanodrop 2000
Spectrophotometer
Thermo Fisher Scientific, Wilmington, U.S.A. 6 IGH Gene Clonality Assay Invivoscribe Technologies Inc., San Diego, USA 7 Thermo-cycler PTC-225 Tetrad DNA Engine PCR System, MJ Research 8 The Mini-Sub cell GT agarose gel
electrophoresis system
Bio-Rad Laboratory Pty., Ltd, NSW, Australia 9 Ultra-pure grade 25X Tris-acetate-EDTA (TAE)
concentrate ready pack
Amresco, Cat No 0912-2PK, Australia 10 Grade Agarose 1 Amresco, Cat No 0710-500G, Australia 11 Safe DNA gel stain in 1 X TAE Invitrogen, Cat No S33111
12 EZ Load 100 bp molecular ruler Bio-Rad, Cat No170-8352 13 Safe imager blue light trans-illuminator Invitrogen, S37102 14 Wizard SV Gel and PCR Clean-Up System Promega, Cat No. A9282
15 25X Tris-acetate-EDTA (TAE) concentrate One foil pouch containing 25X TAE powder was dissolved in 1L Milli-Q water.
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until the solution became very viscous. Protein Precipitation Solution (100 µL) was added to the nuclear lysate and vortexed vigorously for 20 s. The tube was centrifuged at 13,000g for 3 min at RT, and the supernatant containing the DNA was carefully transferred to a new sterile 1.5 mL micro-centrifuge tube containing isopropanol (300 µL). The solution was inverted gently until the white thread-like strands of DNA formed a visible mass, and then centrifuged at 13,000 g for 3 min at RT. After centrifugation, the DNA formed a small white pellet on the side and bottom of the microcentrifuge tube. The supernatant was carefully aspirated and then 70% ethanol (300 µL) at RT was added to the DNA pellet. The tube was inverted several times gently to wash the DNA pellet and the sides of the tube before centrifuging at 13,000 g for 3 min at RT. The supernatant was carefully aspirated leaving a small white pellet of DNA and the tube was inverted onto a clean absorbent paper to air-dry for 60 min. Afterward, DNA Rehydration Solution (100 µL) was added to the DNA pellet and incubated at 65°C for 1 hour or overnight at 4°C. Finally, the rehydrated DNA was quantified using a Thermo Scientific Nanodrop 2000 Spectrophotometer and its software (Thermo Fisher Scientific, Wilmington, U.S.A.).
2.6.2.2. IGH Gene Clonality Assay
Clonal immunoglobulin heavy chain gene (IGH) rearrangements were detected using the IGH Gene Clonality Assay (Invivoscribe Technologies Inc., San Diego, USA). The kit contains six primer master mixes; IGH tube A catalogue no. 2-101-0011, IGH tube B catalogue no. 2- 101-0101, and IGH tube C catalogue no. 2-101-0031, for targeting the framework regions within the variable and the joining regions of IGH, while IGH tube D catalogue no. 2-101-0041 and IGH tube E catalogue no. 2-101-0051, targeted the diversity and joining regions. The last master mix tube is the specimen control size ladder (Catalogue no. 2-096-0021), which targeted multiple genes and generated a serious of amplicons to ensure that the quality and
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quantity of input DNA was sufficient to yield valid results. Positive clonal controls DNA (IVS- 0030, IVS-0019, IVS-0024, and IVS-0008) and negative polyclonal controls DNA (IVS-0000) were also included. Reactions were set up according to the manufacturer's instructions using AmpliTaq Gold DNA polymerase and amplification was performed on a thermo-cycler using the following PCR program,
1 x cycle - Initial incubation at 95°C for 7 min.
35 x cycle - Denaturation at 95°C for 45 s.
- Annealing at 60°C for 45 s.
- Extension at 72°C for 90 s.
1 x cycle - Extension at 72°C for 10 min.
The PCR products were stored at 4°C before analysing them on a 2.5% agarose gel.
2.6.2.3. Agarose Gel Electrophoresis
Agarose gel electrophoresis was performed using the Mini-Sub cell GT agarose gel electrophoresis system, to separate and visualise DNA of various size. For a final concentration of 2.5% agarose gel, agarose (2.5 g) was dissolved in 1X TAE (100 mL) by heating in a microwave oven until the agarose was completely dissolved. SYBR safe DNA gel stain (5 µL) was added to the agarose solution and gently swirled before pouring the gel into a 7 x 10 cm Bio-Rad Mini-Sub cell gel-casting tray. An eight-tooth well-forming comb was inserted, and the gel was allowed to solidify for at least 45 min at RT. The casting tray containing the gel on it was inserted into the electrophoresis box with the sample wells near to the negative black cathode. The box was gradually filled with 1X TAE running buffer until the gel was submerged and the comb was then carefully removed from the solidified gel. EZ Load 100 bp molecular
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ruler (5 µL) was loaded in the first well as a DNA size standard. 6X nucleic acid sample loading buffer (5 µL) was added to 25 µL of amplified DNA sample, and then 20 µL of sample mixture was carefully loaded into agarose well. Electrophoresis was carried out at room temperature at a constant voltage of 110V for 60 min. After electrophoresis, the gel was placed on the Safe Imager blue light trans-illuminator for nucleic acid visualisation and analysis. An image of the gel was collected, and stained agarose gel bands within the valid size range were excised using sterile surgical scalpels and transferred directly into a new sterile 1.5 micro-centrifuge tube for DNA purification.
2.6.2.4. Gel Slice DNA Purification
DNA was purified from gel slices using the Wizard SV Gel and PCR Clean-Up System, according to manufacturer’s instructions. Briefly, membrane binding solution (500 µL) was added to a 1.5 mL micro-centrifuge tube containing the DNA gel slice, vortexed vigorously and incubated at 60°C for 5-10 min until the gel was completely dissolved. The dissolved DNA gel mixture was transferred to a SV mini-column in a collection tube and incubated for 1 min at room temperature. After that, the SV mini-column assembly was centrifuged at high speed (16,000 g) for 1 min, then the flow was discarded and the SV mini-column was washed with membrane wash solution (700 µL) and centrifuged again for 1 min at 16,000 g. Another washing step was performed by adding membrane wash solution (500 µL) followed by centrifugation for 5 min at 16,000 g. After centrifugation, the flow-through was carefully removed, and the SV mini-column in the collection tube was then re-centrifuged for 1 min with the micro-centrifuge lid open to completely evaporate any residual ethanol. The SV mini- column was transferred into a nuclease-free 1.5 mL micro-centrifuge tube and nuclease-free water (30 µL) supplied with the kit was added directly to the centre of the SV mini-column silica membrane without touching the membrane with the pipette tip. The tube was incubated
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at RT for 1 min, followed by centrifuging at 16,000 g for 1 min. The quantity of purified DNA was measured using a Thermo Scientific Nanodrop 2000 Spectrophotometer and its software (Thermo Fisher Scientific, Wilmington, U.S.A.).
2.6.2.5. Sequencing Reaction
Sequencing of the purified fragments was performed at the Australian Genome Research Facility (AGRF) (Westmead Millennium Institute, Westmead, Australia) by high throughput Sanger sequencing using applied bio-systems 3730 and 3730xl capillary sequencer with big dye terminator (BDT) chemistry version 3.1 and standard sequencing protocols
(https://www.agrf.org.au/docs/sanger-sequencing-sample-preparation-guide.pdf ).
Nucleotide sequences were compared with the international ImMunoGeneTics
information system (IMGT) (http://www.imgt.org/) and IgBLAST
(https://www.ncbi.nlm.nih.gov/igblast/) databases and gene-usage and mutational status determined based on the recommended threshold of 2% to distinguish mutated from non- mutated B-CLL cases (Tobin et al., 2005) (Ghia et al., 2007).