Amplification of HBV Pol gene (Kit Biotaq DNA polimerase, 5 U/µl; Bioline/Meridian BioScience)
a) 1st round PCR
1. Put the reagents of the amplification kit at room temperature, shake them slightly, spin them and place them on ice.; Note: the enzyme should only be removed from the freezer at the moment it is going to be used.
2. Mark 0.2 ml microtubes for each of the samples, for the positive control, for the negative control of extraction and for the negative control of PCR.
3. Prepare the mixture for the PCR reaction in a suitable microtube.
PCR mixture (for 1 sample)
Water nuclease-free - 14.45 µl
NH4 buffer (10x) - 2.5 µl
dNTPs mix (100mM) - 0.2 µl
FW_O (10mM) - 1 µl
RV_O (10mM) - 1 µl
MgCl2 (50mM) - 0.75 µl
BioTaq enzyme - 0.1 µl
4. Distribute 20 µl of the mixture into each of the 0.2 ml microtubes (include one extra tube for a negative control of PCR technique).
5. Add 5 µl of each of the extracted samples and controls to the respective tubes. Add 5 µl of “nuclease-free” water into the PCR negative control tube.
6. Place the tubes in a thermal cycler under the following conditions: 94ºC - 4 min; [40 cycles at 94ºC - 45 secs, 55ºC - 30 secs, 72ºC - 1 min (+5 secs/cycle)]; 72ºC - 15 min and 4ºC - ∞
7. After the end of the PCR reaction, save the amplified products at -20ºC or proceed with the nested PCR
b) 2nd round PCR
1. Proceed exactly as for the first PCR, for reagents and microtubes, also using the same amplification kit.
2. Prepare the mixture for the PCR reaction in a 1.5 ml microtube.
PCR mixture (for 1 sample)
Water nuclease-free - 18.45 µl
NH4 buffer (10x) - 2.5 µl
dNTPs mix (100mM) - 0.2 µl
FW_I (10mM) - 1 µl
RV_I (10mM) - 1 µl
MgCl2 (50mM) - 0.75 µl
BioTaq enzyme - 0.1 µl
3. Distribute 24 µl of the mixture into each of the 0.2 ml microtubes.
4. Add 1µl of each of the 1st round PCR products to the respective tubes.
Note: The product obtained for the positive control in the first PCR must be diluted 1:5 (4µl H2O + 1µl product).
5. Put the 0.2ml microtubes in a thermocycler under the same cycling conditions used for the 1st round PCR.
Agarose gel electrophoresis
1. Prepare a 1.5% agarose gel.
2. Fill an electrophoresis cuvette with 1x TAE buffer.
3. Prepare the samples to be applied to the gel, adding 10µl of each to 2µl of Orange G dye.
4. To 0.5µl of the molecular weight marker (GeneRuler 100 bp DNA Ladder, 0,5 mg/ml; ThermoFisher Scientific), 2µl of the dye and 7.5µl of water are added to make a volume of 10µl.
5. Apply the stained samples into the wells of the agarose gel and apply electric current (70 Volts) for approximately 30 minutes.
6. Observe the gel in a transilluminator where a 943 bp band should be visualized for samples and positive control.
PCR products purification using a spin column system (JetQuick PCR Product Purification Spin Kit; Genomed)
1. Place a 1.5 ml microtube with nuclease free water to heat at 65ºC.
2. Add 60µl of H1 kit solution to remaining 15µl of 2nd round PCR product.
3. Mix and transfer the mixture to a purification column previously inserted in a supernatant collection tube.
4. Centrifuge at 15000g for 1 minute.
5. Discard the contents of the collection tube.
6. Add 500µl of H2 solution to the column.
7 Repeat the above centrifugation and discard steps.
8. Centrifuge at 16000g for 1 minute to completely dry the column membrane.
9. Insert the column into a 1.5ml microtube.
10. Add 20-35µl of nuclease free water at 65ºC to each column.
Note: Add a higher volume, up to 50µl, if the band obtained after electrophoresis is too strong.
11. Wait 1 minute.
12. Centrifuge at 16000g for 2 minutes.
13. Store purified products at -20°C or proceed with the sequencing reaction.
Sequencing Reaction (BigDye terminator v.3.1 Cycle Sequencing Kit; Applied Biosystems)
1. Thaw the reagents needed for the sequencing reactions and the purified PCR products:
BigDye terminator v.3.1 (Applied Biosystems)
BigDye terminator 5X sequencing buffer (Applied Biosystems)
Primers Forward and Reverse from the nested PCR (5µM)
Nuclease free water
2. Place the thawed reagents on ice and prepare a mixture for each primers as described below:
Sequencing mixture (for 1 sample)
BigDye terminator v.3.1 - 0.5 µl
Primer (5µM) - 1 µl
Buffer (5X) - 1.75 µl
3. Mix the reagents by pipetting up and down several times, making a spin to the microtube.
4. Distribute 3.25 µl of each mix into the two different tubes.
5. Add between 0.3 - 1 µl of the previously purified PCR product to each tube, according to the intensity of the band observed on the agarose gel.
6. Complete the reaction volume to 10 µl with nuclease-free water and place the samples in the thermocycler under the following conditions:
Sequencing cycle
96ºC - 1 min; [25 cycles at 96ºC - 10 secs, 60ºC - 4 min]; 4ºC- 7 minand 4ºC - ∞
Sequencing products purification through Ethanol/Sodium Acetate
1. Prepare a sodium acetate/ethanol solution by mixing 2 µl of 3M sodium acetate (pH 4.6) with 50 µl of 100% ethanol, per reaction.
2. Add 52 µl of the previous solution to each sequencing reaction and transfer to a 96 well plate.
3. Cover the plate with aluminium foil.
4. Vortex the plate to mix.
5. Centrifuge the plate at 2000g for 20 minutes.
6. Once the centrifugation is finished, remove the aluminium foil carefully trying to not disturb the sediment.
7. Immediately place several absorbent paper towels without residues on top of the plate and invert it.
8. Centrifuge the inverted plate at 150g for 1 minute.
9. Add 150 µl of 70% ethanol, prepared previously, and cover the plate with a new aluminium foil.
10. Centrifuge at 2000g for 5 minutes.
11. When the centrifugation is finished, remove again the aluminium foil carefully trying to not disturb the sediment.
12. Invert the plate over clean towels and centrifuge the inverted plate at 150g for 1 minute.
13. After centrifuging, allow the plate to air dry for approximately 1 minute to eliminate any remaining ethanol.
14. Cover the plate with aluminium foil to store between –15ºC and –25ºC in the dark, but no longer than 2 weeks or add 20 µl of Hi-DiTM Formamide (Applied Biosystems, USA) and move to the processing phase of the sequences in the ABI Prism 3100-Avant Genetic Analyzer according with the equipment manual.