TITLE: Amplification and sequencing of Hepatitis B virus pol gene
AUTHORS: rute.marcelino

[DESCRIPTION]
The Amplification and sequencing of HBV pol gene protocol aim to present all the details from HBV DNA extraction with QIAamp DNA Blood Mini Kit (Qiagen, Werfen) and with a homemade assay for amplification/sequencing of the RT region of the HBV pol gene. This protocol is related to an ongoing publication from PLOS ONE.

[STEPS]
SECTION: HBV DNA extraction and reagents
1. Reagent preparation
Protease: Add 5.5 ml of protease solvent included in the kit (nuclease-free water containing 0.04% sodium azide) to the tube containing the lyophilized protease. Reconstituted protease is stable for up to two months when stored at 2-8°C. Storage at -20ºC is recommended to prolong the life of the protease. To avoid successive freezing and thawing, store protease in aliquots.
Buffer AW1: add 125 ml of 96-100% ethanol to 95 ml of concentrated Buffer AW1 and mix by inversion 5 to10 times.
Buffer AW2: pipette 160 ml of 96-100% ethanol into 66 ml of buffer AW2 and mix by inversion 5 to10 times.

2. HBV DNA extraction
Heat a thermal block to 56ºC.
Shake AL buffer before using it and if there is any precipitate, dissolve it in a bath at 56ºC.
Prepare 350 μl of negative extraction control from a pool of negative plasmas for this virus.
 Pipette 350 μl of HBV plasma sample and positive and negative controls into 1.5ml microtubes.
Add 35 μl of the reconstituted protease into the microtubes. 
 Homogenize the AL buffer and add 350 μl to the same microtubes.
Vortex intermittently for 15 seconds; to promote efficient lysis it is essential that the sample and the AL buffer are mixed until a homogeneous solution is obtained.
Add 3.5 μl carrier DNA to the cap of each tube, close the tubes and mix by inversion.
Note: carrier DNA enables viral DNA binding to the column membrane on the next step. It is not provided in the kit. Here it was used Polyadenylic Acid, PolyA, 10 mg/ml; Amersham; It should be stored at -20°C.
Vortex intermittently for 15 seconds and spin the microtubes in the centrifuge for approximately 4 seconds.
 Incubate the tubes in the thermal block at 56ºC for 10 minutes; the amount of DNA released is maximum after lysis during this incubation step. Longer incubation periods have no effect on the extraction or quality of purified DNA.
Spin the microtubes in the centrifuge for approximately 4 seconds
 Add 402.5μl 96-100% ethanol, vortex intermittently for 15 seconds, and then spin in the centrifuge.
Apply the mixture from the previous step (630μl at a time), carefully, in a QIAamp Mini column incorporated into a 2ml collection tube, without letting the mixture reach the edge of the column, close the lid, and centrifuge at 6000g for 1 minute.
Place the column in a 2ml new collection tube and discard the tube containing the filtrate.
Repeat the two previous steps.
Add 500μl of buffer AW1, close the lid, and centrifuge at 6000g for 1 minute.
 Place the column in a new 2ml collection tube and discard the tube containing the filtrate.
Add 500μl of buffer AW2, close the column cover, and centrifuge at 20000g for 3 minutes.
Place the column in a 2ml microtube (not supplied in the kit) and discard the tube containing the filtrate.
Centrifuge at 20000g for 1 minute to completely dry the membrane.
Discard the 2 ml tube containing the filtrate and introduce the column into a previously identified 2 ml screw tube for elution of virus DNA.
Apply 60μl of buffer AE in the center of the membrane, close the column lid, and incubate at room temperature for 5 minutes.
Centrifuge at 6000g for 1 minute.
Samples can be stored for 24 hours at 2-8°C, at -20°C for longer periods, or can be used immediately.
SECTION: Homemade amplification and sequence assay
3. Amplification of HBV Pol gene (Biotaq DNA polimerase, 5 U/μl; Bioline/Meridian BioScience)

a) 1st round PCR
 Put the reagents from the amplification kit at room temperature, shake them slightly, spin them and place them on ice.
     Note 1: the enzyme should only be removed from the freezer at the moment it is going to be used.
     Note 2: Primer sequences are described in the related article of PLOS ONE.

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.
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

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).
Add 5 μl of each of th e extracted samples and controls to the respective tubes. Add 5 μl of “nuclease-free” water into the PCR negative control.
Place the tubes in a thermal cycler under the following conditions:  

                    94ºC            4 min
                    94ºC            45 secs
                    55ºC            30 secs                         40 cycles
                    72ºC            1 min (+5 secs/cycle)
                    72ºC            15 min
                     4ºC               ∞

After the end of PCR reaction, save the amplified products at -20ºC or proceed with the nested PCR.

b) 2nd round PCR
Proceed exactly as for the first PCR, for reagents and microtubes, also using the same amplification kit.
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 μ
                    RV_I (10mM)                    - 1 μl
                    MgCl2 (50mM)                - 0.75 μl
                    BioTaq enzyme                - 0.1 μl

 Distribute 24 μl of the mixture into each of the 0.2 ml microtubes.
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 at 1:5 (4μl H2O + 1μl product).
Put the 0.2ml microtubes in a thermocycler under the same cycling conditions used for the 1st round PCR.

Agarose gel electrophoresis

Prepare a 1.5% agarose gel.
Fill an electrophoresis cuvette with 1x TAE buffer.
 Prepare the samples to be applied to the gel, adding 10μl of each to 2μl of Orange G dye.
To 0.5μl of the molecular weight marker (GeneRuler 100 bp DNA Ladder, 0,5 g/l;
ThermoFisher Scientific), 2μl of the dye and 7.5μl of water are added to make a volume of 10μl.
Apply the stained samples into the wells of the agarose gel and apply electric current (70 Volts) for approximately 30 minutes.
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)

Place a 1.5 ml microtube with nuclease-free water to heat at 65ºC.
Add 60μl of H1 kit solution to the remaining 15μl of 2nd round PCR product.
Mix and transfer the mixture to a purification column previously inserted in a supernatant collection tube.
Centrifuge at 15000g for 1 minute.
Discard the contents of the collection tube.
Add 500μl of H2 solution to the column.
Repeat the above centrifugation and discard steps.
 Centrifuge at 16000g for 1 minute to completely dry the column membrane.
Insert the column into a 1.5ml microtube.
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. Wait 1 minute.
 Centrifuge at 16000g for 2 minutes.
Store purified products at -20°C or proceed with the sequencing reaction.

 Sequencing Reaction (BigDye terminator v.3.1 Cycle Sequencing Kit; Applied Biosystems)

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
Place the thawed reagents on ice and prepare a mixture for each primer as described below:

               Sequencing mixture (for 1 sample)
               BigDye terminator v.3.1 - 0.5 μl
                     Primer (5μM) - 1 μl
                     BigDye terminator v.3.1 Buffer (5X) - 1.75 μl

Mix the reagents by pipetting up and down several times and make a spin to the microtube.
Distribute 3.25 μl of each mix into the two different tubes.
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.
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
                     96ºC         10 secs
                     60ºC         4 min         25 cycles
                     4ºC            7 min
                     4ºC            ∞
 
Sequencing products purification through Ethanol/Sodium Acetate

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.
Add 52 μl of the previous solution to each sequencing reaction and transfer to a 96 well plate.
Cover the plate with aluminum foil.
Vortex the plate to mix.
Centrifuge the plate at 2000g for 20 minutes.
Once the centrifugation is finished, remove the aluminum foil carefully trying to not disturb the sediment.
Immediately place several absorbent paper towels without residues on top of the plateand invert it.
Centrifuge the inverted plate at 150g for 1 minute.
Add 150 μl of 70% ethanol, prepared previously, and cover the plate with a new aluminum foil.
Centrifuge at 2000g for 5 minutes.
When the centrifugation is finished, remove again the aluminium foil carefully trying to not disturb the sediment.
Invert the plate over clean towels and centrifuge the inverted plate at 150g for 1 minute.
After centrifuging, allow the plate to air dry for approximately 1 minute to eliminate any
 remaining ethanol.
Cover the plate with aluminum 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.