The following protocols describe PCR amplification and polyacrylamide gel electrophoresis conditions for subtype determination of HIV-1 envelope (env) sequences using the Heteroduplex mobility assay (HMA).  PCR primers and reference plasmids containing env genes from different subtypes are provided separately as part of the HMA kit. 

Policy for subtype Determination:

Subtype determination by HMA is based on evaluating the mobility of Heteroduplex formed with DNA fragments from the test samples and the subtype references provided in the kit.  Nested PCR is used to generate either 1.2 Kb, 0.7 or 0.5 Kb env gene fragments from uncharacterized strains of HIV-1. Depending on which fragment you plan to evaluate, subtype reference sequences can be amplified directly from the plasmids provided with the kit without the need for nested PCR amplification.  PCR reactions are then checked for appropriate product length and yields by agarose gel electrophoresis.  Heteroduplexes are then formed by mixing, denaturing and annealing amplified DNA fragment from the unknown with amplified DNA from the reference strains and their mobilities analyze on polyacrylamide gels.  Heteroduplexes formed between the unknown sample and the most closely related sequences are expected to exhibit the fastest mobilities on these gels.  If the closest relatives are all from a single genetic subtype, the likely subtype of the strain is thus determined.  The goal should always be to positively identify the subtype of the unknown.

Extraction of DNA from Peripheral Blood Mononuclear Cells 

DNA is extracted from PBMCs or Whole blood.  DNA can also be extracted from filter paper, paraffin embedded tissue, blood clot, swabs or dried blood spots. Before starting with DNA extraction using QIAGEN kit (QIAamp DNA Blood Mini Kit), following points should be considered.

Notes :

• Equilibrate samples to room temperature
• Heat a water bath to 56^oC
• Equilibrate Buffer AE or water to room temperature for elution in step 10
• Ensure that Buffer AW1, Buffer AW2, and QIAGEN Protease have been prepared according to the instruction manual provided along with kit.
• All centrifugation steps should be carried out at room temperature.

1. Pipette 20 µl QIAGEN Protease into the bottom of a 1.5 ml sterile microcentrifuge tube.
2. Use up to 200 µl whole blood, plasma, serum, buffy coat, or body fluids or up to 5 x 10⁶lymphocytes in 200 µl PBS.
3. Add 200 µl Buffer AL to the sample. Mix by pulse-vortexing for 15 sec.
4. Incubate at 56oC for 10 minutes.
5. Briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid.
6. Add 200-µl ethanol (96-100%) to the sample, and mix again by pulse-vortexing for 15 sec. After mixing, briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid.
7. Carefully apply the mixture from step 6 to the QIAamp spin column (in a 2 ml collection tube) without wetting the rim, close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp spin column in a clean 2 ml collection tube and discard the tube containing the filtrate.
8. Carefully open the QIAamp spin column and add 500 µl Buffer AW1 without wetting the rim. Close the cap and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp spin column in a clean 2 ml collection tube and discard the collection tube containing the filtrate.
9. Carefully open the QIAamp spin column and add 500 µl Buffer AW2 without wetting the rim. Close the cap and centrifuge at full speed (20,000 x g;14,000 rpm) for 3 min.
10. Place the QIAamp spin column in a clean 1.5 ml microcentrifuge tube and discard the collection tube containing the filtrate. Carefully open the QIAamp spin column and add 200 µl Buffer AE or distilled water. Incubate at room temperature for 1 min, and then centrifuge at 6000 x g (8000 rpm) for 1 min.
11. Take OD at 260 nm and 280 nm to check DNA content in 200 µl suspension.

PCR amplification using following any of the two sets of primers:

The primers included in this kit have been chosen because of their success in amplifying HIV-1 env sequences from each of the known subtypes (A-J have been tested) within the main (M) group.  They are not expected to function, however, with sequences of the Outlier (O) and the Non M Non O (N) groups. 

Nested PCR reactions are required for the preparation of sufficient quantities of HIV env DNA fragments from infected human cells to perform 10-20 HMA analyses.  Reference fragments are amplified from 10 ng of plasmid DNA using the second round primers only. Positive (10 copies of Pnl 4-3) and multiple negative control PCR reactions (using reaction mix alone, without template DNA) should be carried through both rounds of amplification.

For single 50 µl reaction only mix :
5 µl 10X PCR Reaction Buffer
6.25 µl 10 mM MgCl2
28.25 µl H2O
5 µl 10X dNTP Mixture
2 µl ED5 (5 pmoles/µl)
2 µl ED12 (5 pmoles/µl)
0.5 µl Taq polymerase (5 U/µl)
Add 0.1 – 2.0 µg infected cell DNA

Amplification conditions:
3 cycles of 94oC for 1 minute, 55oC for 1 minute and 72oC for 1 minute
32 cycles of 94oC for 15 seconds, 55oC for 45 seconds, 72oC for 1 minutes Final extension at 72oC for 5 minutes

Second Round PCR:
For single 50 µl reaction only mix :
5 µl 10X PCR Reaction Buffer
6.25 µl 10 mM MgCl2
28.25 µl H2O
5 µl 10X dNTP Mixture
2 µl ES7 (5 pmoles/µl)
2 µl ES8 (5 pmoles/µl)
0.5 µl Taq polymerase (5 U/µl)
Add 2 l of first round reaction product

Amplification conditions:
3 cycles of 94^oC for 1 minute, 55^oC for 1 minute and 72^oC for 1 minute
32 cycles of 94^oC for 15 seconds, 55^oC for 45 seconds, 72^oC for 1 minutes
Final extension at 72^oC for 5 minutes.
When completed the samples are stored at -20^oC.
After completion of nested PCR, Check Second Round reactions for amplification product by loading 5 µl mixed with 1 µl 5X Ficoll/Loading Dye  on a 1% Agarose Gel  in 1X TBE Gel Electrophoresis Buffer or 1X TAE Gel Electrophoresis Buffer. Electrophoreses at 100 V for 1 hour. Stain the gel with ethidium bromide for 30–60 minutes and detect the DNA by UV transillumination. Agarose gel electrophoresis is conducted to ensure that the correct size DNA fragment was amplified in sufficient amounts (i.e. a bright band should be seen). Detection of multiple bands, especially ones with less than the expected size in agarose gels, is indicative of either the presence of a substantial population of molecules with large internal deletions or products derived from internal priming with Second Round primers. The presence of these molecules occasionally interferes with distance measurements due to the formation of heteroduplexes between DNA strands of substantially different size. Even though such molecules have greatly reduced mobilities, they have not, to date, prevented subtype determination when present as a minority component of the Second Round PCR. However, knowledge of the presence of such deletions is helpful in preventing erroneous conclusions. The majority of the amplified DNA seen on agarose gels may in some cases consist of the smaller fragments, since because of their size, they are preferentially amplified. To remove the smaller products, nested PCR is repeated using serially decreasing amounts of input genomic DNA until only the correct sized DNA fragment is amplified. This dilution procedure can be considered successful if, as in all of the cases we have encountered to date, the full-length fragments represent the majority of the provirus population.

Heteroduplex Mobility Assay in Polyacrylamide Gels :

1. Assess the genetic diversity of each PCR-amplified sample. Mix in a 500 µl Eppendorf tube
1. µl 10X Heteroduplex Annealing Buffer 5 µl H2O
   
           5 µl Second Round PCR reaction (~100–250 ng of DNA)


2. Heat to 94-96oC for 2 minutes in thermocycler (or in boiling water bath)
3. Cool tubes rapidly by transferring to wet ice.  Heteroduplexes can be kept at room temperature before loading or stored at 4oC for several minutes before loading the gel.  This analysis provides a baseline Heteroduplex pattern (resulting from quasispecies diversity in vivo) with which to compare deliberately formed Heteroduplexes experiments, it is therefore important to consistently reproduce the electrophoresis conditions as closely as possible. It is CRITICAL and MOST useful if this sample is run on the same gel as the reactions between references and the unknown, so that bands present due to within quasispecies heteroduplexes can be easily identified.  For this purpose the gel units, plates, acrylamide concentration, buffer, and standard voltage/current conditions should be carefully adhered to in each experiment. Given a particular apparatus and conditions, an equation can be derived to fairly reliably estimate the genetic distance between two DNA fragments from their heteroduplex mobility. Estimates of genetic distances can then be used to infer phylogenetic relationships between multiple sequences without analyzing all of the pairwise heteroduplexes possible. To date we have typically evaluated 25–33% of the [N x (N–1)]/2 possible comparisons (where N = number of sequences being compared). If the electrophoresis apparatus or conditions are changed, the previously determined standard curve is no longer valid and therefore must be reestablished using newly determined heteroduplex mobilities using DNA

General Considerations
The mobility of heteroduplexes is noticeably affected by changes in temperature during electrophoresis, particularly when the reannealed DNA strands are from highly divergent viral strains. The higher the temperature in the gel (i.e. the faster the gel is run) the slower the mobility of the heteroduplexes. Temperature increases result in local increases in duplex melting, thus slowing heteroduplex mobility. In order to compare data acquired across