Protocol for HIV-1 near full-length genome amplification and
sequencing from plasma RNA
1. RNA extraction
2. Amplification in four overlapping fragments by RT-PCR+nested PCR
3. Checking PCR amplification
4. Purification of PCR products
5. Sequencing
1. RNA Extraction
RNA is extracted from 1 ml plasma with NucliSENS® kit (bioMérieux, France),
following the manufacturer’s instructions.
2. Amplification in four overlapping fragments by RT-PCR and nested PCR
Amplification of the HIV-1 genome is done in four overlapping fragments (A, B,
C, and D, with sizes of 2 kb, 3 kb, 2.9 kb, and 1.8 kb, respectively) by RT-PCR
followed by nested PCR using the following reaction mixtures
RT-PCR
H2O (DNAse, RNAse free)
Buffer BioTaq DNA
Polymerase, 10X
MgCl2 (50 mM)
dNTP mix (20 mM) (GE
Healthcare)
Primer sense (20 µM)
Primer antisense (20µM)
rRNasin RNase inhibitor
(40 U/µl) (Promega)
SuperScript III RT
(200 U/µl) (Invitrogen)
BioTaq DNA polymerase
(5 U/µl) (Bioline)
Pfu DNA polymerase
(3 U/µl) (Stratagene)
Premix Vol.
RNA sample
Final Vol.
Nested PCR
X1
14.775 µl
2.5 µl
1 µl
0.25 µl
0.5 µl
0.5 µl
0.125 µl
X ___
H2O (DNAse, RNAse free)
Buffer BioTaq DNA
Polymerase, 10X
MgCl2 (50 mM)
dNTP mix (20 mM) (GE
Healthcare)
Primer sense (20 µM)
Primer antisense (20 µM)
X1
37.9 µl
5 µl
2 µl
0.5 µl
1 µl
1 µl
0.05 µl
0.25 µl
0.5 µl
20 µl
BioTaq DNA polymerase
(5U/µl) (Bioline)
Pfu DNA polymerase
(3U/µl) (Stratagene)
Premix Vol.
5 µl
25 µl
RT-PCR product
Final Vol.
2 µl
50 µl
0.05 µl
0.1 µl
48 µl
X ___
PCR primers
RT-PCR
Fragment
A
B
C
D
Sense
Antisense
Nested PCR
Sense
Antisense
5’R1
PROS2b
3’HIV-OS3
gp41-nefOS-2
PROA
Pol OA1
gp120-OA
Nef-AS-4
5’RU5S
PRTS
3’HIV-OS
gp120S
PRTA
5’HIV-NA
gp41-AS
3’Nef-3
Primers’ sequences and their positions in the HXB2 reference isolate are shown
in table 1.
Thermocycling profiles
RT-PCR:
RT: 55ºC, 30min
Denaturation: 94ºC, 3min
35 PCR cycles:
- Denaturation: 94ºC, 30sec
- Annealing: 55ºC, 30sec
- Elongation: 72ºC, 3 min
Final elongation: 72ºC, 7 min
Hold 4ºC
Nested-PCR:
Denaturation: 94ºC, 3min
35 PCR cycles:
- Denaturation: 94ºC, 30sec
- Annealing: 55ºC, 30 sec
- Elongation: 72ºC, 3 min
Final elongation: 72ºC, 7 min
Hold 4ºC
Although the lengths of amplified fragment are different, the four PCRs can be
performed together with the same thermocycling profile, using 3 min in the
elongation step. If done separately, 2 min elongation can be used for fragments
A and D, and 3 min elongation for fragments B and C.
3. Checking amplification and fragment length
Check the amplification and the length of the amplified fragments by
electrophoresis in a 1% agarose gel.
4. Enzymatic purification of PCR products
Reaction mix:
1 µl PCR product
1 µl shrimp alkaline phosphatase (SAP) (1 U/µl) (GE Healthcare)
0.1 µl exonuclease I (10 U/µl) (GE Healthcare)
4.9 SAP buffer 5x (dilute 1:2 10x buffer)
Incubate at 37ºC, 15 minutes, followed by enzyme inactivation at 80ºC, 15
minutes.
5. Sequencing
Sequencing reaction mix:
5.9 µl H2O (DNAse free)
1.6 µl primer (2 µM)
1.0 µl BigDye terminator cycle sequencing kit v3.1 (Applied Biosystems)
1.5 µl BigDye buffer
1 µl PCR product (previously purified)
Thermocycling profile: 94ºC, 2 min; 25 cycles: 94ºC, 30 sec; 50ºC, 15 sec;
60ºC, 4 min.
Sequencing primers, with their sequences and positions in the HXB2 reference
strain, are listed in table 1. The table includes alternative sequencing primers in
case that good quality electropherograms are not obtained with first-choice
primers.
In case that the sequence is illegible in some fragment, due to length
polymorphisms, end-point dilution RT-PCR of the corresponding fragment can
be done (RT-PCR/nested PCR is done with serial 1:2 RNA dilutions, and
sequencing is done with the PCR product obtained with the highest dilution for
which amplification is positive).
There are different programs to assemble and edit electropherograms. The
program we use is Lasergene’s Seqman (DNASTAR).
References
1. Thomson MM, Delgado E, Herrero I, et al. (2002). Diversity of mosaic
structures and common ancestry of human immunodeficiency virus type 1 BF
intersubtype recombinant viruses from Argentina revealed by analysis of near
full-length genome sequences. J Gen Virol; 83:107-119.
2: Delgado E, Thomson MM, Villahermosa ML, et al. (2002). Identification of a
newly characterized HIV-1 BG intersubtype circulating recombinant form in
Galicia, Spain, which exhibits a pseudotype-like virion structure. J Acquir
Immune Defic Syndr; 29:536-543.
3. Sierra M, Thomson MM, Ríos M, et al. (2005). The analysis of near full-length
genome sequences of human immunodeficiency virus type 1 BF intersubtype
recombinant viruses from Chile, Venezuela and Spain reveals their relationship
to diverse lineages of recombinant viruses related to CRF12_BF. Infect Genet
Evol; 5:209-217.