clotet.qxp 28/1/09 4:45 pm Page 60
HIV and AIDS
Figure 3: Schematic Procedure for 454 Sequencing in HIV
reactions are run in separate wells, always in duplicate and simultaneously
with standards. The copy number of mutant and all (or wild-type) variants is
Adapter A
Target HIV sequence
calculated, and the proportion of mutants is determined with the formula:
Barcode Barcode Barcode
Adapter B Adapter A Adapter B
% of mutants = (copy number of mutants/total copy number) x 100
If a wild-type-specific primer is used instead of a non-specific primer, the
calculation would be:
% of mutants = (copy number of mutants/copy number of WT) x 100
Anneal single-stranded template to an
Emulsify beads and reagents in
excess of DNA capture beads
water-in-oil microreactors. Clonal
Break microreactors
amplification occurs inside In blinded comparisons, ASPCR was the most sensitive method for
Enrich for DNA+
beads Load enzyme
detecting minority variants.
51
It is also the cheapest, fastest and least
Load beads into beads
labour-consuming method. The main limitation is that only one allele can
PicoTiterPlate™
be interrogated per experiment, thereby missing the impact of other
Centrifugation
mutations that could coexist with the target mutation; it is unsuitable for
mutation linkage analysis. Also, polymorphisms at primer sites, particularly
those near the 3’ end, can affect the relative amplification efficiencies of
the two reactions, leading to an underestimation of mutant proportions.
52
This problem is shared by other PCR-based techniques, such as PASS, but
is theoretically cirvumvented by 454 sequencing and LigAmp. Finally, due
to the typical loss of linearity in the measurement of nominal proportions
below 0.1–1%, it may be more difficult to establish clinically relevant
thresholds for minority variants with ASPCR than with other techniques. As
we shall show below, ASPCR remains an important and affordable research
tool to investigate the clinical role of minority variants.
Parallel Allele-specific Sequencing
40
The target sequence is amplified with primers tagged with a sample-specific identification
This assay simultaneously analyses a large number of viral genomes by
four base pair bar code plus an adapter A and B, respectively, that will anneal to 28µM
applying the polony technique.
53,54
A pol gene fragment containing sites
beads. A water/oil emulsion is prepared so each droplet (microreactor) contains one bead
and one amplicon; each amplicon is then clonally amplified by emulsion PCR within the of all major resistance mutations in reverse transcriptase and protease is
droplet. The emulsion is disrupted and 28µM beads are loaded into 44µM wells in
amplified using acrydited primers. Because one acrydited primer becomes
PicoTiterPlates, so, theoretically, there is only one bead per well. Sequencing enzymes
including luciferase and sulfurilase are loaded into each well. Pyrosequencing proceeds by a immobilised by covalently incorporating into polyacrylamide gels during
series of flows of known nucleotides. When a nucleotide hybridises with its complementary,
a pyrofosphate is released; this results in the release of a chemiluminescent signal recorded
the polymerisaton, the PCR products accumulate around individual DNA
by a CCD camera and integrated by a computer. Vertical bars in the sequencing flowchart
templates and form distinct spots (polonies) at the amplification sites.
represent number of bases.
Source: 454 Sequencing website:
www.454.com
After amplification, the solid-phase negative DNA strands hybridise to
complementary sequencing primers whose 3’-end is juxtaposed to the
per sample, although the number of genomes obtained can be increased site where a single-base mutation confers resistance. After single-base
simply by analysing more genomes. extension of this primer in the presence of nucleotides labelled with
different fluorophores, imaging with a microarray scanner can be used to
SGS detects minority variants that are present in at least 2% of the viral distinguish wild-type and mutant populations.
population. Although it is time and labour-consuming, SGS is perfectly
suited for assessing the linkage of several mutations in individual genomes, This technology is more efficient, faster and more sensitive for detection of
which can be of major importance in heavily pre-treated patients and in minor resistance populations than clonal sequencing. The PASS assay
NNRTI-experienced candidates about to receive etravirine. permits a detailed linkage analysis of multiple mutations, allowing study of
the impact of different combinations of mutations existing as minor and
Allele-specific Polymerase Chain Reaction
37–39
major viral populations. In addition, because viral cDNA molecules are
Based on a modification of the amplification refractory mutation system directly embedded into polyacrylamide gel and PCR amplification is carried
(ARMS),
50
ASPCR performs separate realtime PCR amplifications of viruses out at a single-molecule level, artifact sequences that are generated through
containing, respectively, mutant and wild-type alleles of a codon (see Figure recombination or re-sampling during conventional PCR are eliminated.
2). Alternatively, one can amplify the mutant allele and, separately, all However, this technology, is not more sensitive than ASPCR; it is also
variants using a non-specific primer that is identical to the mutant-specific affected by polymorphisms in primer sites, requires a few days to analyse all
oligonucleotide, but has a 3’-end finishing just one base pair before the primary mutations and is more expensive and labour-consuming and
target base. This second method is preferred by the authors because it enables lower throughput than ASPCR.
confers several technical advantages and improves consistency . To increase
the specificity for the mutant allele, the mutant-specific primer usually LigAmp
41,42
contains one intentional base mismatch at the -1 to -1 position of the 3’- The ‘LigAmp’ strategy involves a ligation step followed by an
end. Both the mutant-specific and the non-specific (or wild-specific) PCR amplification-detection step. LigAmp can be used to detect and
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