I"IIJ Applications in Oncology
tion capillary in approximately an hour. A single primer set amplifies all three
templates to assure similar amplification efficiencies. Detection takes place by
analysis of product melting profiles [18,19]. A single fluorescein labeled probe is
used to excite two sensor probes, one labeled with LC-Red 640 detects HPV 18 and
HPV 45 and a second LC-Red705labeled probe detects HPV 16. In the case ofHPV
18 and HPV 45 that are detected by a single probe, the products can be identified
by unique melting profiles. The amplification of the targets cannot be visualized
during the run program because the probes have a lower melting temperature than
the primers.
Materials
Equipment LightCycler instrument (Roche Diagnostics, Mannheim, Germany)
Software version 3
Reagents Human Papillomavirus Detection kit #4 (IT BioChem, Salt Lake City, USA)
Insta-Mini-Prep Kit (Eppendorf- 5 Prime, Boulder, USA)
High Pure Plasmid Isolation Kit (Roche Diagnostics, Mannheim)
KlenTaq (AB Peptides, St. Louis, USA)
TaqStart Antibody (Clonetech Inc., Palo Alto, USA)
lOX Reaction buffer (Idaho Technology Inc., Salt Lake City, USA)
Enzyme Diluent (Idaho Technology Inc., Salt Lake City, USA)
Proteinase K
3M Sodium Acetate pH 5.3
Ethanol (75% and 100%)
10mM Tris pH 8.0
O.lmMEDTA
Procedure
HPV 16 and HPV 18 plasmids were obtained from the American Type Culture
Collection (ATCC, Manassas, VA). We could not obtain an HPV 45 plasmid due to
its low occurrence in nature. We therefore constructed an artificial HPV 45 template by taking advantage of the sequence similarity between HPV 18 and HPV
45 [12]. The first step consisted of making an HPV 45/HPV 18 hybrid sequence by
using primers that were specific for HPV 45, the template being HPV 18. The
primers used were 45art5' and HPV1300r. The second step attached an EcoRI
linker at both ends of the product. The two restriction sites were added for future
cloning purposes and were found on the 45art5'2 and 45art3' primers used during this step. These steps altogether produced a template with primer and probe
annealing sites specific for HPV 45 (see figure 1). Six additional HPV plasm ids
were obtained from ATTC. One plasmid of no risk (HPV2), three plasmids oflow
risk (HPV 6B, HPV 11 and HPV 44) and two plasmids of medium risk (HPV 31
and HPV 56) were used as controls in this study [10]. Plasmids were extracted
tion capillary in approximately an hour. A single primer set amplifies all three
templates to assure similar amplification efficiencies. Detection takes place by
analysis of product melting profiles [18,19]. A single fluorescein labeled probe is
used to excite two sensor probes, one labeled with LC-Red 640 detects HPV 18 and
HPV 45 and a second LC-Red705labeled probe detects HPV 16. In the case ofHPV
18 and HPV 45 that are detected by a single probe, the products can be identified
by unique melting profiles. The amplification of the targets cannot be visualized
during the run program because the probes have a lower melting temperature than
the primers.
Materials
Equipment LightCycler instrument (Roche Diagnostics, Mannheim, Germany)
Software version 3
Reagents Human Papillomavirus Detection kit #4 (IT BioChem, Salt Lake City, USA)
Insta-Mini-Prep Kit (Eppendorf- 5 Prime, Boulder, USA)
High Pure Plasmid Isolation Kit (Roche Diagnostics, Mannheim)
KlenTaq (AB Peptides, St. Louis, USA)
TaqStart Antibody (Clonetech Inc., Palo Alto, USA)
lOX Reaction buffer (Idaho Technology Inc., Salt Lake City, USA)
Enzyme Diluent (Idaho Technology Inc., Salt Lake City, USA)
Proteinase K
3M Sodium Acetate pH 5.3
Ethanol (75% and 100%)
10mM Tris pH 8.0
O.lmMEDTA
Procedure
HPV 16 and HPV 18 plasmids were obtained from the American Type Culture
Collection (ATCC, Manassas, VA). We could not obtain an HPV 45 plasmid due to
its low occurrence in nature. We therefore constructed an artificial HPV 45 template by taking advantage of the sequence similarity between HPV 18 and HPV
45 [12]. The first step consisted of making an HPV 45/HPV 18 hybrid sequence by
using primers that were specific for HPV 45, the template being HPV 18. The
primers used were 45art5' and HPV1300r. The second step attached an EcoRI
linker at both ends of the product. The two restriction sites were added for future
cloning purposes and were found on the 45art5'2 and 45art3' primers used during this step. These steps altogether produced a template with primer and probe
annealing sites specific for HPV 45 (see figure 1). Six additional HPV plasm ids
were obtained from ATTC. One plasmid of no risk (HPV2), three plasmids oflow
risk (HPV 6B, HPV 11 and HPV 44) and two plasmids of medium risk (HPV 31
and HPV 56) were used as controls in this study [10]. Plasmids were extracted