1\'/0 Color Multiplexing and Typing of Human Papillomavirus Types 16,18 and 45 on Ligh tCyc1er pm
45art 5
45art 5' 2
HPV18
HPV 45/18 Hybrid
. .
HPV -45 Artificial
Template
HPVt300r
45art 3
Fig. 1. Construction of an artificial HPV -45 required two amplification steps. The first step produced an HPV45/HPV-18 hybrid sequence. The second consisted in the attachment of EcoRI
linkers to produce the full artificial HPV-45 template. These steps produced a template that has
primer annealing sites for HPV-45
with the Insta-Mini-Prep Kit from Eppendorf- 5 Prime (Boulder, CO) and the
High Pure Plasmid Isolation Kit (Mannheim, Germany). Extraction procedures
were described in the kits. For purification purposes plasmids were Ethanol precipitated.
Thirty-nine clinical samples that had tested positive for the Digene Hybrid
Capture I test (Digene Corp, Bellsville, MD) were DNA extracted using standard
Table 1. Oligonucleotides
(GenBank Accession # 74479)
Primers
Position
Length GC(%)
T m COC)
45art 5'
GATCCAGAAGGTACCGACGGGGAGGGAACG -
74
47.3
82.9
GGGTGTAATGGCTGGTTCTTTGTAGAAACAATTGTAGAGAAAAA
HPV1300r
CCACTTCAGAATTGCCATAGCC
22
50.0
63.6
45art 5' 2
GGGAATTCCAGTAAGCAACAATGGCGGATC47
55.3
81.75
CAGAAGGTACCGACGGG
45art 3'
GGCTTAAGCTTCCACTTCAGAACAGCCATAGCCA
34
50.0
75.7