36
Molecular Techniques
F ig . 3 6 .6 T h e p r o m o te r r e g io n o f a g e n e a n d e x o n s 1 a n d 2 . The
m e th y la tio n status o f th e p ro m o te r correlates w ith g e n e expression.
(A) U n m e th yla te d p ro m o te r: th e g e n e is expressed. (B) M e th yla te d p ro m o te r:
th e g e n e is ina ctivated. A, a ctivator; HAT, histon e acetyltransferase; HDAC,
histon e deacetylase; MBD, p ro te in w ith m e th yla te d D N A -b in d in g d o m a in ; R,
repressor; TF, tra n s c rip tio n factor.
o f w o m e n w ith H SIL, th e s e n s itiv ity o f th e HC 1 assay was n o t
o p tim a l.13
Hybrid Capture 2
A m a jo r technologic breakthroug h was m ade b y launching the
second-generation H yb rid Capture assay (H C 2) on the m arket
in the late 1990s.14 U ndoubtedly, the widespread application o f
this test in H P V detection has contributed m ore than any other
single assay to the ever increasing flo w o f studies reporting H P V
prevalence (lo w - and h ig h-risk types), incidence, and (sem iquan-
tita tive ) vira l loads in divergent target p op ulations in all m a jo r
geographic regions, witnessed b y the scientific c o m m u n ity during
the past 10 years.15,16 This makes a m ore detailed discussion o f
this im p o rta n t technologic in n o v a tio n w arranted in th is context.
Like its predecessor, the H C 2 test is a nonrad ioactive signal
a m p lific a tio n m eth od th a t detects 13 genital h ig h -risk H P V
types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and
5 genital lo w -risk H P V types (6, 11, and 4 2 -4 4 ). The probes fo r
h ig h-risk types and lo w -risk types are in tw o separate reactions
(panel A and panel B). In b rief, the H C 2 p la tfo rm uses specific
R N A probes, h yb rid iza tio n , a ntib od y capture, and signal a m p li-
fica tion to a llo w rapid, standardized testing o f th e genetic m ate-
ria l o f infectious agents in v irtu a lly any lab o rato ry setting. A t the
m om en t, the H C 2 assay is available to detect HPV,
C h la m y d ia
tra c h o m a tis , N e is s e ria g o n o rrh e a ,
and cytom egalovirus, the last in
the tube fo rm a t only.
A
B
Taq DNA
Polymerase
Primer
M1
5‘
<1
Primer
F ig . 3 6 .7 (A) Q u a n tita tive m e th yla tio n -sp e cific polym erase ch ain reaction (QMSP) run sh o w in g a b e r r a n t m e th y la tio n o f C D K N 2 A in bron ch ial aspirates
o f tw o tu m o r p atie n ts (TU1 and TU2) b u t n o t in th o se o f n o n tu m o r (NT) patients. A fte r 30 polym erase ch ain reaction cycles, th e fluorescence em ission o f F1
show s an e xp o n e n tia l increase. (B) P rinciple o f QMSP (see te xt). Q, q ue n ch er; R, reporter.
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