36
Molecular Techniques
healthy tissue showing
two distinct peaks
corresponding to
both alleles
Two independent
amplifications of a
microsatellite region from
tumor tissue showing
only one peak indicating
the loss of one allele
Molecular size marker
4200
4400
4600
4800
5000
F ig . 3 6 .4 Exam ple o f lo s s o f h e te r o z y g o s ity o f a m icrosa te llite in tu m o r
cells o f a ca nce r p atient.
is unchanged despite b isu lfite treatm ent. T he u nm ethylated
p rim e r set anneals to D N A th a t is n o t m ethylated in the same
p rim e r-b in d in g site and w ill therefore have th y m in e in place
o f cytosine in the p rim e r sequence. T he PC R products resulting
fro m b o th p rim e r sets are visualized in p arallel using an agar-
ose gel. M SP m ay be sensitive to 0.1% m ethylated alleles o f a
given CpG island locus. However, M SP does n o t appear to be
h ig h ly reproducib le fo r som e samples th a t m ay have lo w levels
o f m eth yla tion .
Nested MSP
T he ap p lication o f a nested, two-stage PCR approach im proves
the sen sitivity o f M SP to a m a jo r extent. Thereby, p ro m o te r
h yp e rm e th yla tio n can be detected in cytologic specimens w ith
very rare tu m o r cells o r even in the setting o f tu m o r cell screen-
ing. Stage 1 o f the nested M SP am p lifies a p ro m o te r sequence
ind ep end ently fro m the m e th y la tio n status o f the target gene.
Stage 2 u tilize s m ethylation-sp ecific prim ers lik e conventional
MSP. W ith th is nested MSP, it is possible to detect aberrant p ro-
m o te r m e th y la tio n in cytologic specimens th a t contain 1:10 000
o r even few er m ethylated alleles. However, due to the q u alita -
tive nature o f the assay, the influence o f aging, e n viro n m e n t, o r
d iet o n D N A m e th y la tio n m ay c on fou n d results i f even a m in o r
fra ctio n o f n o rm a l cells are m ethylated at the gene o f interest in
cancer-free ind ivid uals.
Quantitative MSP
Q u a n tita tiv e m ethylation-sp ecific polym erase chain reaction
(Q M SP, M e th yL ig h t) applies rea l-tim e PCR technolog y to MSP.
As it detects m e th y la tio n at a q u an titative level, it allow s adjust-
m e n t o f the assay's perform ance characteristics. T he Q M SP
assay uses fla n kin g m ethylation-sp ecific prim ers lik e conven-
tio n a l M SP (Fig. 3 6.7). In a d d itio n, a central olig onucleotid e
m arked b y tw o fluorochrom es is applied. This TaqM an probe,
as w e ll as the prim ers, is com p lem entary to p ro m o te r sequences
th a t develop fro m m ethylated CpG islands after b isu lfite con-
version. T he tw o fluorochrom es are called rep orter (R) and
quencher (Q ). The fluorescence em ission o f th e rep orter (e.g.
FAM ) is suppressed by the quencher (e.g. TA M R A ) due to the
fluorescence energy resonance transfer. Since the em ission fre-
quency o f the rep orter (F1) matches the excitation spectrum
o f the quencher, the energy fo r excitation o f the rep orter does
n o t result in lig h t o f wavelength F1 b u t is d irectly transferred to
the quencher. T he la tte r transform s it in to lig h t w ith the wave-
length F2. D u rin g the synthesis o f a new D N A strand, the D N A
polym erase separates the rep orter fro m the quencher due to its
5' to 3' exonuclease activity. T his makes th e fluorescence signal
o f the rep orter (F1) detectable fo r the real-tim e PCR device.
W h ile F1 p ro p o rtio n a lly increases w ith the fo rm a tio n o f PCR
product, the signal F2 em itted fro m the quencher decreases.
O n ly i f p rim e r and probe b in d specifically does the ra tio o f F1:
F2 increase. Therefore the Q M SP shows a hig h er specificity and
a m ore th an 10-fold hig h er se n sitivity as com pared w ith con-
ve n tio n a l MSP.
In parallel to the target gene, an in te rn a l reference gene is
investigated to c o n tro l fo r in p u t D N A . M yogenic d iffe re n tia -
tio n antigen-1 (M Y O D 1 ) and P-actin (ACTB) represent inte rna l
reference genes w ith a well-established Q M SP protocol. The
m e th y la tio n level is defined as a ra tio o f m easurem ents derived
fro m the target gene and reference gene:
m e th y la tio n level (% ) = (target gene/reference gene) x 100.
This allow s q u a n tific a tio n and com parison o f d iffe re n t PCR
runs. The c u to ff values m ay be chosen e ith er in a w ay th a t
exceeding them can be tig h tly correlated w ith gene ina ctivatio n
o r in order to o p tim ize specificity and se n sitivity o f the assay fo r
tu m o r diagnostics. T he Q M SP represents a precise and reliable
procedure fo r the detection o f p ro m o te r m e th y la tio n suited fo r
h ig h -th ro u g h p u t m easurem ents. C o m b in in g d iffe re n t m arker
genes m ay im p rove the se n sitivity o f the assay.
Hybrid Capture System
Digene Corp. (G aithersburg, M D ) is one o f the m ost active com -
panies engaged in developing h u m a n p a p illo m a viru s (H P V )
tests fo r clinical use over the past decades. The firs t tw o dia-
gnostic tests provided b y Digene were ViraPap (fo r H P V screen-
ing) and V iraTyp e (fo r H P V typ ing ), w h ic h have been w id e ly
adopted in H P V research and ro u tin e clinical diagnosis since the
early 1990s. It was soon realized, however, th a t b o th tests shared
several shortcom ings in h e re n t to d o t b lo t h yb rid iz a tio n .10
Hybrid Capture 1
T he V ira P A P and V iraTyp e tests w ere fo llo w e d b y th e la u n c h -
ing o f th e D igene H y b rid C apture assay, w h ic h is based o n a
s o lu tio n h y b rid iz a tio n p rincip le. T his first-g en era tio n H y b rid
C apture assay (H C 1 ) soon o b tained FD A approval and was
w id e ly used in H P V research and c linica l diagnosis d u rin g the
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