PART THREE
Special Techniques in Cytology
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F ig . 3 6 .3 P rin c ip le s o f p o ly m e ra s e c h a in re a c tio n (PCR). The te m p la te D N A is heated to 94°C, w h ic h separates th e tw o strands o f th e d o u b le helix.
W h e n th e te m p e ra tu re is lo w e red to 55°C, th e prim ers can anneal to th e ends o f th e ta rg e t sequence. O nce th e prim ers have annealed to th e te m p la te , th e
te m p e ra tu re is increased to 72°C. This is th e o p tim u m te m p e ra tu re fo r th e Taq polym erase to e lo n g a te D N A using th e prim ers as sta rtin g p o in t. The n ew ly
synthesized D N A strands serve as te m p la te fo r th e next PCR cycle.
hund red o f genes m ay be m ethylated at th e ir p ro m o te r in a
single tu m o r. Since p ro m o te r h yp e rm e th yla tio n is stable even in
fixed specimens, it represents a p rom ising source fo r th e devel-
o p m e nt o f biom arkers.
T he developm ent o f key m olec ular technologies enabling
sensitive and rather sim p le detection o f m e th y la tio n changes
has d ram atically advanced progress in th is fie ld .9 For diagnostic
purposes especially, techniques lin k e d to b isu lfite conversion
have been proven useful. T his section aim s to present som e
w ell-established m ethod s fo r diagnostic m e th y la tio n analysis o f
selected genes.
Bisulfite Conversion
T he
chem ical
m o d ific a tio n
o f
D N A
k n o w n
as
b isu lfite
conversion plays a key ro le in th e detection o f p ro m o te r m eth yla -
tio n . Acid-catalyzed conversion o f genom ic D N A w ith b isu lfite
deam inates cytosine to uracil, whereas 5 -m ethylcytosine rem ains
unchanged. T he resulting change o f base sequence depends on
the m e th y la tio n status and m ay be detected w ith PCR-based
m ethod s such as sequencing o r d iffe re n t types o f m e th yla tio n -
specific polym erase chain reaction (M S P ). B isu lfite treatm ent
creates a m o s tly three-base D N A nearly devoid o f cytosine. This
red uction in sequence c om p le xity m ay lead to a reduced specifi-
city o f h yb rid iza tio n , w ith prim ers o r probes lim itin g th e spe-
c ificity o f MSP.
Conventional MSP
C o nve ntio na l M SP is a w id e ly used technique th a t gives a q u a li-
tative estim ate w h ethe r aberrant m e th y la tio n is present o r not.
Tw o p rim e r sets are designed to anneal to a region containing
CpG dinucleotides. The m ethylated p rim e r set assumes the
CpG dinucleotides are fu lly m ethylated, i.e. the D N A sequence
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