A nonradioactive, fluorescence-based telomeric repeat amplification protocol to detect and quantitate telomerase activity

A nonradioactive,

fhorescence-based

telomeric

repeat

telomerse

amplikation

protocol

to detect

and quantitate

activity

100

50 A_

‘Ob

___

_

._L,-_A.

150

Sze marker

\

@P)

(1) - i,

--__--~

0)

Telomerase signals

t

I

Internal telomerase asszy standard

No Internal

standard

Tws

TECHNICAL

X to : ~1of11~ IYXpnxluct

2 ..~&I ~imntn~ twfk~ ( 1 I-H’.) gel npp3r,ltus of the DNA sequencer. .i 5I:x tvith 1 pl of loading buffer consisting of 9G% fonnilmide and 10% 131ucdextnn (Pharmacia ). .d ~cn:~ruw for -1min :u 9ST. rhcn chill quickly on ice tid samples on tlw 8% denaturing ge!, L’w 100. 150 and EOOirp tlurwc*wcnt wc mnrken tPhorm:~cra Biotechl. 5 Kun the D.SA scqucncer 0 I?IC Iluorw~nr gel cl&~ ohtained hy the AL.F DNA .quencer Ii are collected and analyzed automatically by the Fnwment ~anagc~ progt~m (Phannacia Hiotech). E..:h fluowscent peak is quantitated in rerms of size tbp,, peztk height and peak area. In the ~3.w of twcrJmplil?ed pnxluc~s which would give unrelizhle area value. an aliquot of the ICR products should he diluted In dlsttlllccl wstcr and reanalyzed

R’c thank Alan J. Kinniburgh. Roswell Park Cancer Institute. Buffalo. NY, USA for his critical reading of this manuscript and ~;en ~+s+x Pharmrlcia Biotwh Japan. Tokyo, for helpful suggestions.

Sam&a 1 1

3

5

L---_-----TIC; 0UOW.K 1996 VW.. 12 NO. 10

396

1”

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1

Sample 2 n 3

5

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2”

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d&500 do&

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- 200 -100

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