15 Preparation of Samples for CGH by OOP-PCR from microdissected cells
191
Subprotocol 2
Alternative protocols for DOP-PCR amplification of released nuclear DNA
Procedure
G·······················~rrnT!T!TlTiTITEJ S'- ~ :~~~~q~~: ~
mi~1fu~t~~W -3'
D stringent primer binding region
~ variable binding region
[J main binding region
Sequence and structure of the original DOP-PCR primer,
developed by Telenius et aI., 1992
1st reaction step under low stringency conditions: Variable and main primer binding
regions anneal to the template DNA and initiate first replication products
~f?Vl22Zd.:.:.:.:.:.:.:1
~f?Vl22Zd.:.:.:.:.:.:.:1
In the following cycles variable and main primer binding regions bind also to products of first
cycle and initiate the replication of DNA products with DOP-primer complementary regions
on 3"-ends
..
I!:::::!:!:!:!:WI/)?ZA~
2nd reaction step under high stringency conditions: Complete DOP-primer is binding to
its complementary regions on 3'-ends of replicated DNA fragments
,::.:::.:.:!:j:V/WZlA ..••..•1
f.·.·.· .V/WZlAf:.:j:.:::!:!:1
...
After complete DOP-PCR reaction, a high amount of similar DNA fragments results with
DOP-primer sequences on both ends
wm
f.·.·.· .VI/2?Z&I.:.:.:.:.:::.:1
Fig. 2. DOP-PCR reaction mechanism
1.:.:.:.:.:.:.:Vd/Z/4.·.·.· .1