A new method for optimizing multiplex DNA microsatellite analysis in low quality archival specimens.
BACKGROUND
Testing microsatellite instability seems to be a useful tool for the initial screening of putative non-polyposis colorectal cancer (HNPCC), preceding analysis of germ-line mutations of DNA mismatch repair genes. However, diagnosis of microsatellite instability becomes complicated when highly-damaged DNA from formalin-fixed paraffin-embedded tissue specimens has to be investigated.


METHODS
A new methodical approach was established based on special multiplex PCR regimes (e.g., on touch-up cycling conditions), allowing both sufficient, as well as specific, amplification of the Bethesda reference panel loci with low quality DNA as template.


RESULTS
By applying our new method, microsatellite instability could be analyzed successfully in 75 out of 84 investigated tumors (89%), whereas, by using standard PCR protocols, microsatellite analysis failed in 36% of the investigated cases.


CONCLUSIONS
Our new methodical approach should be recommended for the use of archival material since it allows an efficient and accurate amplification of the Bethesda marker fragments and is less dependent on the DNA quality.
Drobinskaya
Irina
I
Department of Surgery, Heinrich-Heine-University of Duesseldorf, Duesseldorf, Germany.
Gabbert
Helmut Erich
HE
Moeslein
Gabriela
G
Mueller
Wolfram
W
D048868:Adaptor Proteins, Signal Transducing; D022981:Allelic Imbalance; D002352:Carrier Proteins; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D005557:Formaldehyde; D006801:Humans; D007150:Immunohistochemistry; D018895:Microsatellite Repeats / Q000235:genetics*; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics; D016707:Tissue Fixation
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein