Successful pyrosequencing of GC-rich DNA sequences by partial substitution of deoxyguanosine with deoxyinosine.
We experienced significant problems while developing a PyrosequencingTM (Biotage, Uppsala, Sweden) assay to characterize the rifampin resistance-determining region of Mycobacteriumtuberculosis, a target with high guanosine-cytosine content. This paper describes the successful use of a modified pyrosequencing protocol through partial substitution of deoxyguanosine triphosphate with deoxyinosine triphosphate in the polymerase chain reaction.
Bravo
Lulette Tricia C
LT
Departments of Infectious Disease, Cleveland Clinic, Cleveland, OH, USA. bravot@ccf.org
Tuohy
Marion J
MJ
Shrestha
Nabin K
NK
D001426:Bacterial Proteins / Q000235:genetics; D001482:Base Composition; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003849:Deoxyguanosine / Q000378:metabolism*; D006801:Humans; D007288:Inosine / Q000031:analogs & derivatives* / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics; D057089:Pathology, Molecular / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*; D013548:Sweden
C119094:ARR-2 protein, Bacteria; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D007288:Inosine; D003849:Deoxyguanosine; C012271:deoxyinosine