33. Specific requirements for PCR amplification of long mitochondrial A+T rich DNA

RONDAN DUEÑAS JC; PANZETTA DE DUTARI GM; GARDENAL CN

BIOTECHNIQUES

INFORMA HEALTHCARE

Año: 1999 vol. 27 p. 266 - 266

0736-6205

olymorphic gene markers are powerful tools in phylogenetic and population genetic studies. Several approachessuch as restriction fragment-lengthpolymorphism (RFLP), sequencing, alkaline phosphatase polymerase chainreaction (AP-PCR) and random-amplified polymorphic DNAs (RAPDs) havebeen used in different taxa (9). At thepopulation level, the analysis of markers present in the noncoding control region of the mitochondrial genome isparticularly useful (10). In invertebrates, this fragment is known as ?A+Trich? because its adenine and thyminecontent can be >90% (2,3,6). It is highlyvariable, in sequence and length, andprovides an important source of polymorphic markers for closely relatedtaxa (8). The ability to amplify A+Trich regions in vitro facilitates their further characterization by direct sequencing and restriction enzyme analysis(2,11). However, we have found thatfragments larger than 1.5 kb are almostimpossible to amplify under standardreaction conditions. Here, we demonstrate t