A-overhang-dependent repeat expansion determination (ADRED).
In this study we present a quick and easy method for counting trinucleotide repeats by de-oxyadenosine overhang (A-overhang)-dependent repeat expansion determination (ADRED). During standard Taq DNA polymerase-based sequencing reactions, the unterminated sequencing products of short PCR fragments are tagged with a 3'-end A-overhang that is visible as an intense peak in an electropherogram; this allows for easy and precise determination of the fragment length and thus the extent of repeat expansions. ADRED has clear advantages over existing methods, because repeat numbers of both normal and pathogenic (expanded) alleles can be analyzed without using labeled primers or labeled DNA standards. Because ADRED includes a sequencing step, disease-relevant polymorphisms (e.g., CAA interruptions in spinocerebellar ataxia type 2) can simultaneously be detected.
Achmüller
Clemens
C
Institute of Biochemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck, Austria. clemens.achmueller@uibk.ac.at
Köhler
Andrea
A
Bösch
Sylvia
S
Schneider
Rainer
R
D000483:Alleles; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D003839:Deoxyadenosines / Q000378:metabolism*; D006801:Humans; D006816:Huntington Disease / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA; D020754:Spinocerebellar Ataxias / Q000235:genetics*; D013132:Spinocerebellar Degenerations / Q000235:genetics*; D019914:Taq Polymerase / Q000235:genetics; D019680:Trinucleotide Repeat Expansion / Q000235:genetics*; D018911:Trinucleotide Repeats / Q000235:genetics
D003839:Deoxyadenosines; D004247:DNA; D019914:Taq Polymerase