A reproducible assay of polymerase chain reaction to detect trinucleotide repeat expansion of Huntington's disease and senile chorea.
A simple and reproducible method of polymerase chain reaction (PCR) assay was established to detect trinucleotide repeat expansion for Huntington's disease (HD) using a new DNA polymerase and buffer system. The system consists of an extremely heat stable DNA polymerase (Pfu), and a buffer supplemented with ammonium sulfate and dimethyl sulfoxide. Previous methods to amplify expanded alleles for HD have been very complex in PCR conditions, but the reproducibility was sometimes very low because of repetitive sequences around the primer sequences. With the present method, strong bands for the disease alleles were reproducibly visible in a conventional agarose gel stained with ethidium bromide without using isotopes. Three cases with sporadic HD and a case with senile chorea showed expanded alleles for HD with smaller sizes of the expansion than cases with typical HD. These results showed that the present method provides a simple and reproducible way to detect HD allele, and some cases with sporadic HD and senile chorea had expanded HD alleles.
Watanabe
M
M
Department of Neurology, Gunma University School of Medicine, Maebashi, Japan.
Abe
K
K
Aoki
M
M
Kameya
T
T
Itoyama
Y
Y
Shoji
M
M
Ikeda
M
M
Iizuka
T
T
Hirai
S
S
D000328:Adult; D000368:Aged; D001483:Base Sequence; D004247:DNA / Q000097:blood; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D003704:Dementia / Q000235:genetics*; D005091:Exons; D005260:Female; D006801:Humans; D006816:Huntington Disease / Q000235:genetics*; D008214:Lymphocytes; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012016:Reference Values; D015203:Reproducibility of Results; D018911:Trinucleotide Repeats
D017931:DNA Primers; D004247:DNA; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase