Detection of mutations in whole genome-amplified DNA from laser-microdissected neurons.
Laser microdissection allows removal of individual cells for DNA extraction, but obtaining reliably amplified DNA from the small numbers of cells that survive neurodegenerative diseases can be difficult. We therefore tested a recently-available technique to amplify genomic DNA to see if it could reliably detect a mutation in nervous system cells. Fifty cortical motor neurons were removed by laser microdissection from cases of motor neuron disease both with and without known mutations in the gene for superoxide dismutase 1 (SOD1). DNA was extracted and amplified with a linear genomic amplification kit (GenomiPhitrade mark). The PCR product of exon 4 of SOD1 from this DNA was then sequenced. The GenomiPhi kit amplified the extracted DNA approximately 70 times. When exon 4 of SOD1 from this DNA was amplified by PCR the E100G SOD1 mutation could be identified on sequencing. No errors in replication were found. In conclusion, the GenomiPhi method of genomic DNA amplification appears to result in reliable replication of mutations in neurons. This technique can be used to obtain increased amounts of genomic DNA to study mutations within cells of the nervous system.
Pamphlett
Roger
R
Academic Units of Neurology, The University of Sheffield, UK. rogerp@med.usyd.edu.au
Heath
Paul R
PR
Holden
Hazel
H
Ince
Paul G
PG
Shaw
Pamela J
PJ
D004247:DNA / Q000032:analysis*; D004252:DNA Mutational Analysis / Q000379:methods*; D016678:Genome; D006801:Humans; D007834:Lasers; D042282:Microdissection / Q000379:methods; D016472:Motor Neuron Disease / Q000235:genetics / Q000473:pathology; D009154:Mutation; D009474:Neurons / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D012333:RNA, Messenger; D004247:DNA