Non-radioactive direct sequencing of PCR products amplified from neuropathological specimens.
We have developed a simple, rapid and relatively inexpensive protocol for direct non-isotopic cycle sequencing of DNA amplified using the polymerase chain reaction (PCR). PCR is performed on routine and archival neuropathological tissue. For sequencing, a 5'-digoxigenin end-labelled oligonucleotide primer is annealed and extended during thermal cycling, sequencing reactions are separated on a standard sequencing gel and the gel is contact-blotted to a nylon membrane. Sequenced DNA is visualized using immunological detection of digoxigenin.
Kösel
S
S
Laboratory of Molecular Neuropathology, Ludwig-Maximilians-University, Munich, Germany.
Graeber
M B
MB
D001483:Base Sequence; D004076:Digoxigenin / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D009422:Nervous System Diseases / Q000473:pathology*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic
D015345:Oligonucleotide Probes; D004076:Digoxigenin