Open AccessNon‐Radioactive Direct Sequencing of PCR Products Amplified from Neuropathological Specimens
Author(s) -
Kosel Siegfried,
Graeber Manuel B.
Publication year - 1993
Publication title -
brain pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.986
H-Index - 132
eISSN - 1750-3639
pISSN - 1015-6305
DOI - 10.1111/j.1750-3639.1993.tb00770.x
Subject(s) - polymerase chain reaction , digoxigenin , microbiology and biotechnology , multiple displacement amplification , dna sequencing , oligonucleotide , biology , primer (cosmetics) , applications of pcr , dna , sanger sequencing , chemistry , dna extraction , genetics , in situ hybridization , multiplex polymerase chain reaction , gene , messenger rna , organic chemistry
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.