Innovations in non-isotopic DNA sequencing: using an electrotransfer unit to blot sequencing gels and an automated membrane processor for detecting DNA sequences.
As alternatives to radiolabeled DNA sequencing, chemiluminescent and chromogenic sequencing methods can be comparable in both sensitivity and resolution. Chemiluminescent/chromogenic detection procedures are safer because they completely eliminate the handling and use of radioisotopes. One method involves standard dideoxy DNA sequencing reactions that are initiated with biotinylated primers, separated by gel electrophoresis, transferred onto nylon membrane and detected utilizing chemiluminescent 1,2-dioxetane substrates for alkaline-phosphatase. Alkaline phosphatase is linked to the biotinylated sequencing products by a streptavidin/alkaline phosphatase conjugate (SAAP). In this paper we describe an optimized procedure for transferring sequencing gels. The procedure is based on a semidry method developed at Hoefer Laboratories using the GeneSweep Sequencing Gel Transfer Unit. Transfer is rapid, uniform and reliable from gel to gel. We also describe automation of the development process using a fully programmable Gel/Membrane Processor that automates delivery, incubation and disposal of reagents. All crucial points for electrotransfer of sequencing gels and the detection of biotinylated DNA sequencing reaction products are discussed.
Patel
A
A
Hoefer Scientific Instruments, San Francisco, CA, USA.
Nash
B
B
D000469:Alkaline Phosphatase; D001710:Biotin; D015139:Blotting, Southern / Q000379:methods*; D002863:Chromogenic Compounds; D004247:DNA / Q000302:isolation & purification; D001330:Electronic Data Processing; D004586:Electrophoresis / Q000379:methods; D006571:Heterocyclic Compounds; D006573:Heterocyclic Compounds, 1-Ring; D008163:Luminescent Measurements; D008567:Membranes, Artificial; D009757:Nylons; D017422:Sequence Analysis, DNA / Q000379:methods*
D002863:Chromogenic Compounds; D006571:Heterocyclic Compounds; D006573:Heterocyclic Compounds, 1-Ring; D008567:Membranes, Artificial; D009757:Nylons; C079142:1,2-dioxetane; D001710:Biotin; D004247:DNA; D000469:Alkaline Phosphatase