DETECTION AND ANALYSIS OF NUCLEIC ACIDS - A Guide to Methods in the Biomedical Sciences - page 35

Figure 2. DNA Sequencing
Automated DNA sequencing
Automated sequencing uses the dideoxy sequencing method, but in-
stead of using a radioactive label fluorescent ddNTPs are used, each
labeled with a different fluorophore. Once the reactions are completed,
they are resolved on a PAGE gel. Because the laser in the automated
reader can distinguish the 4 different fluorescent labels, the sequencing
can be accomplished by running all 4 reactions in a single lane. Au-
tomated sequencing is not only more efficient (more reactions can be
loaded on each gel), but generally provides more sequence information
per reaction, generating 600-1000 bp or more of readable sequence
versus the 300-400 possible with manual sequencing methods.
D. Detection and quantitation of RNA
A number of methods have been developed to identify RNA species
and quantify a specific RNA transcript, specifically messenger RNA
(mRNA) within total cellular RNA, or total RNA isolated from tissues.
mRNA is the “+” or “sense” strand of RNA that is transcribed and pro-
cessed from DNA encoding a gene that is subsequently translated into
protein. The following represent the more commonly employed methods.