In order to understand what is going on you need some basic information about genetics and molecular biology.

tools of the trade-- cloning vectors, restricition enzymes, gel electrophoresis, PCR
 

cloning	overview of cloning and cloning vectors
PCR	nontechnical description animation -- requires shockwave
restriction enzymes	simple description from Kimball's Biology pages
Gel Electrophoresis	animated tutorial of restriction enzymes and gel electrophoresis -- requires FlashPlayer
Southern Blot DNA fingerprinting	animation -- requires shockwave
DNA sequencing	cycle sequencing animation -- requires shockwave
RFLP	an example of a genetic analysis using RFLPs from University of Arizona's Biology Project
FISH	http://www.accessexcellence.org/AB/GG/fish.html

Sequencing Basics

A gene is a discrete sequence of nucleotides -  Cold Spring Harbor DNA Learning Center chapter 22
-- contains a nice animation of the chemistry of dideoxysequencing (also called chain termination or Sanger method)
-- this animation shows manual sequencing:reactions incorporate a radioactive nucleotide fragments separated on polyacrylamide gel and visualized by autoradiography

cycle sequencing animation -- Cold Spring Harbor Animation Library
--demonstration of automated sequencing: reactions incorporate fluorescent tag, fragments separated on a polyacrylamide gel, fluorescence detected by computer which yields electrpherogram (chromatogram)

Sequencing the Genome -- sequencing primer from Genome News Network
-- comparison of the two approaches to sequencing genomes. Since this was originally published at the Celera education web site, they focus on the advantages of the shotgun method.  Nice figures.

Acquiring the sequencing information is only the first step.  Whether the data is generated by manual or automated sequencing, the sequence data requires some editting.
In the case of manual sequencing,  the bands on the gel need to be interpretted and converted to a sequence of A's, C's, G's and T's (with an occassional N where the data is inconclusive).
For automated sequencing,  the computer generates an electropherogram which correlates the peak fluorescence with the presence of a given base in a process called base calling:

see samples of automated sequencing output from PE biosystems.  You can click on the some of the figures to see larger versions.  Note that close to the primer the peaks are sharp and well defined.  Further away from the primer the peaks get broader and more difficult to define accurately.

Obtaining the sequence of bases is just the beginning.

Subsequent steps are focused on assembly and annotation of the sequence -- putting it in context and gaining useful information from the sequence.
 

More advanced information
Analyzing Prefinished Sequence Data --
Overview of the analytical steps involved in Prefinishing-- figure from '"An Effective Approach to Analyzing 'Pre-finished' Genomic Sequence Data" by P.M. Kuehl, J.M. Weisemann, J.W. Touchman, E.D. Green and M.S. Boguski

© 2001 Donna E. Crone, PhD, Clinical Assistant Professor, Dept of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180 croned@rpi.edu
last revised Aug 24,2001