catholic university of Louvain school of medicine (Brussels)
sponsored by PROMEGA NL.
DNA sequencing (July 2005)
(workshop of molecular genetics SBIM 2520)
Jean-Pierre HERVEG

go back to information, general biology


DNA pol-1

is an enzyme which is used in sequencing to extends the 3' end of a primer along a ssDNA template using dNTPs and ddNTPs.

DNA pol-1 doesn't make the difference between some modified ddNTPs, such as for example a dideoxynucleotide (ddNTPs) made fluorescent by addition of chemical groups.

The enzyme will place either a dNTP or a fluorescent ddNTP. It will place a ddNTP depending on the concentration ratio of ddNTPs/dNTPs

Each time the enzyme place a ddNTP the sequence will be "terminated", because ddNTPs don't have a 3' end.

primer extension

In a 400 µl microcentrifuge tube mix the reagents

primer

dNTPs and fluorescent ddNTPs

DNA pol and the buffer

Incubate the tube at the right temperature. In our example, DNA pol-1 will produce the 21 terminated sequences.

polyacrylamide denaturing electrophoresis
(the gel could be loaded in capillaires)

These 21 sequences are separated in a denaturing polyacrylamide electrophoresis. (denaturing, to separate the template).

Each sequence will move in the gel as a function of its size, the shortest first.

They will pass according to their sizes in front of a excitatory laser beam and a fluorescence detector. As a sequence passes through the excitatory laser beam, it fluoresces according to its terminal ddNTP. The successsion of the fluorescences is recorded.

There are two types of automated sequencing machine in our Faculty. The are produced by Perkin-Elmer and Beckman-Coulter.

sponsored by PROMEGA NL.
DNA sequencing (July 2005)
(workshop of molecular genetics SBIM 2520)
Jean-Pierre HERVEG

go back to information, general biology

catholic university of louvain la neuve school of medicine (brussels)