DNA sequencingcapillary electrophoresis

by Jean-Pierre Herveg and a lot of friends atthe Brussels Branch of the Ludwig Institute for Cancer research (Licr) and the Christian de Duve*

Institute for cellular Patholgy (ICP).

April 2006Université Catholique de Louvain

Avenue E. Mounier, 1200 Brussels (Belgium)

----------------------*Christian de Duve got the Noble Prize in medicine in 1974. He and his team discovered

both lysosomes and peroxysomes.

questions

1. Explain the sequencing with capillary electrophoresis.2. When do we have to insert the DNA to be sequenced into a vector ?3. What’s a contig ?4. Draw a ddNTPs

DNA sequencing

plan:

To sequence a DNA molecule we need:

1. the moelcule to be sequenced (in a vector)the DNA pol2. Taq DNA pol3. a mix containing the dNTPs and the fluorescent ddNTPs4. a Thermocycler5. a “sequencer” (a device where capillary electrophoresis can

be done)

6. las moléculas à secuencar (un vector)7. la ADN pol8. La mezcla de dNTPs y ddNTPs. 9. el estiramiento a ± 70°C en un termociclador mediante la ADN pol y los nucleótidos10. la electroforésis denaturante en el secuenciador los "secuenciadores" en el comercio.

Para secuenciar, se necesita primero la molécula de DNA a secuenciar,un vector de secuencaje, cebadores, una ADN pol, dNTPs, ddNTPs fluorescentes y un secuenciador automático.

---------------------------QuestionExplain the sequencing with capillary electrophoresis.

1. sequencing vector

In most of the case the fragment to be sequenced is unknow.It is inserted into a sequencing vector. Usually it’s a phage.This phage is prepared as dsDNA, placed in a bacteria which secretesone or the other ssDNA in the medium.This method allows the sequencing of an unknown DNA fragment, the primersare complementary to the vector the sequence of which is very well known

To sequence a genome or simply a chromosome, or the insert of a BAC, the total DNAis ultrasonicated in small pieces, with a mean length of 3000 bp. The ultrasonication hed tobe standardized before. The operation is fully automatic.All the sequences are put in between the arms of a vector (phage or plasmid).All the operation is automated. So, each ssDNA fragment, sens or antisens is sequencedwith a vector primer (sens or antisens).

The automatic sequencers are unable to read more tha 500 nt at both ends. So we should useseveral genomes to cover every sequences.

A huge computer group sequences in what is called a contig.

Most of the time, a contig ends when there is a repetitive élément like an Alu sequence.

3. a mix of dNTPs and ddNTPs

ddNTPS ar dNTPs without a 3’ OH (see the picture below)The DNA pol is unable to discriminate between dNTPs and ddNTPs, so it puts ddNTPs at random.The number of time the enzyme put a ddNTP is a function of the ddNTPs/dNTPs ratio.Once the enzymes has put a ddNTPs, The strand is “terminated”, because it lacks the3’ OH required to elongate the strand.The ddNTPs are fluorescent. Each ddNTPS has it own fluorescence.So the “treminated” strand has a fluorescence depending of the ddNTPs which ends it.As the fluorescent molecule added to the ddNTPs are somewhat huge, a special Taq DNA polhas been engineered (with a bigger catalytic site).

4. a thermocycler

5. a sequencer

applied biosystem

beckman