Transcript DNA sequencing

DNA sequencing
plan:
To sequence a DNA molecule we need:
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the moelcule to be sequenced (in a vector)the DNA pol
Taq DNA pol
a mix containing the dNTPs and the fluorescent ddNTPs
a Thermocycler
a “sequencer” (a device where capillary electrophoresis can be done)
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las moléculas à secuencar (un vector)
la ADN pol
La mezcla de dNTPs y ddNTPs.
el estiramiento a ± 70°C en un termociclador mediante la ADN pol y los nucleótidos
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.
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 secretes
one or the other ssDNA in the medium.
This method allows the sequencing of an unknown DNA fragment, the primers
are 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 DNA
is ultrasonicated in small pieces, with a mean length of 3000 bp. The ultrasonication hed to
be 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 sequenced
with a vector primer (sens or antisens).
The automatic sequencers are unable to read more tha 500 nt at both ends. So we should use
several 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 the
3’ 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 pol
has been engineered (with a bigger catalytic site).
4. a thermocycler
5. a sequencer
applied biosystem
beckman