Presentation on theme: "DNA sequencing. plan: To sequence a DNA molecule we need: 1. the moelcule to be sequenced (in a vector)the DNA pol 2. Taq DNA pol 3.a mix containing the."— Presentation transcript:
plan: To sequence a DNA molecule we need: 1. the moelcule to be sequenced (in a vector)the DNA pol 2. Taq DNA pol 3.a mix containing the dNTPs and the fluorescent ddNTPs 4. a Thermocycler 5. a sequencer (a device where capillary electrophoresis can be done) 1. las moléculas à secuencar (un vector) 2. la ADN pol 3. La mezcla de dNTPs y ddNTPs. 4. el estiramiento a ± 70°C en un termociclador mediante la ADN pol y los nucleótidos 5. 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 its 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).