Sequencing

  1: What is the DNA sequence?
  2: Why do we sequence DNA?
  3: How do we sequence DNA?
  4: What is the assembly?
  5: Why were Genome Centers created?

There are 4 distinct types of nucleotides, which are the basic units of DNA, A,T,C and G. In a fragment of DNA to be sequenced, hundreds of nucleotides are present in a specific order. Sequencing of a DNA fragment consists in determining this order, or sequence, which is a long word written in an alphabet which consists of only 4 letters.

The prinicple of sequencing is to produce, from a fixed point, partial copies of the DNA molecule (the template) which are of variable length because they are interrupted randomly. The interruption is provoked by the random incorporation during copying, of a nucleotide analog which blocks the reaction. Starting from a fixed point, all possible partial copies are obtained.

These partial fragments are then separated according to their size by allowing them to migrate in a porous gel subjected to an electric field. The smallest DNA fragments will migrate fastest. This technique, called electrophoresis, can separate two consecutive incomplete copies which have a size difference of only one nucleotide. If we can identify the nucleotide where the copy has been interrupted (the terminal nucleotide) for each of the incomplete copies, from the smallest to the largest, we can then reconstitute the succession of nucleotides along the length of the copy.

In practice, how are the terminal nucleotides identified? Four series of copies are done in parallel, each of which includes an analog of one of the 4 types of nucleotides A,T,G or C, for example, all the partial copies of a series terminated by an A. Moreover, the compound which provokes the interruption is fluorescent: the fragment copied can therefore be detected automatically by an optical system at the end of its migration in the electrophoresis apparatus, which is called a sequencer. The signal detected as the partial copies finish their migration is interpreted by a computer programme which reconstitutes the original sequence of the DNA fragment analysed. For each operation, or “read”, an automatic sequencer can determine the order of 500 to 1000 bases (a “raw” sequence, also called a read). List of questions.