DNA sequencing

DNA sequencing

1. Intro

   • "process of determining the order of nucleotides in a DNA molecule"
   • HGP was used to alucidate numerous mutations that are linked to diseases >> provides a probabilistic risk of develop disease or anabling drug treatment

2. The Sange or "chain terminator" method

   • Elements require
     • DNA polymerase + dideoxy nucleotides (ddNTPs + deoxyNTPs (dNTPs)
     • Primer (labelled and annealed to the sequence of interest that is being sequenced)
   • Process
     • 1. sepatation of double strand by applying heat
     • 2. Bind prime
     • 3. Reaction mixture reparated into 4 tubes which contain all 4 dNTP and 1 ddNTP
     • 4. When the polymerase extends the sequence in tube A, it incorporates the dNTPs as it goes along the sequence. Incorporation either a dd or d when encounters a T (dd stop extension)
     • 5. generation of strands of diff lengths generated
     • 6. DNA separated by size (electropheresis)
   • effiicient on short parts of sequence
   • in order to sequences the all genome >> it is cut into pieces using restriction enzymes

3. Other methods

   • sequencing by synthesis
     • labeled bases are incorporated one at a time, with the unattached bases washed between cycles. The fluorescent signal provides the indication that one base has been added (or multiple).
   • pyrosequencing:
     • bases are unlabeled but the incorporation of the bases is detected via the pyrophosphate that is released. A variant of this method can detect the hydrogen ion H+ released instead
   • nanopore sequencing
     • DNA molecules are passed through a nanometer-sized pore, one base at a time. Upon passing through, the base triggers a signal (electrical current for example) that is detected. The pores can be solid state (small transistors) or biomolecules (alpha-hemolysin).