DNA sequencing

DNA sequencing

1. Maxan and Gilbert method

   • 1 - double stranded DNA is labeled using radioactive phosphate (phosphate 32) at the 5' end of each strand
     • the attachment of Phosphate 32 is done using a polynucleotide kinase enzyme and 32P-dATP
   • 2 - the double stranded DNA is treated with dimethyl sulphoxide and heated to 90 degree centigrade to separate the strands
     • these strands are then purified
   • 3 - the strands are treated with various chemicals which cause base modification
     • formic acid modifies purines (A and G)
     • dimethyl sulphate modifies guanine
     • hydrazine modifies pyrimidines
       • adding NaCl prevents modification of thymines
   • 4 - the altered/modified bases are removed, hot piperidine is used to cleave the sequence at modified bases
     • the remaining strands of DNA undergo gel electrophoresis, through these results we can work out what the DNA base sequence is
       • Maxam and Gilbert

2. Sanger method

3. next generation sequencing

   • sequencing by synthesis
   • sequencing by ligation
   • single molecule sequencing

4. is finding the order of nucleotides in a piece of DNA

   • sequencing can be done on anything which contains genetic information
     • animals
     • plants
     • bacteria
     • archaea
   • depending on the method used you may be provided with DNA or RNA sequences

5. sequencing can be used to determine the sequence of

   • individual genes
   • large genetic regions such as gene clusters or operons
   • chromosomes
   • entire genomes

6. the results of sequencing may be used by

   • researchers to further scientific progress
   • by medical proffessionals to aid treatment decisions and genetic counselling