Extraction and Purification

• The first step to sequencing DNA involves extracting it from the cells that contain it. There are several methods that can be used to extract DNA. There are many commercial kits available from biological suppliers that make extraction quick and easy. After the DNA has been isolated, purify it if needed by using a commercial spin column kit. This is important for sequence validation purposes. If the sequencing procedure will not be performed immediately after DNA extraction, store the DNA at 4 C.
  
  

Sequencing Using Sanger Technology

• The most popular method for sequencing DNA has historically been the Sanger method. This method uses the incorporation of chain-terminating (di-deoxy) nucleotides into a complementary strand of DNA that is synthesized by using the extracted DNA as a template. Four reactions must be set up so that each reaction contains only a single chain terminating nucleotide. The special nucleotides are tagged with a marker that is visible as a band when the newly synthesized DNA is denatured on a polyacrylamide gel. The pattern of bands that appears on the gel represents the sequence of nucleotides that make up the DNA. The results must be manually decoded from the band pattern into a lettered sequence.
  
  

Sequencing Using 454 Technology

• A recent advance in sequencing technology is gaining popularity. Pyro-sequencing, or 454 sequencing as it is sometimes called, is a faster and less labor-intensive method for sequencing DNA; so much so that it is making sequencing on the order of the genomic level an everyday occurrence. Pyro-sequencing is so called because it involves the incorporation of nucleotides that release an inorganic pyrophosphate when added to the DNA chain. This chain is a complementary strand of DNA synthesized by using the extracted DNA as a template. With pyro-sequencing, only one reaction needs to be set up and the sequencer records the order in which special nucleotides are added to the nascent strand as it happens. Simply observe the data compiled by the machine after sequencing is complete.