Monday 19 November 2007

DNA SEQUENCING

5. DNA SEQUENCING

This refers to the conversion of template DNA into small fragments. This is achieved through chemical methods and/or enzymatic reactions. The template DNA is denatured to single strands, where short oligonucleotides anneal to the single strands, (specifies the start of the DNA polymerization). Elongation subsequently follows and a specific terminator is added in the reaction. Primarily, there are 4 reactions for each ddATP, ddTTP, ddGTP, and ddCTP, and 4 lanes are available for each lane to measure the strands. This is termed as the classical way. In principle, the shortest fragment is positioned at the bottom/end of the plate.

It is noteworthy that for detection of the fragments, one must label them by use of fluorescent labeling using a fluorescent molecule, and/or by terminator modeling where a terminator nucleotide is labeled by a fluorescent molecule). In the former, each terminator can be labeled using 4 different color signals and one reaction can be done in one single tube; while the latter, one lane is sufficient for the reaction as each reaction is labeled.

Capillary electrophoresis: An alternative method is use of capillary (30-70 cm long) known as capillary electrophoresis, where a very high voltage is applied to have a faster flow of DNA and a detector is placed at the end of the capillary tube which automatically detects the color and reads simultaneously. This method requires 2 hours to read bases of up to 1000.

Cycle sequencing system: this system is similar to the PCR technique as the template is denatured and a primer molecule can use the same template to start elongation again. Elongation can be repetitive using a single primer only, and the template can be used many times. The double-stranded DNA can also be used as a template directly because they will eventually separate into single strands.

The experiments in this topic focused on:

  1. The cycle reaction with 4 color terminator labels and
  2. Capillary electrophoresis

Purification of the reaction products: this involved the precipitation of DNA fragments.

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