[From The North Carolina Bionetwork]

Every strand of DNA is made up of pairs of molecules called nucleotides strand together in a long chain called the double helix. Each pair of nucleotide is held together by its complimentary bases: Adenine sticks to Thymine, and Guanine sticks to Cytosine. To sequence the segments of DNA is to figure out the exact order of these base pairs in a DNA chain.

The first step in DNA sequencing is the separation of the double helix, dividing the molecule into two long strands.

The single strands are then put into four buffer solutions that contain free nucleotides and an enzyme to help the nucleotide attached to the strands and rebuild the double helix. However, each of the four buffer solutions also contains a dideoxyribonucleotides which stops the chain from being rebuilt any further. The result is a collection of DNA strands, each as long as it is allowed to grow until randomly encountering a dideoxyribonucleotide.

For example, in the solution with Adenine Dideoxyribonucleotide, the strands have been built to every possible length that ends with Adenine. The solutions are done the same, for Cytosine, Guanine and Thymine. When the solutions are loaded into an electrophoresis gel, the rebuild chains arrange themselves by length. Because we know which base ends in the solution, we can read the gel base by base, from shortest to longest. This tells us the order of bases in the chain – the DNA sequence.

• This experiment begins with four test tubes that contain the DNA segments we want to sequence, already denatured in the single strand.
• Add to this, the reaction buffer which include a number of the necessary components:
  1. First it contain all four free floating nucleotides: Adenine, Cytosine, Guanine, and Thymine, which will attach to the single strands to reform the DNA.
  2. Also included are DNA polymerase, an enzyme that helps nucleotides attach to the strand.
  3. And a primer which is a short strand of nucleotide already in the right order that will attach to the strand and get the process started.
  4. Most importantly, each of the four buffers contain one type of free floating dideoxyribonucleotide. When one of these attaches to the chain, it will prevent it from growing any longer.
• Once all the components are combined, put them on a thermocycler time to undergo  PCR. Repeated cycle of heating and cool will help build and amplify a new various links of DNA.
• When they are ready, add 5 microliter of gelloid solution.
• To settle the contents to bottom of each tube, run the centrifuge.

Agarose gel electrophoresis is a common method of isolating regions of DNA. A gel trace is prepared with DNA samples placed on one side. DNA molecules have a strong negative charge, so if an electric field is applied in the tray, they’ll move toward the positive electrode. Agarose gels have microscopic pores which act as a filter when they try to move through them. Smaller molecules will move through them more quickly than larger molecules. Those closest to the positive electrodes are the smallest and those farthest away are the largest.

To prepare a vertical electrophoresis gel:

1. First, carefully clean the plate with alcohol. Use petroleum jelly to seal spacers between the two plates.
2. Clamp the plates together and tape the bottom edge to prevent leaking.
3. Elevate the open end of the plates and pour agarose gel solution between them.
4. Insert the coomb into the top of the gel and allow it to set.
5. When the gel is ready, remove the clamps and tape and place it in an electrophoresis tower.
6. Fill the reservoirs at the top and bottom with buffer and low the wells.
7. Connect the power source and run the electrophoresis for a time specified by your instructor. The time required will vary depending on the voltages but it should be several hours.

Because the amount of DNA at each position in the sequence is very small, the loading solution usually contains a radioactive nucleotide that will mark the positions of the band when placed against x-ray films. This makes it possible to read from bottom to top, the sequence of nucleotide in the segment of DNA.

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