Ooooc

Termination Coloring Pages For Therapy

Figure 3. Chromatogram produced by an Applied Biosystems automated DNA sequencer. DNA sequence is read by a computer based on the fluorescent dye color associated with each band (represented as a peak) on the gel.

taining all the components described above, but with no radioisotopes. In the "A" reaction, the primer carries a dye at one end that fluoresces green when struck with a laser. In the "C" reaction, the primer carries a dye that fluoresces blue. The "G" reaction's primer has a yellow dye on its end and the "T" reaction's primer carries a red dye.

Termination events in the "A" reaction tube result in fragments with a green dye at one end and a dideoxyadenosine at the other. Termination events in the "C" reaction result in the formation of fragments having a blue dye at one end and a dideoxycytidine at the other. Similar relationships hold in the "G" and "T" reactions. When incubation is complete, the four reactions are combined and electrophoresed in a single gel lane of an automated sequencer. A laser, shining at the bottom of the gel, excites the dyes on the DNA fragments, causing them to fluoresce as they pass. The instrument's optics system detects the fluorescent colors during elec-trophoresis and a computer then translates the order of colors into a base sequence (see Figure 3).

In the second approach to fluorescent DNA sequencing, the dye terminator method, dyes are attached to the dideoxynucleotides instead of to the primers. The DNA fragment in the sequencing reaction becomes dye-labeled when a dideoxynucleotide is incorporated. The dye terminator method uses a single reaction tube (rather than four) because each dideoxynucleotide is associated with a different dye. The cost and time for sequencing are therefore reduced, making this approach the preferred method used by most laboratories.

Manufacturers of DNA sequencing reagents now provide kits that contain all the components necessary for a sequencing reaction in a "master mix" format. For the dye terminator approach, a master mix can be purchased that contains DNA polymerase, the four nucleotides, buffer, magnesium, and the four dye-labeled dideoxynucleotides. The addition of template and primer to the master mix completes the reaction.

In addition to the widespread use of sequencing kits, other improvements have been made. Enhanced signal strength and improved sensitivity have been achieved through the development of stronger fluorescent dyes and the exploitation of heat-stable DNA polymerases that allow for repeated cycling of the sequencing reaction. see also Automated Sequencer; Cycle Sequencing; DNA; DNA Polymerases; Gel Electrophoresis; Human Genome Project; Nucleotide; Restriction Enzymes.

Frank H. Stephenson and Maria Cristina Abilock

Figure 3. Chromatogram produced by an Applied Biosystems automated DNA sequencer. DNA sequence is read by a computer based on the fluorescent dye color associated with each band (represented as a peak) on the gel.

a, a template a master copy

This automatic DNA sequencer performs "capillary electrophoresis." This system enables the electrophoresis to complete separations faster since higher voltages are used.

Bibliography

Bloom, Mark V., et al. Laboratory DNA Science: An Introduction to Recombinant DNA Techniques and Methods of Genome Analysis. San Francisco: Benjamin Cummings Publishing, 1995.

Maxam, Allan M., and Walter Gilbert. "A New Method for Sequencing DNA." Proceedings of the National Academy of Sciences 74 (1977): 560-564.

Sanger, Frederick, S. Nicklen, and Alan R. Coulson. "DNA Sequencing with Chain-terminating Inhibitors." Proceedings of the National Academy of Sciences 74 (1977): 5463-5467.

stem cells cells capable of differentiating into multiple other cell types

Severe Combined Immune Deficiency

The development of the immune system is a very complicated process. Stem cells in the bone marrow continually give rise to the white blood cells responsible for producing antibodies (B-lymphocytes), recognizing and destroying foreign cells (T-lymphocytes) and performing immune surveillance for cancer and foreign cells. Immunodeficiency results when the normal complex interactions of the immune system required to do this fail, resulting in susceptibility to infections or cancer.

Was this article helpful?

0 0
How To Bolster Your Immune System

How To Bolster Your Immune System

All Natural Immune Boosters Proven To Fight Infection, Disease And More. Discover A Natural, Safe Effective Way To Boost Your Immune System Using Ingredients From Your Kitchen Cupboard. The only common sense, no holds barred guide to hit the market today no gimmicks, no pills, just old fashioned common sense remedies to cure colds, influenza, viral infections and more.

Get My Free Audio Book


Post a comment