DNA SequencingDNA sequencing is the process used to determine the sequence of DNA, made up of the four nucleotides. If you have family members suffering from hereditary disease, it is advisable to get tested yourself. There are many known diseases which can be determined by DNA sequencing. Furthermore, you can use this information to seek effective treatment, thus preventing the disease from affecting you. DNA sequencing can also be used for maternity or paternity tests, should the father or mother of a child be in question. How was the human genome sequenced?
The most common way to sequence DNA is to take advantage of three things: the way DNA replicates, Polymerase Chain Reaction (PCR), and electrophoresis. It is common knowledge that DNA is a double helical structure, made up of two complement strands of nucleotide bases. DNA replication relies heavily on the base pairing. Among the nucleotides, adenine pairs with thymine and cytosine pairs with guanine. This complement base pairing means that if one side of DNA is known, the other is known as well. PCR is used to artificially replicate DNA. DNA from a specimen is extracted and then heated to make the two strands separate. These strands are exposed to various chemicals including the four bases: sugars, phosphates, and the enzyme DNA polyermase. DNA polymerase essentially uses the other chemicals to synthesize the other side of DNA. The PCR procedure is modified when used in DNA sequencing. A single strand of DNA of an unknown sequence is used as a template. The new strand being synthesized is called the primer. The template is exposed to four different chemical mixtures. They all contain the four nucleotide bases, sugars, phosphates, DNA polymerase, and a variable chemical. This variable chemical is either ddATP, ddCTP, ddGTP, ddTTP. The chemicals mentioned are the four bases with a methyl group attached to it. DNA is synthesized as it would normally, with one difference. When the DNA polymerase uses a base attached to a methyl, it cannot sequence the DNA any further. What results are many fragments of DNA of different sizes. For example, the sequence may be GATTACA and the complement is ACATTAG. Let’s say the methyl base used is ddATP (represented by A’). The resulting fragments are A’, ACA’, and ACATTA’. The last step uses gel electrophoresis. Gel electrophoresis implements electricity to move fragments of DNA within some medium, usually an agarose gel. The DNA is placed in little wells on one side of the gel. DNA has a negative charge and when an electrical current is run through, the DNA moves toward the anode. Smaller fragments of DNA can move through the gel more easily, while larger fragments stay closer to the well. The DNA in the four different chemical mixtures are placed in their respective wells. Electrophoresis is performed. The end result is a gel with lines at different lengths of the gel. These lines can be read directly off the gel to show the DNA sequence. Usually, an X-ray photograph is taken for easier reading. The X-ray is analyzed by a computer to give an accurate description of the sequence. Additional Resources and LinksDOEgenomes.org--genome programs of the US Department of Energy National Human Genome Research Institute - Home Automatic DNA and Genome Sequencing (PDF fil, requires Adobe Acrobat Reader) ReferencesCampbell, N., & Reece, J. (2002). Biology: Sixth edition. San Francisco: Benjamin Cummings. Facts about genome sequencing . (2004, September 15). Retrieved September 17, 2004, from Human Genome Project Web site: http://www.ornl.gov/sci/techresources/ |