To make a genetically modified organism, three main components are required: the gene you want to transfer, the organism you want to put it into (target species), and a vector to carry the gene into the target species cells.
The steps in making a GMO are relatively straightforward, but can be technically challenging. The gene to be transfered (trans-gene) must be cut out and isolated from the original organism. This is usually done by restriction enzymes, which are like molecular scissors, that recognize specific sequences in the DNA and cut it at those places.
A restriction endonuclease is an enzyme that cuts strands of DNA at a specific point. It scans the DNA for a specific target sequence, and when it finds that target sequence it cleaves the DNA. Target sequences are relatively short. For instance, the common restriction enzyme EcoR1 only has a 6 basepair target sequence. To date, thousands of restriction endonucleases (RE) have been isolated, mostly from bacteria. Bacteria use these enzymes as a defense mechanism because the can recognize and cleave foreign (virus) DNA.
Restriction endonucleases can cut double-stranded DNA in a few different ways. Sometimes it cuts both strands at the same position, which causes blunt ends. Other times it cuts each strand at a different point causing overhangs to occur. An overhang means that one strand is longer than the other, and sometimes people refer to this as having sticky ends. See the diagrams below for examples of blunt and sticky ends.
Below are two animations that illustrate how restriction endonucleases cleave DNA.
|There is an online video of how restriction endonucleases cut DNA by McGraw-Hill Higher Education: http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf|
“Restriction Endonucleases.” Biology 7th Edition. 2005. McGraw-Hill Higher Education. 8 July 2009. http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf
|The Dolan DNA Learning Center also has an online animation about restriction endonucleases cut DNA: http://www.dnalc.org/ddnalc/resources/restriction.html.|
“DNA Restriction.” DNA Learning Center. Cold Spring Harbor Laboratory. 12 December 2008. http://www.dnalc.org/ddnalc/resources/restriction.html
To test for a mutation using restriction endonucleases, the segment of the patient’s DNA containing the target sequence is mixed with restriction enzyme. If the target sequence is present, the enzyme is able to cut the DNA (this in turn means that the patient is carrying the HNPCC mutation). If the target sequence is not present, the enzyme will not be able to cut the DNA (meaning the patient does not carry the HNPCC mutation).
Whether the DNA has been cut or not can be determined through a technique called gel electrophoresis.