Gene Splicing

Living organism and involves the production and use of recombinant DNA. Recombinant DNA (rDNA) is DNA that has been genetically altered through a process known as gene splicing. In gene splicing, a DNA strand is cut in half lengthwise and joined with a strand from another organism or even another species. DNA is cut into shorter fragments through the use of restriction enzymes – restriction enzymes are a group of enzymes that catalyze the cleavage of DNA at specific sites to produce distinct fragments. The ends of these fragments are attracted to complementary ends on other DNA fragments and will seek those out in the target DNA.

By looking at the size of the fragment created by a restriction enzyme, investigators can determine whether the gene has the proper genetic code. Gene splicing is the first step in two other genetic techniques – gene transfer, or incorporation of new DNA into an organism’s cells and gene therapy. In this lab, you will be taking a fragment of DNA from one organism and splicing it into another organism.

A. Go to: http://www. mhhe. com/biosci/genbio/virtual_labs/BL_22/BL_22. html

B. Read question material carefully.

C. Review diagrams under “diagrams” icon at bottom of webpage.

D. Follow directions and complete at least 5 transgenic organisms (use the “help” icon) if needed.

E. Answer questions below – please provide enough details to thoroughly answer each question:

1. DNA is made up of two separate strands of base sequences. The same sequence is found on both strands, but running in opposite directions. What word describes this characteristic? anti-parallel

2. What does the term “sticky ends” refer to in gene splicing? “Sticky ends” occur when a plasmid is cut with a restriction enzyme and is transformed into a linear molecule with a specific base sequence that can bind by base pairing with a complementary sequence. These “sticky ends” form hydrogen bonds with one another .

3. What is a plasmid? How is a plasmid used in gene splicing? A plasmid is an extra-chromosomal double stranded DNA molecule that can replicate independently from chromosomal DNA. It is usually found in bacteria. It is used in gene splicing as a vector. A piece of DNA can be inserted into a plasmid if both the circular plasmid and the source of DNA have recognition sites for the same restriction endonuclease. The plasmid and the DNA are combined at their complementary ends and are recombined by DNA Ligase to create a new plasmid that contains foreign DNA.

4. What types of vectors are used to carry DNA from one species into the DNA of another species? Give examples. Most plasmid vectors are from E. coli and yeasts and contain little more than the essential nucleotide sequences required for their use in DNA cloning: a replication origin, a drug-resistance gene, and a region in which exogenous DNA fragments can be inserted.

5. What is a “transgenic organism”? Give examples. A living organism that has had a foreign gene added to it by means of genetic engineering. Examples are medical applications; (gene therapy, bacteria modification to produce insulin for diabetics), production of agriculturally advantaged plants; (insect resistant, incresed nutrition or improved shelflife), and animals used as experimental models: (fruit flies used as human disease models, fish production increases by growth hormones).

6. Why is it essential that the same restriction enzyme be used to cleave (cut) the DNA of both organisms used to create a transgenic organism? If two different DNA molecules are cut with the same restriction enzyme, both will produce fragments with the same complementary sticky ends, making it possible for DNA chimeras to form. So, if both vector DNA and donor DNA are cut with EcoRI, the sticky ends of the vector can bond to the sticky ends of a donor fragment when the two are mixed.

7. Are there any factors other than technical ones that might slow — or even prevent — the use of bioengineering? There are some concerns as to whether the use of bioengineered foods is safe. People are worried that genes inserted into food producing crops, or the proteins they make may have an effect on the people eating them. Also the use of antibiotic resistance marker genes in crops could pose a public health concern.

Graph of base pairs against the migrated distance of the fragments of DNA Conclusion: The fragments that moved more across the gel were the ones that had less base pairs because their mass is smaller and they can move across the gel …

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