AP_Bio_-_dna_technology_notes_and_essay_ques - DNA...

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DNA Technology Restriction enzymes: nucleases which cut up foreign DNA in bacteria. There are a couple hundred that we know about, and a few dozen commercially used. Blunt Ends- cut straight through the DNA Sticky ends- cut in a zig-zag, thus there are overlapping ends. These are the most useful because two pieces of DNA cut with the same R.E. will have matching ends they can be combined. Names always include _____________ Cloning Vector: Any DNA molecule which can carry foreign DNA into a cell and replicate/ translate it. (Viruses and Plasmids) Plasmids: The use of a double marker is best. pBLU has both the ampR gene, and lacZ gene. Thus it can live on ampicillin treated plates, and will normally grow a blue colony on an X-gal treated plate. The Lac Z gene is known to have multiple restriction sites. Thus if you want to cut the gene from any strand of DNA and insert it into the lacZ gene there are several enzymes to choose from. The advantage is knowing that the bacteria have the engineered plasmid in it. If a colony can grow on amp, it has the plasmid, and if it remains white on the X-gal plate, then something must be inserted in the lacZ gene, which knocks it out. Once you know that there is something in the plasmid there are two ways to test for it. 1) If it translates into a specific protein or enzyme test for it. 2) use a Gene (DNA) “chip.” or a nucleic acid probe. Which includes finding a match between two pieces of denatured DNA. If you know the DNA that you are looking for, you can make sure that you have a complement to match any denatured DNA. (Fig 20.4) Inserting Eukaryotic genes into Prokaryotes. 1) Requires the correct promoters. “Expression Vector” has a prokaryotic promoter just upstream of the restriction site. Thus when the new DNA is inserted the promoter region is right in front of it.
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2) Requires removal of Introns! Thus you can not directly take DNA from the nucleus to insert into a vector. Use mRNA. It already has the Introns removed. Thus mRNA exposed to reverse transcriptase can produce DNA with the Introns removed. The new DNA can then be exposed to DNA polymerase to produce a double strand. This is called complementary DNA (cDNA) Because the mechanisms for Prokaryotes and Eukaryotes is different, it is better to grow recombinant DNA in a Eukaryote. Yeast is eukaryotic, and it has plasmids. Some plasmids can be grown in either Yeast or bacteria.
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This note was uploaded on 11/16/2011 for the course BIO 101 taught by Professor Martin during the Fall '08 term at Rutgers.

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AP_Bio_-_dna_technology_notes_and_essay_ques - DNA...

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