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3. biotech tools student (1)


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3. biotech tools student (1)

  1. 1. Genetic Biotechnology Tools
  2. 2. Restriction Endonucleases: p. 278-281 • Bacterial enzymes, known as restriction enzymes or molecular scissors that cut DNA into fragments at specific sites (recognition sites) • The recognition sites are usually palindromic and range from 4 to 8 nucleotides in length • Each RE has a specific sequence unique to it – Ex. EcoRI is a restriction enzyme that has the following recognition site: G A T G A A T T C A GA C T A C T T A A G T C T • The enzyme scans the DNA and stops when it reaches its recognition site, binding onto the DNA and disrupting the bonds between the A and G on both strands, producing fragments of DNA that have unpaired bases, forming sticky ends
  3. 3. • If ______________________was cut from another organism, using the _________________________, it could be joined to this fragment, producing __________________________________ • Some RE produce ____________________, but these are not as useful because it is ______________________________________ • Recognition sites of _______________ are usually more useful than 4 or 8 base pairs since a 4-base pairing would occur too often (cutting the gene in question into pieces) and an 8-base pairing wouldn’t occur often enough (including more than the gene in question) • The reason bacteria have RE is to cleave or cut foreign DNA that make it into the bacteria, rendering them useless • There are about ___________________, of which 200 are used by scientists Restriction Endonucleases: (cont’d)
  4. 4. Methylases: p. 281 • Enzymes in prokaryotes and eucaryotes • In prokaryotes they add a methyl gene to the recognition site of its DNA preventing the restriction enzymes from cutting its own DNA • Foreign DNA in the bacteria are not protected and will be cut • Ex. EcoRl methylase places a methyl group onto the second A in the EcoRl recognition site, thereby preventing EcoRl from cutting the DNA G A A T T C C T T A A G • Methylases are useful to molecular biologists when working with prokaryotes because they can use them to protect a gene fragment from being cleaved in an undesired location
  5. 5. DNA Ligase: p.281-282 • An enzyme that ____________________ broken by the restriction enzymes, allowing the fragments to be connected • The __________________________________between the bases of sticky ends, but the __________________________ between the nucleotides will _________________________
  6. 6. Gel Electrophoresis: p. 282-284 • After the fragments of DNA have been spliced, you want to isolate only the fragment with the specific gene you want • The different fragments can be separated using the chemical and physical properties of DNA: ______________________ __________________ • The __________________ is mixed with a ____________ so it can be seen and so it will sink to the bottom of the ____________________ • The gel is made of _______________ and is ____________________ of electrolytes and is attached to a power supply • After the DNA fragment solution is placed into each well, an electric charge is passed through the gel • Once the gel electrophoresis is complete, the DNA fragments in the gel are ______________________________ • The desired fragment can then be cut out of the gel and cleaned to reveal the DNA with the gene you want
  7. 7. Plasmids: p. 285-287 • Small circular pieces of DNA found in bacteria • They can be easily extracted from and inserted into other bacteria • Each plasmid possesses a 'copy number' that determines how many copies of that plasmid exist in the cell • Once a fragment of DNA that carries a specific gene is isolated , it can be inserted into a plasmid and then inserted into a bacterium • The newly formed plasmid (recombinant DNA) will then be replicated using the bacterium’s replicating materials and multiple copies of the protein coded for by the gene are made • If the gene is inserted into an operon of the plasmid, then the production of that protein can be regulated • Human insulin is now being made by bacteria this way
  8. 8. Transformation: p. 287-288 • The process of a __________________taking in a ________________ • Bacteria that readily take up foreign DNA are called ______________ – if they are not naturally competent, they can be _______________ under specific conditions • The host cell is put into a _________________________ • The Ca+2 are attracted to the _______________________________ of the membrane stabilizing them and the low __________________ __________________ making it more rigid • The vector plasmid is then inserted into the solution and the Ca+2 attract to the _____________________of the plasmid • The solution is then subjected to a _________________________ for 90 seconds, ___________________(__________________________) that sweeps the plasmid into the cell through the permanent pores • Then the cells are allowed to ________________________________
  9. 9. Selective Plating: p. 288 • Used to determine if the transformation of the bacteria worked – are the bacteria ‘_____________________’? • Usually the plasmids (vectors) being transformed not only contain the _______________________(new gene inserted), but also an _________________________________ • The bacteria are grown on plates of ________________________and other plates of ______________________________________ • The resulting colonies will determine if the plasmid is present
  10. 10. Questions: p.281, 282, 284, 287, 289