Genetic Biotechnology Tools

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

• 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)

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

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 _________________________

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

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

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 ________________________________

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

Questions: p.281, 282, 284, 287, 289