Bio 205, Chapter 9

What do you need to make a useful gene product?

- Restriction enzymes - Vectors (plasmids) - PCR - Transformation - Selection of gene of interest

Where do restriction enzymes come from? What's their role in nature? What's their role in a lab?

- Restriction enzymes come from many bacteria, where they are isolated to use in a lab to make rDNA (recombinant DNA) - In nature, restriction enzymes function as part of a bacteria's immune system. When a bacteriophage infects a bacterium, restriction enzymes may protect the bacterium by hydrolyzing phage DNA.

Restriction Enzymes

1. Also called restriction endonucleases 2. Destroy bacteriophage DNA in bacterial cells --> Methylated cytosines in bacteria protect their own DNA from digestion 2. Most create stagged cuts known as sticky ends

Describe a restriction enzyme's role in making rDNA (recombinant DNA)

1. Restriction enzyme cuts double-stranded DNA at its particular recognition sites 2. These cuts produce a DNA fragment with two sticky ends 3. DNA from another source like a plasmid is cut with the same restriction enzyme 4. When two such fragments of DNA are cut by the same restriction enzyme, they can come together by base-pairing. 5. The joined fragments will usually form either a linear or a circular molecule. 6.The enzyme DNA ligase is used to unite the backbones of the two DNA fragments, producing a molecule of recombinant DNA.

Be able to explain how you clone a gene of interest. That includes PCR, restriction digests, sticky ends, transformation, antibiotic selection, and blue/white screening. Whew!

1. Take a vector like a plasmid and isolate it by using a restriction enzyme to cut its DNA. 2. Take the DNA containing the gene of interest and cleave it using the same restriction enzyme you used to isolate your vector. 3. Because the same restriction enzyme was used,

Shuttle vector (pUC19)

Plasmids that are capable of existing in several different species.

Clone

Population of genetically identical cells arising from one cell; each carries the vector

Vector

Self-replicating DNA molecule used to transport foreign DNA into a cell

Blunt ends vs. Sticky ends

Sticky ends are more useful in recombinant DNA because they can be used to join two different pieces of DNA that were cut by the same restriction enzyme. - The sticky ends "stick" to stretches of single-stranded DNA by complementary base-pairing.

Recombinant DNA technology

The insertion or modification of genes to produce desired proteins

Biotechnology

The use of microorganisms, cells, or cell components to make a product - Food, antibiotics, vitamins, enzymes