DNA Technology & Genomics

DNA Technology & Genomics Chpt. 20 The use of recombinant DNA technology has already impacted your life in ways that you might not expect.

How has recombinant DNA affected your life? • The popular stonewashed denim look is actually achieved by treating denim with cellulase enzymes which partially break down the cotton fibers of the denim. This gives stonewashed jeans their soft texture when compared to regular jeans. Many different cellulase enzymes have been discovered in microorganisms. Recombinant DNA technology is used to clone the genes encoding these enzymes so that large quantities of enzyme can be produced and sold to textile manufacturers.

How has recombinant DNA affected life? • Insulin from an animal source, such as pigs has traditionally been used to treat diabetics. Insulin from these animals is similar but not identical to human insulin. Because of this, many patients develop allergic reactions. Recombinant DNA tools have enabled researchers to locate and clone the gene for human insulin, ensuring an ample supply of insulin that does not cause allergic reactions.

How do we do this? • The Nobel Prize in Physiology or Medicine 1978. • "for the discovery of restriction enzymes and their application to problems of molecular genetics"

How do we do this? • The Nobel Prize in Physiology or Medicine 1978. • "for the discovery of restriction enzymes and their application to problems of molecular genetics" Swiss American American

WHAT… is a RESTRICTION ENZYME??

phage • Bacteria are under constant attack by bacteriophages (viruses). phage phage phage phage

To protect themselves, many types of bacteria have developed a method to chop up any foreign DNA, such as that from attacking phages.

These bacteria create endonucleases--(restriction enzymes) • an enzyme that cuts DNA that enters the bacterium via. the phage

. The endonucleases are termed "restriction enzymes" because they restrict the infection of bacteria phages.

restriction enzymes do not attack their own bacterial DNA b/c they have a gene that prevents the r.e. from attaching to their chromosomal DNA

Restriction Enzymes • Restriction enzymes are enzymes isolated from bacteria that recognize specific sequences in DNA • and then cut the DNA to produce fragments called restriction fragments.

Restriction Enzymes • Different restriction enzymes recognize and cut different sequences of DNA.

Restriction Enzymes GA A T T C C T T A A G

So, how can we use these?

Restriction Enzymes • If we are able to locate a eukaryotic “gene of interest”. ex. Insulin gene

Restriction Enzymes • And that gene of interest is “downstream” from a restriction site…

Restriction Enzymes • We are able to cut the gene of interest out of a eukaryotic genome… and “attach” it to the prokaryotic genome

Restriction Enzymes “glue” together w/ LIGASE

Restriction Enzymes • The “recombined” genome of the prokaryote will now be placed BACK INTO a prokaryote

Restriction Enzymes • The bacteria will produce insulin right along with the replicating bacteria!!

Restriction Enzymes: restrictionsite - area where DNA is cut usually only 4 - 6 bp’s long -it is a palindrome -some DNA molecules have many of these specific sites… some have none

Naming the R.E.’s - ex. BamH I B = genus Bacillus am = speciesamyloliquefaciens H = strain (kind) I = order order inwhich this R.E. from this species of bacterium was isolated

So, how does this work?

Gene cloning via. a Bacteria 1) Get a plasmid/ find your “gene of interest” in the eukaryote~ cut both with restriction endonuclease

Gene cloning via. a Bacteria 2) Using ligase, combine “gene of interest” into plasmid RECOMBINANT!

Gene cloning via. a Bacteria 3) Put RECOMBINANT plasmid back onto bacterial cell TRANSFORMATION

Gene cloning via. a Bacteria 4) Grow transformed host cell in culture~ forms many cloned genes of interest

Gene cloning via. a Bacteria 5) Protein harvested

Gene cloning via. a Bacteria 6) Gene inserted into other organisms

Gene cloning via. a Bacteria~ a bit more complex lacZ gene codes for an enzyme that hydrolyzes lactose & X-gal (a lactose mimic) when X-gal (a lactose mimic) is hydrolyzed , a blue product is formed Notice there is a lacZ gene and right in the middle is the restriction site!!

Gene cloning via. a Bacteria~ a bit more complex Many eukaryotic genes get excised, only one of those carries the gene of interest

Gene cloning via. a Bacteria~ a bit more complex add ligase The thing is…many other recombinants will form~ think about how many sites the human DNA was cut. How many carry the gene of interest… ONE

Gene cloning via. a Bacteria The recombinant plasmids… are mixed with bacteria that has a mutation in their lacZ gene.

Ampicillin w/ kill any bacteria without the resistance gene with lacZ gene intact -> X-gal will cause the bacteria to turn blue

So, how do you find the colony with the gene of interest?

Nucleic Acid Hybridization Colonies containing gene of interest Solution containing probe Radioactive single-stranded DNA Master plate Probe DNA Gene of interest Master plate Filter lifted and flipped over Filter Hybridization on filter Single-stranded DNA from cell After the developed film is flipped over, the reference marks on the film and master plate are aligned to locate colonies carrying the gene of interest. The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography). A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter. The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter.

Nucleic Acid Hybridization Colonies containing gene of interest Solution containing probe Radioactive single-stranded DNA Master plate Probe DNA Gene of interest Master plate Filter Hybridization on filter Single-stranded DNA from cell After the developed film is flipped over, the reference marks on the film and master plate are aligned to locate colonies carrying the gene of interest. The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography). Single stranded, radioactive probe added A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter. The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter.

Nucleic Acid Hybridization Colonies containing gene of interest Solution containing probe Radioactive single-stranded DNA Master plate Probe DNA Gene of interest Master plate Filter lifted and flipped over Filter Hybridization on filter Single-stranded DNA from cell Colonies can now be isolated After the developed film is flipped over, the reference marks on the film and master plate are aligned to locate colonies carrying the gene of interest. The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography). A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter. The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter.

So, how do we store these once we find them?

Genomic Libraries

http://plantandsoil.unl.edu/croptechnology2005/crop_tech/animationOut.cgi?anim_name=genecloning.swfhttp://plantandsoil.unl.edu/croptechnology2005/crop_tech/animationOut.cgi?anim_name=genecloning.swf

Would this take a LONG time???

What if I only have a little sample?

DNA Technology & Genomics