DNA Technology & Genomics

DNA Technology & Genomics

Chpt. 20The use of recombinant DNA

technology has already impacted your life in ways that you might

not expect.

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How has recombinant DNA How has recombinant DNA affected your life?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.

• 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 has recombinant DNA affected life?

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"

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Swiss American

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WHAT… is a RESTRICTION ENZYME??



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

phage

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

G A 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 Restriction EnzymesEnzymes::restrictionrestriction sitesite - -area where DNA area where DNA

is cut is cut usually only 4 usually only 4 - 6 bp’s long- 6 bp’s long

-it is a palindrome-it is a palindrome-some DNA molecules -some DNA molecules have have manymany of these of these specific sites… some specific sites… some have have nonenone

Naming the R.E.’sNaming the R.E.’s --ex. BamH Iex. BamH I

B = genus B = genus BBacillusacillusam am = species= species amamyloliquefaciensyloliquefaciensH = strain (kind)H = strain (kind)I = orderI = order order order inwhich inwhich this this R.E. from this species R.E. from this species

of bacterium was of bacterium was isolatedisolated

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




2) Using ligase, combine “gene of interest” into plasmid

RECOMBINANT!




3) Put RECOMBINANT plasmid back onto bacterial cell

TRANSFORMATION




4) Grow transformed host cell in culture~ forms many cloned genes of interest




5) Protein harvested




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!!


Many eukaryotic genes get excised, only one of those carries the gene of interest

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

add ligase



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

Master plate

Filter

Solutioncontainingprobe

Filter liftedand flipped over

Radioactivesingle-strandedDNA

ProbeDNA

Gene ofinterest

Single-strandedDNA from cell

Hybridizationon filter

Master plate

Coloniescontaininggene ofinterest

A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter.

The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography).

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 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.


Master plate

Filter




ProbeDNA

Gene ofinterest



Master plate







Master plate

Filter



ProbeDNA

Gene ofinterest



Master plate






Single stranded, radioactive probe added


Master plate

Filter




ProbeDNA

Gene ofinterest



Master plate







Master plate

Filter




ProbeDNA

Gene ofinterest



Master plate






Colonies can now be isolated


Master plate

Filter




ProbeDNA

Gene ofinterest



Master plate






So, how do we store these once we find

them?

Genomic Libraries



http://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?

PCR - Polymerase Chain ReactionA DNA sample, target DNA, is obtained (crime scene?)

PCR - Polymerase Chain Reaction

DNA denatures by heating at 98oC for 5 minutes


The sample is cooled to 60oC / DNA PRIMERS are bonded to each strand


Free nucleotides, and the enzyme DNA polymerase are added / complementary strands synthesized!


There are now two copies of the original strand


Process repeated… now four strands


LOTS of strands in very little time


They key… heat-stable DNA polymerase

- this comes from thermophillic bacteria…


They key… heat-stable DNA polymerase

- this comes from thermo bacteria…

This does not denature in high heat. Benefit - keep heating the test tube up after each round.

PCR - Polymerase Chain Reactionhttp://www.youtube.com/watch?v=ZmqqRPISg0g

http://www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html

"Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents and a source of heat. The DNA sample that one wishes to copy can be pure, or it can be a minute part of an extremely complex mixture of biological materials. The DNA may come from a hospital tissue specimen, from a single human hair, from a drop of dried blood at the scene of a crime, from the tissues of a mummified brain or from a 40,000-year-old wooly mammoth frozen in a glacier."

http://www.youtube.com/watch?v=ZmqqRPISg0g




Dr. Kary Mullis awarded Nobel Prize 1993 for discovery of PCR in 1983… Hobby was a Freshman @ Miami U. that day he

invented it!! Probably, I had no idea of this happening…Hmmm, what will happen when YOU are Freshman that you wont realize

until later??

DNA Fingerprinting


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• Murder. A body lies on the sidewalk near an alley. It looks like the victim fought off her attacker, leaving blood and tissue under her fingernails. There is also a pool of blood on the sidewalk next to the victim. Two suspects have been picked up just a block away with fresh scratches on them. Could one of them be the killer?

• The lead detective on this case will be using DNA fingerprinting to determine if one of the suspect's DNA matches DNA found at the crime scene.

DNA Fingerprinting




Lets Practice …

DNA Fingerprintinghttp://www.pbs.org/wgbh/nova/sheppard/analyze.html



GEL ELECROPHORESIS



http://www.npr.org/templates/player/mediaPlayer.html?action=1&t=1&islist=false&id=5534279&m=5534280

GEL ELECROPHORESIS



GEL ELECROPHORESIS



How will we use restriction enzymes?



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RFLP ~ Restriction Fragment Length Polymorphisms

• Method for detecting minor differences in DNA structure between individuals.


• individuals inherently have differences in their fragment lengths b/c of individual insertions, deletions etc.


• RFLP’s can be used to genetically tell individuals apart.

• RFLP’s can also show the genetic relationship between individuals, because children inherit genetic elements from their parents.


1. Digest DNA with restrictive enzymes.2. Separate pieces by Gel Electrophoresis3. Identify sequences with identifying probes.

• converts a GAG codon (for Glu) to a GTG codon for Val

• abolishes a sequence CTGAGG, recognized and cut by one of the restriction enzymes.




• converts a GAG codon (for Glu) to a GTG codon for Val

• abolishes a sequence CTGAGG, recognized and cut by one of the restriction enzymes.




Southern Blotting• Makes RFLP fragments

that are different IDENTIFYABLE.

• With just the gel electro… you would not really be able to identify where the SPECIFIC difference was!

Southern Blotting

• Move fragments from the gel to a nylon sheet.

• Make a DNA fragment that is complementary to the “area of interest.

Southern Blotting

• Put a radioactive PROBE on the DNA fragment of interest

Southern Blotting

• Wash the membrane “paper”

Southern Blotting•Only your “area of interest” shows up well!Only your “area of interest” shows up well!

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