4/26/2010 BIOTECHNOLOGY

Introduction Biotechnology: Genetic Engineering: any technological application that uses living systems and organisms to make useful products Molecular biologists use living biological organisms or biological molecules as tools Using these tools, molecular biologists can cut, join and replicate DNA! Genetic Engineering: Modifying the characteristics of an organism by manipulating its DNA

Introduction DNA is the genetic material of all living things and all organisms use the same genetic code (same bases: A,T,C,G) Because of this, genes from one organism can be transcribed and translated when put into a different kind of organism! For example, human and other genes are routinely put into bacteria in order to synthesize products for medical treatment and commercial use Examples include: human insulin, human growth hormone, and vaccines

Recombinant DNA

Recombinant DNA Recombinant DNA: a fragment of DNA composed of sequences originating from at least 2 different sources Individuals that receive genes from other species are considered transgenic organisms Why would we want to give an organism DNA from another species? ex: Agricultural Benefits Imagine a plant that has high crop yields or resistance to disease or crop longevity If we can isolate the genes responsible for these traits, we could insert them into many types of plants so that they too would express these desirable traits

Vectors

Vectors Vectors are a source of DNA that is used as a vehicle to transport foreign genes into another cell A vector must be capable of self replicating inside a cell Examples of vectors include bacterial plasmids and viruses

Vectors Plasmids Small circular pieces of DNA that can exit and enter bacterial cells Naturally exist in the cytoplasm of many types of bacteria Bacterial cells are capable of taking up DNA from their environment. This process is called transformation Plasmids are independent of the chromosome of bacterial cells Often carry genes for resistance to chemicals, herbicides, antibiotics

Vectors Molecular biologists can cut open a plasmid, insert a desired gene, and reseal it. Then the plasmid can be taken up by a bacterial cell (lab procedures such as exposure to chemicals and heat can be used to make bacteria more permeable to plasmids) Once inside the cell, the bacterial machinery will then be able to replicate, transcribe and translate the plasmid DNA (with the new, foreign gene) The gene is now cloned

Vectors Example, human insulin is produced this way At one time diabetics depended on insulin extracted from other animals but this could cause allergic reactions Now the human gene for insulin can be put into a plasmid

Vectors Viruses Contain genetic material (DNA or RNA) but are not alive This is because they need to be inside a host cell to be able to reproduce (replicate their DNA) They use the host cell’s machinery to replicate their own DNA to make new viruses! Viruses can also be a vector They can accept larger amounts of DNA than plasmids A foreign gene can be inserted into a virus and the virus is then allowed to “infect” a host cell Once inside, the host cell’s machinery (i.e. polymerase, ribosomes) will be able to replicate, transcribe and translate the viral DNA, including the inserted gene (gene is now cloned)

Restriction Endonucleases

Restriction Endonucleases Also known as Restriction Enzymes are “molecular scissors” that can cleave (cut) double stranded DNA at a specific base pair sequence Where do these enzymes come from? Restriction enzymes were discovered in bacteria (use them as a defense mechanism to cut up the DNA of invading viruses known as bacteriophages)

Restriction enzymes act as an immune system in the bacterium Restriction enzymes act as an immune system in the bacterium. When a bacteriophage (a virus) tries to inject its DNA into the bacteria, restriction enzymes cut up the bacteriophage DNA into many fragments – thus, preventing it from doing any harm to the bacterium.

Restriction Endonucleases Restriction endonucleases must be able to distinguish between foreign DNA and their own DNA otherwise they would cut up their own DNA!! METHYLASES are enzymes that modify a restriction site by adding a methyl group thus preventing the restriction endonuclease from cutting it. This prevents the cell from cutting its own DNA. methylation Foreign DNA in a cell will not be methylated and therefore may be broken down by the enzymes

Restriction Endonucleases Hundreds of different restriction enzymes have been isolated. Each one cuts DNA at a specific sequence Recognition Site: the specific sequence where the restriction enzyme makes its cut.

Restriction Endonucleases both strands have the same sequence (when read in the 5’ to 3’ direction) For example, the restriction enzyme called EcoRI always cuts DNA at GAATTC. The cut is made in a very specific way as shown below 5’ 3’ 3’ 5’

Did you know? Where do they come up with these strange enzyme names? Name of enzyme is based on the bacteria they come from Example: EcoRI E = Escherichia (genus name) CO = coli (species name) R = strain I = first endonuclease isolated from E. Coli

Restriction Endonucleases Example 1: consider the following DNA sequence. Let’s cut this sequence using the enzyme EcoRI

STICKY ENDS

Restriction Endonucleases Example 2: Consider the following DNA sequence. Let’s cut this sequence with the enzyme SmaI BLUNT ENDS

Restriction Endonucleases STICKY ENDS: both fragments of the newly cleaved DNA have DNA nucleotides lacking complimentary bases. BLUNT ENDS: The ends of the DNA fragments are fully paired.

Restriction Endonucleases Restriction enzymes that produce sticky ends are more useful biological tool (than blunt ends) because these DNA fragments can easily be joined to other DNA sticky end fragments made by the same restriction enzyme. Can easily be used to create recombinant DNA

Restriction Endonucleases

Restriction Endonucleases The same restriction endonuclease (enzyme) must be used on both DNA otherwise the 2 DNAs won’t be able to bind together – there needs to be complimentary base pairing (the sticky ends must match up) Two complimentary strands will naturally anneal and form hydrogen bonds between the base pairs but an enzyme is required to form the phosphodiester backbone of the DNA

Restriction Endonucleases DNA Ligase is used to join the cut fragments of DNA together via a dehydration synthesis (condensation) reaction. Phosphodiester bonds are reformed between adjacent bases T4 DNA Ligase – is specific enzyme that is especially useful for joining blunt ends together

Restriction Endonucleases Recognition Sites for restriction enzymes are usually about 4 to 8 nucleotides long This results in a low frequency of cuts along a piece of DNA For example, if the recognition site was 6 nucleotides long, then the enzyme would cut once every 4096 nucleotides (46 = 4096) If the site was only 2 nucleotides long, then the enzyme would cut once every 16 nucleotides (42= 16) This would be a problem for a molecular biologist because the DNA to be too fragmented Furthermore, the gene they are trying to remove may get cut up!

Animation http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

Real Life Application: INSULIN Insulin is a hormone that regulates blood sugar levels by converting excess glucose into glycogen for long term storage. Diabetics do not make sufficient amounts of insulin. Diabetics may be required to take insulin injections.

Real Life Application: INSULIN Using restriction enzymes, the gene for synthesizing insulin is cleaved out of DNA and inserted into the DNA plasmid of a non harmful bacteria. The plasmid containing the human insulin gene is then put into a bacteria DNA ligase is added to the bacteria. The bacteria is also given the necessary amino acids. The bacteria can then express the gene as it transcribes and translates the gene to make human insulin protein The insulin that can be collected from the bacterial cell and administered to diabetics