Genetic Lecture 7 Flashcards
DNA Technology The Genetic Revolution
How humans can interfere and make changes to DNA
Plasmid (a.k.a. Vector)
A small, circular piece of DNA that is replicated in a
bacteria.
Gene of interest
A gene that encodes a protein that could be useful.
Foreign DNA
The DNA we want to isolate and make changes if we need to, and to make more protein.
Recombinant DNA
Molecules of DNA from two different sources that are
combined together in the lab. It’s called this because you are recombining two things that very often aren’t possible in nature.
Recombinant Bacterium
A bacterium host with a recombinant DNA plasmid.E. coli is the bacteria that is used most often in these experiments.
When did cloning a gene first happen?
In 1975, seemed unnatural and a lot people talked about the ethics of it.
Steps of cloning a gene?
Gene inserted into a plasmid. Plasmid put into a bacterial cell. Host cells grown in culture to form a clone of cells that contain the cloned gene of interest. Basic research and various applications.
Where is the bacteria typically grown in?
A flask that has a medium good for the bacteria. These bacteria have a new generation every 20 minutes.
Two uses of cloning a gene?
Isolating the placid and taking out the genes of interest. This can be used for gene for pest resistance inserted into plants, or gene used to alter bacteria for cleaning up toxic waste. Another way is to harvest the protein expressed from the gene of interest. This can be used as human growth hormone treats stunted growth or protein dissolves blood clots in heart attack therapy (mostly in pharmaceutical ways).
Restriction Enzymes
Cut DNA into fragments based upon recognizing a
specific sequence of nucleotides.
Restriction site
Read same in both directions (antiparallel). Where the divide occurs. It can be recognized by some enzymes that will be able to cut right at the specific spot.
Sticky Ends
Single-stranded ends left hanging after the DNA is cut. The free base pairs can hydrogen bond with
complementary sticky ends.
Steps for Making Recombinant DNA with Restriction
Enzymes?
Restriction enzyme cuts the sugar-phosphate backbones at each arrow. DNA fragment from another source is added. The fragment from different DNA molecule cut by the same restriction enzyme. Base pairing of sticky ends produces various combinations. DNA ligase seals the strands.
DNA ligase
Catalyzes the formation of the covalent bonds
that close up the sugar- phosphate backbones.
Plasmid maps
Are graphical representation of plasmids, that show the locations of major identifiable landmarks on DNA like restriction enzyme sites, gene of interest, plasmid name and length etc. Circle of DNA.
Amplicillain resistance (antibiotic)
So when you introduce the bacteria with this plasmid in it, it will make it resistant to ampicillin. By adding ampicillin in your medium to kill off everything else that doesn’t have your plasmid.
Multi cloning site
Different restriction sites that have been put there by people. To give flexibility in the lab to put your piece of DNA in. The number next to the enzyme is its position.
The first restriction enzyme discovered?
EcoR1
LacZ arrow
If they added a piece of DNA in the multi cloning sites, it will stop LacZ from working. Gives a chemical that turns blue when lacZ is working. Want it to not be blue, because it means the bonds are broken.
DNA Gel Electrophoresis
To visualize and purify the DNA. Separates DNA of different sizes.
Gel electrophoresis steps?
In the tube a bit of restriction enzyme, plasmid, and DNA are put in the DNA that you want to insert into the plasmid, close that up and put into the incubator for a few hours. Then mix with your bacteria, then go to the plate and see if you get blue or white colonies.
What is the jelly in gel electrophoresis made of?
Agarose gel
What are the wells?
Where the DNA is placed in, it’s made by the comb.
What is in the tube?
A mixture of DNA molecules of different sizes.
The charge of DNA?
Negatively because of the phosphate groups. The DNA is placed on the negative side and then moves to the positive electrodes.
Cathode
Negative end
Anode
Positive end
What molecules move faster through the gel?
The shorter molecules.
What does higher density gel do?
Used to separate smaller molecules. All the molecules will move slower because there is more molecules stoping them (that they have to move through).
What happens if the gel isn’t dense enough?
The shorter ones will just go right through it.
How can people take just one piece of the DNA out of the gel?
By cutting it out with a blade.
PCR (Polymerase Chain Reaction)
To copy (amplify) a DNA sequence. It’s very sensitive and uses enzymes from nature to our advantage to amplify DNA. Works with a target sequence. A lot of ideas came from DNA’s own replication.
Taq polymerase
An unusual heat- stable DNA polymerase from Thermus aquaticus. With this they could isolate the protein with DNA polymerase from the organism to use in the PCR reactions.
Steps of PCR?
Denaturation (95 degrees C) separates the DNA strands. Annealing (60 degrees C) the DNA primers are added (use them instead of RNA because they are more robust and won’t get degraded). Extension (72 degrees C) where new nucleotides can be added on the 3’ end.
How many does each cycle produce in PCR?
Cycle 1 is 2 molecules
Cycle 2 is 4 molecules
Cycle 3 is 2 of the 8 molecules match the target sequence and are the right length.
The DNA amount doubles at each cycle.
Results of PCR?
After 3 cycles, two molecules match the target sequence exactly. After 30 more cycles, over 1 billion molecules match the target sequence.
