03 - DLA Flashcards
Restriction endonucleases II
Recognize and cut DNA within a segment of DNA
What is a palindrome?
5’-3’ (left to right, top) matches the 5’-3’ (right to left, bottom)
DNA ligase?
Catalyses the formation of phosphodiester bond
Four general types of DNA polymerase?
DNA polymerase, Klenow fragment DNA polymerase, Reverse transcriptase, Taq polymerase
What is the most commonly used vector?
Prokaryotic plasmids
What are the three important features of cloning vectors?
- self replicate
- Multiple Cloning Site (MCS)
- Selectable marker (usually it is a gene for an enzyme that is not found in the host)
Cloning process?
Cleave the plasmid and the DNA with the same restriction endonuclease creating complementary sticky ends, DNA ligase joins the plasmid and DNA of interest = recombinant DNA molecule, the recombinant DNA is introduced into the bacterial host by transformation, bacteria are grown in the presence of antibiotics, the bacteria are lysed and the plasmids are cut with restriction endonuclease releasing many copies
The steps to PCR?
Denature the DNA at 95 into two strands, anneal primers to flanking regions at 45-65, chain growth moves in at 72, two new double stranded DNA molecules can be denature and the process is repeated
What happens with each cycle of PCR?
Each cycle doubles the amount of DNA
What do enzymes do?
Decrease the activation energy
What are apoenzymes?
Enzymes without the coenzyme
What are coenzymes?
Also called cofactors and they can be vitamins or minerals
Path to Trypsin?
The inactive trypsinogen is synthesized in the pancreas, released into the pancreas and then activated into trypsin once in the Duodenum
How can allosteric enzymes change their affinity?
Feed forward activation (binds to allosteric site which increases the affinity for substrate)
Feedback down-regulation (The enzyme knows it is done when something binds to the allosteric site)
What shifts the Km to the left?
feed forward activator
- shift to the left resulting in a smaller Km
