Session 7 Flashcards
Briefly explain DNA gel electrophoresis.
Fragments of DNA are placed in wells at the cathode of the gel.
A current is applied through the cel from the cathode to the anode.
DNA fragments move from the cathode to the anode because they are negatively charged.
Smaller fragments move further through the gel so the DNA fragments separate based on size.
What is required for DNA gel electrophoresis to occur?
A gel, buffer, power supply, stain/other detection method.
What are common features of restriction endonucleases?
Cut at a palindromic site on DNA.
Cut the DNA to leave sticky ends.
Explain DNA cloning.
DNA of interest is cut and joined into a vector (usually a plasmid).
Vector is introduced to host cells for replication to occur ( usually bacteria).
Clones containing the DNA of interest are identified and isolated.
Why is DNA cloning useful?
It can be used to produce useful proteins, e.g. Insulin.
Allows the function of a gene to be established.
For genetic screening.
May be used in gene therapy.
Briefly explain PCR.
Requires DNA, free nucleotides, buffer, Taq polymerase, primers.
First heated to 95 degrees to denature DNA.
Cooled to 50 degrees to allow primers to bind to DNA and prevent it from annealing.
Temperature raised to 72 degrees (optimum for Taq) to allow it to synthesis complimentary DNA to the DNA strands.
Cycle is repeated to exponentially increase the amount of DNA present.
On what basis are proteins separated in gel electrophoresis?
Size, shape or charge.
What is SDS-PAGE?
Method of separating proteins on the basis of size.
What is isoelectric focusing?
Method of separating proteins on the basis of charge.
Proteins migrate until they reach a pH equal to their pI.
What is 2-D electrophoresis?
A method to separate proteins based on pH (isoelectric focusing), then by size (SDS-PAGE). Useful for separating complex mixtures of proteins.
What is Western blotting used to detect?
Proteins
Briefly describe Western blotting.
A nitrocellulose replica of a gel electrophoretorgram is created.
Primary antibody is bound to the protein of interest on the nitrocellulose.
A second enzyme-linked antibody is bound to the primary antibody.
Allows detection of the protein of interest because it will present as an immunoblot on the nitrocellulose.
What enzymes are markers for liver damage and liver disease?
Aspartate transaminase (AST) and alanine transaminase (ALT).
What enzymes are markers for pancreatitis?
Amylase and lipase.
What are enzyme assays used for?
Used to diagnose disease for serum enzymes or metabolic disorders in tissues.
