Molecular Diagnosis Flashcards
What is special about the restriction sites where endonucleases act?
Palindromic
Describe the basis of DNA gel electrophoresis
When fragments of DNA are placed in a magnetic field, they will separate due to size and charge. The negative DNA will move towards an anode.
What are the 4 things required for gel electrophoresis?
Gel
Buffer
Power supply
Stain/detection
Give 3 reasons to do a gel electrophoresis
See the size of the DNA fragments
See mutations
See DNA variation
Clone DNA
Why are plasmids used in gene cloning?
Able to carry genes and replicate independently. They can transfer between bacteria and can carry genes for antibiotic resistance
Give 2 uses for human gene cloning
Proteins made externally
Find the functions of genes
Make probes
Advances in gene therapy
What is PCR used for?
Used to amplify target DNA
Outline the steps of PCR
DNA is heated
Strands separate
Primers attach to the strands
Taq polymerase copies DNA
What is a primer?
Short, one-stranded pieces of DNA that are complementary to the target DNA.
On what properties can proteins be separated by gel electrophoresis?
Size, shape, charge
What 4 things are required for protein gel electrophoresis?
Gel
Buffer
Power supply
Stain
Outline the steps of Isoelectric Focusing (IEF)
pH gradient established in a tube
Protein added and electric field applied
Proteins move until they reach a pH equal to their pK (ie. their pI)
What types of enzymes do bacteria produce which cut DNA?
Endonucleases
How are proteins identified?
Digest protein. Mass Spectrometry. List of peptide sizes. Use database
Outline the steps of ELISA?
Specific antibody binds to an antigen
An enzyme-linked antibody then binds to the specific antibody
A substrate is added and converted by the enzyme into a coloured product. The rate of colour transformation is equal to the amount of specific antibody
