Lecture 14- Introduction To Molecular Diagnosis Flashcards
How could you analyse DNA at the nucleotide level?
DNA sequencing
Analysis of DNA at gene level?
Souther hybidisation
Microarray
PCR variations
Analysis of DNA at chromosome level?
Karyotyping
FISH
What are endonucleases?
Enzymes that chop specific DNA sequences- used as restriction enzymes (molecular scissors)
Recognise and degrade foreign DNA
How odes gel electrophoresis work?
DNA negatively charged due to phosphate groups. Travels from negative to positive electrode. Fragments separated according to size. Can use ethicist bromide, fluoresce under UV light to see bands
Why use restriction analysis?
To investigate size of DNA fragments
To investigate mutations
Investigate DNA variation
To clone DNA
What are plasmids?
Circular bacterial DNA. Can be used as vectors
Why clone human genes?
To make useful proteins
To find out what genes do
Genetic screening
Gene therapy eg cystic fibrosis, replace defective gene with a cloned normal gene
Polymerase chain reaction (PCR)
Uses Taq polymerase which is stable at high temperatures
Function of primers in PCR?
They define what part of the DNA to be amplified
How does PCR nbraodly work?
DNA heated to denature it. Primers then bind and DNA is cooled so that it renatures. DNA strand synthesised. One two stranded DNA becomes two two stranded DNA molecules. repeat multiple times for exponential increase in DNA
Uses of PCR?
Single base mutations
Small deletions or insertions
Variation and DNA profiling
Protein Gel electrophoresis?
Separation on the basis of size, shape or charge. Move from negative to positive through a gel
Isoelectric focusing?
Proteins separate based on PI value. Will migrate until they reach PH equal to PI value
SDS PAGE
Like gel electrophoresis but uses SDS to add negative charge and denature and B mercaptoethanol
