Molecular Techniques Flashcards
What methods of DNA testing are there at the gene level?
Restriction enzymes mapping DNA gel electrophoresis PCR Southern blotting/ DNA hybridisation Microarray RT- PCR Probes
What methods of DNA analysis are there at the nucleotide level?
(sanger) DNA sequencing
Restriction mapping
Electrophoresis can see single nucleotide changes
Probes may work but not very specific
What methods of DNA analysis are available for chromosome level mutations?
FISH
Karyotyping
What methods are available for analysing proteins?
Protein electrophoresis (SDS- PAGE, Isoelectric focussing, 2D- PAGE)
Immunoassays (ELIZA, RIA, Westernblotting)
Enzyme assays
What neumonic is used to remember the uses for southern, northern and western blotting?
SNoW DRoP
Southern= Look at Dna w/ Dna probes
Northern= Look at Rna w/ Dna probes
Western= Look at Proteins w/ antibodies
How does restriction mapping work?
Restriction enzymes will recognise specific sequences (often palindromic) that are 4-8 bp long, they will break the hydrogen bonds between those bases and the phosphodiester bond at either end. If a mutation changes the recognised sequence, the DNA will not be cut here and different sized fragments will be produced. Gel electrophoresis is needed to determine the lengths of the fragments.
Describe the process of southern blotting.
- DNA extracted from cell and all/ specific region amplified using PCR, an cut into managable fragments using restriction enzymes
- Gel electrophoresis used to separate fragements
- Overlay nitrocellulose gel, alkali (to denature) and then paper towel above that
- Add heavy weight to top, and fragments will be drawn onto the nitrocellulose gel
- Add solution containing fluorecesnt/ radioactive probe
- Wash to remove unbound probe
- Detect using xray film/ UV light
What are the uses of southern blotting
- find large deletions/ duplications (size of fragments band)
- finds number of triplet repeats (length of fragment)
- find mutations across restriction enzyme site (fragments present)
- Able to detect small amounts of DNA not visible after electrophoresis alone
- Sifts through large quantities of DNA to find useful bit.
What two enzymes indicate a myocardial infarction? How can they be detected?
cardiac troponin 1- ELISA test
Creatine Kinase- Protein electrophoresis/ Enzyme assays (?)
What are the notable characteristics of probes?
- They will anneal even if the sequences only match 80%
- They will partially bind to a probe (If the enzyme cuts in the middle of the probes site, the probe may still bind to both fragments produced)
- They do not affect the position of the DNA on the gel
Describe how gel electrophoresis works
The DNA is placed in a well in a gel matrix. A current is passed through it. The DNA migrates to the positive anode because they’re negatively charged. Smaller fragments travel further because they can move through the gel more easily. Stain the gel to detect where the DNA went.
Describe the process of PCR and how allele specific probes can allow us to identify which probe is present in a individual.
- Heat dsDNA to 95 C to denature DNA
- Cool to 50C to allow primers complimentary to 5’ and 3’ ends of the DNA to anneal
- Heat to 72C to allow taq. Polymerase to extend the ds sections
- repeat, after 4 cycles a pure ds section of the DNA of interest will be produced
- Allele specific probes will not be complimentary to one allele at the 3’ end of the primer and so the DNA and probe will not anneal here and so polymerase will not extend the DNA and duplicate it.
Describe how microarray works to look at differential expression of genes.
- Genome is digested and different genes put onto different places on microscope slide by pins
- mRNA taken from normal and disease state cells
- Reverse transcriptase used to convert to cDNA.
- label cDNA of diseased cell with red tag and blue tag for normal cells.
- Mix both cDNAs and hybridise to microarray
- if both cells expressing gene equally red and green mix to produce brownish colour, if more in diseased cell then that gene will turn up more red
Describe how microarray can be used to look at differential amounts of a certain gene. (Look at genome wide changes)
- Do not use mRNA but instead digest all DNA from a diseased and a normal cell and compare them using microarray as before
Describe process of RT PCR and its used
Used to look at gene expression in a cell.
- take mRNA from cell
- Add DNA primer complimentary to poly A tail (TTTTTT)
- Add reverse transcriptase to get cDNA strand (Will be bound to old mRNA strand)
- Add RNAse to digest mRNA
- Add forward primer and taq. polymerase to make dsDNA
- Add forward and backward primers (complimentary to gene of interest) and PCR as normal. If gene expressed this will duplicate some mRNA if not then nothing will be duplicated.
