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.
Describe how sanger sequencing is carried out
- Have 4 test tubes each with AMP, TMP, CMP and GMP in.
- In each test tubes add one of the dideoxynucleotides.
- Add the sample DNA and PCR each test tube in turn
- Add DNA from each test tube into a different well and do gel electrophoresis
- The shortest fragments (and so first nucleotide in the sequence) will travel furthest, then work back to find complete sequence.
What is the currently used method of DNA sequencing?
Fluorescent dideoxy DNA sequencing
How does FISH and chromosome painting work?
FISH:
- Make probe with sequence specific to target sequence and add a fluorescent marker
- Add to DNA in metaphase so chromosomes condensed.
- Wash and if sequence is present it will fluoresce. The number of times it fluoresces and location will give you more info on genotype and translocations ect
Chromosome painting:
- Have many probes to one chromosome and give each probe for the same chromosome the same colour
How is karyotyping done?
Prepare cells in metaphase and stain them so that you can see chromosomes and banding within them under a microscope.
Identify each chromosome and its pair, see if there is missing or extra material by banding.
Describe what a chromosome report should look like
chromosome number, sex complement, structural changes. NO SPACES ONLY COMMAS
46,XX
47,XY+21
What does protein gel electrophoresis separate based on?
How can it be used to show serum levels?
Size and Shape
More dense bands suggest more protein, could indicate disease levels (Creatine Kinase= MI)
What does SDS- PAGE separate based on?
How is it done?
Polypeptide length
B-ME and SDS added to breakdown secondary and tertiary structure and to give all proteins a - charge. Then do gel electrophoresis
What does isoelectic focussing separate proteins based on?
How is it done?
Based on charge/ pI
Place in test tube of gel with pH gradient along it and a current is applied. Proteins will migrate towards the positive electrode until they reach the pH that equals their pI. The protein will then loose its charge and so stop migrating to the electrode.
What does 2D page separate based on?
How is it done?
Size and charge/ pI
combines IEF and SDS- PAGE- first IEF is done horizontally to separate based on charge and then SDS page is done to separate by size vertically.
How is western blotting done?
Do gel electrophesis to seperate proteins on size.
Transfer to nitrocellulose replica (by blotting)
Add primary antibody complimentary to protein
Add secondary antibody complimentary that’s to the 1st and has a marker
Wash and see if it lights up
How is an ELISA test done?
What can it be used to detect?
- Coat well in antigen
- Add sample of serum from pt (contains the antibodies) and wash
- Add 2nd antibody complimentary to first which contains an enzyme and wash
- substrate added (or serum sample containing substrate)
- enzyme converts substrate to coloured substrate. If antibodies present substrate coloured. If low levels of substate colour is weak
Can therefore be used to test for antibodies or proteins such as cortisol, insulin, TSH, T3/3, troponin
What is the name for an ELISA test that used a radio labeled primary antibody?
RIA - radioimmunoassay
How are levels of clinically important enzymes detected?
Enzyme assays- add substrate as see rate of reaction, faster= more enzymes
Detect product change using spectrophotometry, chemoluminescence, radioactivity, chromatography
What clinically important serum enzymes can be detected using enzyme assays?
AST/ ALT- marks liver damage
Amylase/ Lipase- pancreatitis
y- glutamate transferase- liver damage by alcohol
Creatine kinase 2- MI
Cardiac troponin 2-MI (detected by ELISA)
How can important metabolites such as glucose be measured?
Enzymes:
glucose oxidase converts to gluconolactone and H2O2 which will change the colour of a dye (Used in urine strips)
How is DNA finger printing done?
- DNA cut eitherside of minisatallites (non coding regions of repeating DNA that varies in length)
- Gel electrophoresis to see size of minisatallites
- There are so many minsatallites that they will be unique in length to an individual
- However they will be common with parents
What’s the difference between fingerprinting and profiling?
- Profiling uses VNTRs
- Profiling uses real time PCR to see how long each sequence is and how many fragments there are
