Methods in detecting DNA damage Flashcards
Types of DNA damage and the repairs associated with it
Bulky adduct-NER
single strand break-BER
Double stranded break-recombinant repair
insertion/deletion-mismatch repair
Describe comet assay technique
It detects and compares the cleaved DNA fragments in the sample by applying an electric field that will make them migrate from the nucleoid on the other hand intact cells won’t migrate much and there won’t be along tail
The electric current pulls the negaitively charged DNA from the nucleoid towards the anode.
Alkali assay
pH >13, t converts the apyrimidic and apurinic sites to breaks so can detect BER intermediates by cleaving the glycosylate site
Why is SYBR better than ethidium bromide?
It is safer and more sensitive
What parameters are measured in the comet assays?
Tail length, FLUORESCENCE in head and tail,tail moment
What are some applications of comet assays?
Heterogenity of response tumour cells, or rsponse indifferent cell types Assess food sfety Assess tumour response to treatment Biomonitoring Assess repair
What are the advantages and disadvantage?
Advantage: biodistribution sensitive easy, cheap,fast any eukaryotic cell small cell sample data Disadvantage: technical variability single cell data cant generalize interpetation
What is pulse gel electrophoresis?
Same as conventional electrophoresis except it alternates electric field on and off, when it’s on it enlongates and align with new field when off it relaxes. Relaxation rates differs with DNA size
Why can’t we use conventional electrophoresis for large DNA? How does it seperate ?
Will co-migrate as one long band. Through the mass-charge ratio when electric field is applied moves towards anode
What are the advantages and disavantages of pulse field gel electrophoresis?
+:Can measure many cells and take the average DNA damage
Multiple samples can be loaded and compared to each other
Can detect double stranded breaks by blotting(PGFE)
Seperates well
Single measurement
-: High viscous solution
Large DNA fragments
Can be cleaved through shearing
How to measure adducts?
32 post labelling, immunohstochemistry, MS spectroscopy, comet assay
How does 32 post labelling work? What does it measure?
They hydrolyze using enzymes, add nuclease, enrichment of adducts, radiolabel 32P and see using autoradiography
It seperates, detects and quantifies them.
What does LS-MS measure DNA adducts? What is it’s limitation?
By seperating them and then analyzing the DNA adducts isotopic content.
It’s sensitivity limited by the background isotope of normal DNA
Advantages and disadvantages of immunohistochemistry
+: It is easy, cheap, can be done on cells or tissues
-: There isn’t always an antibody against the thing you are studying
How to detect adduct using comet assay
Add specific glycosylase against the specific adduct will cleave it and leave the AP site, the AP sites can be converted to SSDNA by running in alkali conditions and detecting it through comet assay
What is an indirect way to detect DNA damage?
By unsing antibodies against phosphorylated H2AX that is only there during double stranded DNA breaks
How do we detect mutations? How are they seperated ?
Single stranded conformation polymorphism using PCR or denturing/ temperature gradient, protein truncation test, real time assay
What are the advantages and disadvantes of SSCP?
+:It’s easy, versatile, detects small mutations
-: Can have more than 2 bands per sequence: because of isoconfomers or TAQ DNA pol adds an extra nucleotide
How does the denaturing/temperature gradient detect mutations?
First they are seperated based on molecular weight but after they reach a higher gradient they start to move according to their sequence composition and a mobility shift si observed.
What are the advantages/disadvantages of denaturing/temperature gradient?
+:Little as small mutation can be observed
-:There can be bands because of heteroduplexes or GC clamps
Only suitable for small fragments
What does HPLC measure?
It seperates homoduplex and heteroduplex strands
What are the advantages of HPLC?
High thoroughput
Efficient
How does proetin truncation test work? Adavntages? Disadvantages?
It is in vitro translation and transcription of PCR amplified coding regions
+:It detects large fragments in one assay and can detect disease causing mutations such as frameshift, splicing, nosense
-:It can’t detect missense, polymorphism, or silent mutation
How does real time allele specific assay?
They block the sequence of where the mutation occurs in wildtype so it’s not amplified but the mutant sequence is able to amplified
How is high resolution melting used?
After PCR detects through different melting points can detect variation n nucleotide sequences.
How does primer extension assay work?
They only use ddNTP so it’s not enlongated but once it detects the base emits signal, each base has a different fluorescent label, can compare different samples together
What is something ypu need to know before you find the mutations in all the real time assays?
The sequence position of the mutation
