Methods in detecting DNA damage

Question 1

Types of DNA damage and the repairs associated with it

Answer 1

Bulky adduct-NER
single strand break-BER
Double stranded break-recombinant repair
insertion/deletion-mismatch repair

Question 2

Describe comet assay technique

Answer 2

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.

Question 3

Alkali assay

Answer 3

pH >13, t converts the apyrimidic and apurinic sites to breaks so can detect BER intermediates by cleaving the glycosylate site

Question 4

Why is SYBR better than ethidium bromide?

Answer 4

It is safer and more sensitive

Question 5

What parameters are measured in the comet assays?

Answer 5

Tail length, FLUORESCENCE in head and tail,tail moment

Question 6

What are some applications of comet assays?

Answer 6

Heterogenity of  response tumour cells, or rsponse indifferent cell types
Assess food sfety
Assess tumour response to treatment
Biomonitoring
Assess repair

Question 7

What are the advantages and disadvantage?

Answer 7

Advantage: biodistribution 
sensitive
easy, cheap,fast
any eukaryotic cell
small cell sample data
Disadvantage:
technical variability
single cell data cant generalize
interpetation

Question 8

What is pulse gel electrophoresis?

Answer 8

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

Question 9

Why can’t we use conventional electrophoresis for large DNA? How does it seperate ?

Answer 9

Will co-migrate as one long band. Through the mass-charge ratio when electric field is applied moves towards anode

Question 10

What are the advantages and disavantages of pulse field gel electrophoresis?

Answer 10

+: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

Question 11

How to measure adducts?

Answer 11

32 post labelling, immunohstochemistry, MS spectroscopy, comet assay

Question 12

How does 32 post labelling work? What does it measure?

Answer 12

They hydrolyze using enzymes, add nuclease, enrichment of adducts, radiolabel 32P and see using autoradiography
It seperates, detects and quantifies them.

Question 13

What does LS-MS measure DNA adducts? What is it’s limitation?

Answer 13

By seperating them and then analyzing the DNA adducts isotopic content.
It’s sensitivity limited by the background isotope of normal DNA

Question 14

Advantages and disadvantages of immunohistochemistry

Answer 14

+: It is easy, cheap, can be done on cells or tissues

-: There isn’t always an antibody against the thing you are studying

Question 15

How to detect adduct using comet assay

Answer 15

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

Question 16

What is an indirect way to detect DNA damage?

Answer 16

By unsing antibodies against phosphorylated H2AX that is only there during double stranded DNA breaks

Question 17

How do we detect mutations? How are they seperated ?

Answer 17

Single stranded conformation polymorphism using PCR or denturing/ temperature gradient, protein truncation test, real time assay

Question 18

What are the advantages and disadvantes of SSCP?

Answer 18

+: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

Question 19

How does the denaturing/temperature gradient detect mutations?

Answer 19

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.

Question 20

What are the advantages/disadvantages of denaturing/temperature gradient?

Answer 20

+:Little as small mutation can be observed
-:There can be bands because of heteroduplexes or GC clamps
Only suitable for small fragments

Question 21

What does HPLC measure?

Answer 21

It seperates homoduplex and heteroduplex strands

Question 22

What are the advantages of HPLC?

Answer 22

High thoroughput

Efficient

Question 23

How does proetin truncation test work? Adavntages? Disadvantages?

Answer 23

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

Question 24

How does real time allele specific assay?

Answer 24

They block the sequence of where the mutation occurs in wildtype so it’s not amplified but the mutant sequence is able to amplified

Question 25

How is high resolution melting used?

Answer 25

After PCR detects through different melting points can detect variation n nucleotide sequences.

Question 26

How does primer extension assay work?

Answer 26

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

Question 27

What is something ypu need to know before you find the mutations in all the real time assays?

Answer 27

The sequence position of the mutation