Quantitation Of Nucleic Acids Flashcards
What wavelength is DNA maximally absorbed ?
260nm
What is the simplest method to quantify nucleic acids ?
Spectrophotometry
An absorbance of 1 at 260nm in a 1cm path length indicates what DNA concentration ?
50micrograms/ml
What is ten chemical constituent of DNA that contributes to the absorbance at 260nm ?
The bases because they are hexagonal and symmetrical so they have delocalised electrons around the ring which can absorb the UV light
What molecules might contaminate a DNA sample ?
RNA because it also contains bases
Certain aromatic proteins such as tryptophan, tyrosine and phenylalanine - absorb light around 280nm but they also show significant absorbance at 260nm - the aromatic side chain at absorbes he uv light
How are contaminants removed from a DNA sample ?
RNases remove RNA by degrading them but this causes th addition of protein
PolyT tails can be used to attach to the polyA tails of mRNA molecules to pull them out
To remove protein phenol and chloroform are added to remove the protein into a precipitate at the phenol water barrier
Or the DNA igself could be extracted using ion exchange binding resins
What effect does adding phenol to DNA sample have ?
It is an aromatic protein so it can absorb light at 270nm which would interfere with the assay so even though it is necessary for DNA purification it has to be applied with chloroform so it can be extracted
How do you concentrate DNA in a dilute sample ?
Ethanol precipitation
Cooling it enhances the precipitation and then centrifuging it to get a pellet
Absorbance methods are not actually that sensitive, what is another way of quantifying DNA?
Fluorescence methods
Adding a fluorescence dye such as sybr green because it is able to intercalate between the bases of double stranded DNA and then they can fluoresce
What wavelength does protein absorb UV light ?
280nm absorbs it more strongly than DNA
What is the A260:A280 ratio for pure DNA ?
2
What is the A260:A280 ratio for pure protein ?
0.57
Why does the A260:A280 ratio decrease as protein concentration increases ?
Because proteins absorb light more at 280nm so if more protein is present the ratio will be closer to the pure protein ratio because more light will be being absorbed at 280nm
What are restriction endonuclease?
They are enzymes which break down DNA into smaller fragments
They recognise specific sequences in the DNA sequence at cut and their specific recognition site
Allowed the discovery of recombinat DNA technology and are useful for cloning genes and disease diagnosis
Which direction does DNA travel in ?
Travels towards the positively charged anode because DNA is negatively charged due to its phosphate group
What is translation and transcription?
Transcription - production of an mRNA strand from DNA
Translation- production of a protein at a ribosome from the mRNA strand
What are transcription factors ?
They control production of mRNA by binding to specific promoter regions
What does controlling the abundance of mRNA allow ?
Regulate process such as development
Respond to stress
Adapt to altered circumstances
What has to be extracted to measure the transcript abundance of a specific gene ?
Total RNA
What is total RNA made up of ?
Ribosomal and transfer RNA -96%
mRNA - 4%
Why is there more ribosomal RNA compared to mRNA ?
Because ribosomal population is much more fixed and static because we need lots of ribosomes to produce proteins
A single mRNA strand can be amplified by many ribs poems making a massive amplification of the signal
Is RNA a stable molecule ?
No
Because it contains ribose sugar which has a hydroxyl group that acts as a nucleophile, increasing the hydrolysis of the molecule
How is RNA degraded ?
By RNases
They are everywhere so upon extraction of RNA it must be protected to prevent its degradatio.
What are protein denaturants and give examples
They destroy RNases
Phenol and guanidinium thiocyanate
How is mRNA separated from rRNA?
mRNA has a polyA tail which means it has lots of As on the end of it
Therefore it can be hooked out with an oligonucleotide T
Oligonucleotide Ts bund to the As allowing the mRNA strand to be core over from the rRNA and tRNA
What precautions should be taken when handling extracted RNA ?
Wear gloves to cover hands because they will have RNases on the,
All equipment must be decontaminated
Tissue could be frozen as enzymes don’t work as well at low temperatures
During processing of the sample it should remain on ice
How can you measure the amount of a transcript fro a specific gene ?
Complementary sequences of nucleotide
What techniques allow the interrogation of the expression of all gene at the same time ?
Microarrays
Throughput sequencing
What is a microarray ?
An array of many thousands of spots each with a DNA probe for a single gene - DNA probe hybridises the product of a single mRNA exclusively
Why are microarrays useful to scientists ?
They can compare the relative expression levels of genes in a healthy cell and a diseased cell or against a cell that’s had a drug administered to it
Explain the microarray process for comparing genes in a cancer cell and a non cancer cell
Isolate RNA from both cells
RNA is reverse transcribed to produce cDNA probe in the presence of fluorescently labelled nucleotide (Cy3 and Cy5)
Fluorescently labeled cDNAs are mixed together so they hybridise on the microarray
Microarray can then be scanned and the fluorescence emissions at specific wavelengths can be quantified
What is the levels of expression of a gene not an indication of ?
Not an indication of its biological importance
What does the magnitude of changes to the expression of a gene not proportional to ?
The effects of those changes
What does northern blotting measure ?
Measure mRNA transcript abundance
What does southern blotting measure ?
Used to identify specific sequences of DNA
Explain the process of northern blotting
Total RNA is run on agarose/polyacrylamide gel to separate RNA on basis of size
RNA fixed onto nitrocellulose membrane by cross linking
mRNA sequence of gene of interest will be sliced without its introns and compleat art probe produced
Probe is labelled with 32P-dCTP to cytokines with radioactive phosphorous
Probe is hybridised to the membrane and then exposed to film to produce black marks representing where the probe has bound to the mRNA species for the gene of interest
In northern blotting what does the size and intensity of the marks indicate ?
Strong indication of relative quantities of mRNA
What is normally used as a loading control in northern blotting ?
Actin because it tends to remain constant independent of changes to condition s
What is e purpose of the control ?
To ensure the same amount of total RNA is loaded to each well
If there are changes in the mRNA transcript but actin stays the same then you know that the amount of mRNA for the gene of interest has changed
But if you see changes in the intensity of the actin band then you known the total RNA may not have been prepared properly
What is reverse transcriptase PCR?
Process to amplify mRNA from small samples
Involves using a viral enzyme to produce a DNA transcript of mRNA
Explain the cycle in PCR
Heating the double stranded RNA to separate it
Cooling it to allow the primers to bind
Allowing sufficient time to allow the DNA polymerase to extend the primer to produce a new full strand
