RR1 Nucleic Acids: Detection and Quantification Flashcards
what is qualitative analysis?
the nature of the molecules
size
nucleotide composition
conformation/composition
structure
what is quantitative analysis?
determine the levels of gene products
how can molecular probes be used to find a molecule in a mix?
- complex mix of macromolecules is separated on a gel (that acts like a sieve) according to size
- they are bound to a membrane (nitrocellulose or nylon) which keeps the relative placements together, provides a record of the separation
maintains the protein on a solid surface so that it can be analysed - probe specific to the target you are looking for (complementary base pairs) is introduced to the mix
- non specific molecules are remove
- to visualise the final target, label the probe in some way
only the final molecule will show up in analysis
how can probes be labelled and generated?
obtain known sequence that corresponds to a gene product of interest (DNA or RNA)
synthesize an oligonucleotide that has the reverse complementary sequence
polynucleotide kinase (PNK) will phosphorylate nucleotides by transferring the gamma (radioactive) phosphate of ATP to the free hydroxyl at the 5’ end of the synthetic oligonucleotide
the 5’ end becomes very highly radioactive, very efficient and effective
is now labelled
how can PCR be used to make labelled DNA probes?
introduce dNTPs that carry an isotopic radiolabel on the alpha phosphate into the PCR amplified DNA
unincorporated radioactive dNTPs are removed
what must happen to the radioactively labelled PCR product before it can be used?
has to be denatured (boiled), and has to be sticky
how can be DNA be analysed by transferring to solid state support?
DNA is cut with restriction enzymes (too long) and run through agarose gel (electrophoresis) that reflects aspects of DNA sequence
put gel on alkaline solution, and on top of that your membrane (nylon or nitrocellulose)
alkaline solution denatures DNA and makes it single stranded
DNA is transferred onto membrane by capillary action
it can be kept there permanently with UV
how can RNA be analysed by transferring to solid state support?
RNA is run through denaturating gel (formaldehyde), because RNA folds in 2° structures, not good in gel because it will separate by structure and not size
run through electrophoresis
put on transfer buffer and transfer to solid state support by capillary action
what happens after the DNA/RNA is transferred to solid state support?
nucleic acids bind strongly to the membrane
can be permanently bound by UV crosslinking
permanently records levels (abundance) and position (size) of molecules after gel separation
the support, or “blot” can be hybridized with probes for any sequence of interest
washes remove non specific signals and only complementary sequences are detectable on blot after radiography (X-ray)
what are the names for the different blots depending on the substance?
DNA southern blot
RNA northern blot
protein western blot
what information can southern analysis give us?
DNA detection: probe and target
molecular identification of polymorphisms
can analyse alleles present by probind them
figure out from the individuals affected by diseases which alleles are responsible and where it came from
relatedness and/or diagnostic
how can polymorphisms be detected without probes?
there is a simpler way
on the normal gene there are the restriction sites for the different enzymes (in this example EcoRI)
if we amplify the gene cut with EcoRI the fragments show up in gel
but if there something wrong in the sequence, the EcoRI doesn’t cut, only one fragment shows up in the gel –> something is wrong, something changed, there was a mutation
what does northern analysis give us?
levels of genes in different tissues
If you know the radioactivity of the probe, you can know the abundance of the gene, assuming the probe is in excess
can give us tissue specific and stage specific (temporal) expression (human vs embryo)
how can quantitative RT-PCR (RT-qPCR) be used to determine mRNA levels?
- prime the reverse transcriptase reaction with polydT (all mRNAs have polyA tail so will all be subjected to RT)
- RT reaction will convert of mRNA to cDNA
- sequence subjected to PCR reaction (with sequence specific primer) that includes an intercalating fluorescent dye that emits signal when incorporated into the growing DNA polymer (doesn’t fluoresce on its own)
- the more DNA is produced, the more fluorescence signal is detectable
- this can be measured in real time, watch the formation of DNA
assesses the amount of mRNA in the sample
what are the phases of PCR reactions and how can they be used to determine mRNA levels?
all PCR reactions have an exponential phase, a linear phase and a plateau
this plateau is reached faster (fewer cycles) in samples with greater amounts of starting material (cDNA, and indirectly RNA)
amount of starting material can be calculating by monitoring number of cycles it takes to reach the plateau phase
more cDNA = faster
proportional to the levels of mRNA in original sample
compare the quantitative cycle (cq is when it takes off) with standard curves of known concentrations
