Northern blotting & mRNA detection by qPCR - part...
Transcript of Northern blotting & mRNA detection by qPCR - part...
Northern blotting & mRNA detection by qPCR - part 2
“It has not escaped our notice that the specific pairing we have postulated immediately suggests a copying mechanism for the genetic material.”
Central Dogma of Molecular Biology:���Information flows from DNA to RNA to protein
Principle of gene expression:���From DNA to RNA to protein
DNA Base Pairing: also works for RNA
Classic procedure:
Radioactive labellingof complementaryRNA
Probe is either a complementaryRNA sequenceor a DNA strand(RNA happily formsa ds heteroduplexwith DNA).
RNA/DNA hybridization is influenced by…
Probe concentration
���Sequence (GC bonds more stable than AT)
Stringency
• Probe concentration: • The higher the probe concentration, the faster
the hybridization occurs. However, the higher the probe concentration, the higher the blot background (i.e. probe will stick everywhere).
• Low probe concentrations and long incubations (overnight) usually produce the best results.
• Stringency
• Stringency is a key concept in all nucleic acid detection experiments
• We can adjust the stringency so that only exact DNA or RNA sequences match. Or, we can allow some mismatching. We do this mostly by modifying wash conditions, especially salt.
• Salt: • High salt wash, hybrids are more stable, more RNA stuck,
blot is less stringent. • Low salt wash, hybrids are less stable, blot is more stringent.
• Temperature:• nucleic acids denature (helix melts) at high temperatures. High
temperature, less stable, more stringent
Quantitative PCR
• We know the sequence of the target gene, X (more on this later in the course).
• Another way to measure the amount of mRNA present is to use a PCR reaction.
• The idea of specific base pairing to detect the sequence is exactly the same
RT-PCR• Wait, how come PCR works on RNA?• It doesn’t.
• We have to convert our mRNA to DNA first. mRNA that has been converted to DNA is called cDNA (copy DNA)
• We do this using a REVERSE TRANSCRIPTASE. This is an enzyme from a retrovirus that copies RNA into DNA. This does not normally happen in a cell.
• We follow the accumulation of the PCR product using a fluorescent dye (SYBR green) that only fluoresces in the presence of dsDNA (not primers or cDNA).
• We use a special thermal cycler to do this that incorporates a laser and PM tube
• We’ll talk more about PCR next week and visit the machine later in semester.