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.