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.