PCR and Primer design

Amplicon

A piece of DNA or RNA that is the source and product of replication, for example from PCR. The head to tail repeat sections.

Primer

Short, single-stranded fragment (15-30 bases) that binds to the DNA sequence to allow RNA polymerase to bind and start transcription.

Becomes a part of the amplicon.

The length of the primer ensures adequate specificity and appropriate Tm

What prime does forward transcription go to?

5’ to 3’

What prime does the complement reverse on a single strand of DNA go from?

3’ to 5’

What is Tm?

Melting temperature of the hydrogens between base pairs - displayed as midpoint of sigmoidal curve

Typically only theoretical Tm calculated and this is normally 50-60 deg. C

What is Ta?

Annealing temperature - the temperature at which the hydrogen bonds between bases form (annealing is opposite of curve for melting)

Typically 3-5 deg. C below Tm - this ensures that primers will not melt the template during the annealing step (thermodynamic stability)

3 methods to find template sequence?

1) Sequence to be found in a database

2) Use a homologous sequence - evolutionarily related by sharing a common answer 

3) Can create a mix of primers with all possible sequences to narrow down potential sequences

Method for cloning the amplicon

• inset amplicon into cloning vector e.g. plasmid 

• use restriction endonucleases to cut sticky/cohesive ends (more efficent than blunt ends)

• use DNA ligase to join and the ends in ligation

What is a MSC or polylinker?

A section of the plasmid that contains multiple restriction sites which can be cleaved to allow a sequence to be inserted in ligation

Promoter

Specific gene sequence found upstream of the gene, serves as a binding site for RNA polymerase and other transcription factors

What factors is Tm dependent on?

• length of primer (longer = higher Tm)

• G+C content (higher = higher Tm)

• salt concentration (more cations = higher Tm)

How can Tm be adjusted so Ta is just below Tm?

• tweak 5’ pre-restriction site bases

• tweak 3’ ends using GC clamping

How to incorporate restriction sites?

• add restriction site to the 5’ end

• ensure result is ‘in-frame’, can add a few extra bases to make cutting more efficient

What is a primer dimer?

Unintended by-product formed in PCR when two primers anneal rather than to target DNA

How to design the 3’ end?

• primer extension occurs at this end so more critical than 5’

• no degenerate bases at 3’ end

• no design mismatches

• try to have a G or C at the 3’ end - ‘GC clamp’