The specificity of the PCR depends crucially upon the primers. The following factors are important in choosing effective primers

Primers should be 17 to 30 nucleotides in length

A GC content of about 50% is ideal. For primers with a low GC content, it is desirable to choose a long primer to avoid a low melting

 temperature.

Sequences with long runs (i.e., more than three or four) of a single nucleotide should be avoided.

TLS Online TPP Program

#Id: 2917

#Unit 3. Fundamental Processes

Input of energy in the form of ATP is required at several stages for the prepriming reaction, and it is required for unwinding DNA.

TLS Online TPP Program

#Id: 2918

#Unit 3. Fundamental Processes

Once the prepriming complex is loaded onto the replication forks, the next step is the recruitment of the primase, DnaG, which is then loaded onto the DnaB hexamer.

TLS Online TPP Program

#Id: 2920

#Unit 3. Fundamental Processes

This entails release of DnaC, which allows the DnaB helicase to become active. DnaC hydrolyzes ATP in order to release DnaB. This step marks the transition from initiation to elongation.

TLS Online TPP Program

#Id: 2923

#Unit 3. Fundamental Processes

Seq A binds to hemi methylated DNA and is required for delaying rereplication. 

TLS Online TPP Program

#Id: 2924

#Unit 3. Fundamental Processes

SeqA may interact with DnaA.

TLS Online TPP Program

#Id: 2925

#Unit 3. Fundamental Processes

As the origins are hemimethylated they bind to the cell membrane and may be unavailable to methylases.