Sequencing Primer Analysis

Sanger Sequencing Primer Analysis
by Ivano Zara

This application allows a thermodynamic analysis of the sequencing primers
the program uses by default the parameters (annealing temperature and component concentrations) related to the Sanger sequencing reaction. The user can modify them using 'Advance setting'
In addition to the temperature, it also returns the possible secondary structures (hairpin-loop, self-dimeri) that the primers form at the annealing temperature (chosen by the user).
The primer sequences are also analyzed for the base content, for the presence of similar base stratches and for the stability of the 3'
The program will show as output, a penalty to evaluate the 'goodness' of the sequence and possibly the percentage of primers forming structures.
By clicking on the appropriate links, you can view the details of the penalty and details of any facilities.
The percentages of primers forming structures are calculated with thermodynamic hybridization parameters at the annealing temperature. For any secondary structures at 3', a lower temperature is adopted.
The structures are determined using a special program that thermodynamically analyzes the hybridization of short sequences.

Tip: If you're working with the human sequence, maybe you might be interested in apps in Human variant Tab
OR to the app: Create PCR Primers on regions without polymorphism

Input Primer Sequence

Show / Hide Advance setting

Advance Setting (Under costruction)
To assign penalties, the application uses a sigmoid curve with 2 characteristic parameters: the point where the 50% penalty will occur and the slope of the curve at that point.
For each type of penalty these parameters will be assigned as specified in the table below.

Temperature to analyse structure 3' Extention

Annealing temp:
Cold_temp:

Cold temperature to analyse hybrid for 3'extention

Range Optimal Primer Melting Temperature:
Ann.Temp.+ °C to Ann.Temp.+ °C (Enable: )
If the Melting Temperature falls between these values, there is no penalty

Instead
For lower temperatures the Point with 50% penalty is: ΔT= Sloop is: )
For higher temperatures the Point with 50% penalty is ΔT= Sloop is:

Concentrations

[Primer]: µM

[Na+]: mM

[Mg++]: mM

[dNTPs]: mM

Minimum fraction of
hybrid in the structures

f_thermo_min:

Dimeri
(*) Amplify_dimer_conc:
Extention factor at Low Temp.:
Extention factor at Annealing T.:

penalty_dimer_cutoff:	duplex	Dimer fraction cutoff	penalty parameter: point with 50% penalty Dimer fraction: sloop:
penalty_dimer_cutoff:	3' extention	Dimer fraction cutoff	penalty parameter: point with 50% penalty Dimer fraction: sloop :

Hairpin
Extention factor at Low temp.:
Extention factor at Annealing T.:

penalty_hairpin_cutoff:	duplex	Hairpin fraction cutoff	penalty parameter: point with 50% penalty Hairpin fraction: sloop :
penalty_hairpin_cutoff:	3' extention	Hairpin fraction cutoff	penalty parameter: point with 50% penalty Hairpin fraction: sloop :

Vedi grafico sigmoide-penalità

Base content weight (Enable: )

penalty_cutoff

weight_imbalance

weight_stretch

weight_gc_at_stretch

weight_stabiliy_3p

Note:
(*) 'Amplify_dimer_conc' represents a forcing to highlight any formation of 'small' primer dimers
These seem to be, in theory, thermodynamically difficult to form, under reaction conditions. But it also seems, from some papers,
that any 'small' primer dimers can influence sequencing and PCR reactions. This parameter is simply a multiplication factor of the primer concentration.