Primer Structure Analysis
(Release 2011_01)
(hairpin-loop, dimer, bases penalty and melting temperature)
by Ivano Zara
Tip: If you're working with the
human sequence
, maybe you might be interested in apps in
Human variant Tab
An example is:
Shows the Genomic Regions with polymorphisms
Help - Overview
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This application allows a rapid and complete thermodynamic analysis of a pair of primers
For each primer, returns, in addition to the melting temperature, the possible structures (hairpin-loop, self and hetero dimers) 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.
Primer Structure and Analyse (Release 2011_01)
Primer (1) sequence
5'
3'
Primer (2) sequence
(5'->3')
As strand '+'
Annealing temperature
°C
Attention, April 2024: different data for the PCR reaction could be obtained compared to the old version.
In fact, based on our experiences and to highlight the formation of any dangerous dimers or hairpin-loops, we have modified some default parameters, which can be modified in 'advance setting' relating to the formation of dimers and hairpin-loops of the primers.
In particular, the default data has been changed:
- Range Optimal Primer Melting Temperature --> For higher temperatures the Point with 50% penalty is ΔT changed from 10.0 to 5.0 ; Sloop is: changed from 0.08 to 0.1
- Dimers -> Extention factor at Annealing T. changed from 1.5 to 10.0
- Dimers --> penalty parameter: point with 50% penalty --> 3' extention -> Dimer fraction: changed from 0.15 to 0.12
- Hairpin --> Extention factor at Annealing T changed from 1.0 to 5.0
If you want to get the same data as the old version, change the values in 'advance setting'
Cold temperature to analyse hybrid for 3'extention
°C
(only if not checked DNA Polymerase Hot Start)
DNA Polymerase Hot Start
DNA Polymerase Proofreading
(3' &rarr 5' exo-activity)
Concentrations
[Primer]
µM
[Na++]
mM
[Mg++]
mM
[dNTPs] total
mM
If you found some bugs or error, please write to ivano.zara.bio @ gmail.com
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.
Annealing temperature (Ann.Temp.):
the one entered in the submission form
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:
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:
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 :
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.
