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Overview Primer Structure Analysis

This software allows a quick and complete analysis of some parameters that characterize primers in PCR reactions.
For each primer, it returns, in addition to the melting temperature, also the possible structures (hairpin-loop, self and hetero dimers) that the primers form at the annealing temperature (chosen by the user). The sequences of the primers for the content in bases, for the presence of stretch of similar bases and for the stability of the 3 'of the primer are also analyzed. The formation of secondary DNA structures and the formation of DNA duplexes are determined by calculating the thermodynamic parameters, entropy and enthalpy, in the hybridization reaction, applying the Nearest Neighbor method and using the basic parameters of SantaLucia modified with the parameters suggested by Zuker . The fraction of duplexes are determined with an appropriate equation (see 'Oligo Melting')).

In summary:

• a) Melting temperature it should result in some degrees (normally 4 or 5) greater than the annealing temperature of the PCR reaction. It is calculated with the functions of the software 'Oligo Melting', present in this same platform.


• b) Bases contained in the primer:
  - Its base composition (contained in GC and AT) must be balanced - No stretch of equal bases or dinucleotides (eg GC or AT) must be present because, in addition to destabilizing the duplex locally, they may be nonspecifically hybridized (in the DNA there are often regions with repeated bases or dinucleotides). - the 3' end (the last 4 or 5 bases) must be 'balanced': in the duplex, the 3' ends of the two complementary strands do not have to be very strongly bound (eg with many GCs) as they would be amplified nonspecific, they must not be tied too weak because it would make it difficult to start the primer. .


• c) They must not form particular secondary structures:
  - They must not be able to form 'important' hairpin-loops. Since this is a unimolecular reaction (the primer folds in on itself) it can also be formed with a few complementary bases. - They must not be able to form 'important' dimers (self and hetero). This is a bimolecular reaction (occurs between two different molecules) and is more difficult to form than hairpin-loops. - Any hairpin-loops and dimers should not affect the 3 'end of the primers because the DNA polymerase enzyme could stretch the primers rendering them useless.

In detail

b) Composition analysis in bases:
The software analyzes 4 different aspects, assigning to each of them a possible penalty if the relative value deviates from the optimal.
For each penalty there is a weight set by the user in 'advance setting' (default = 1.0)..

-  InBalance bases Penalty 
	penalties given for the unbalance of content in GC and AT):
    penalty = Inbalance_base_weight x 200 x {f (GC) -f (AT)} ^ 2
    where:
   GC = number of G or C bases present in the primer
   AT = number of A or T bases present in the primer 
   f(GC) = Fraction of GC = GC / length in bases of the primer.
   f(AT) = Fraction of AT = AT / length in bases of the primer.
   A penalty is only awarded if f (GC) and f (AT) exceed 0.6 or is less than 0.4.

- Stretch penalty (stretch of equal bases)
   penalty = stretch_weight x [(n-4) x 10 + (n-2) ^ 2];
     where n is the length of the stretch
     VA penalty is assigned only if there is a stretch of 4 or more equal bases.

- Stretch bases type penalty (stretch of similar bases (stretch of G or C and stretch of A or T)
    penalty = GC / AT_stretch_weight x [(n-3) x 3 + (n-4) ^ 2];
   where n represents the stretch length (in bases) An additional penalty is assigned if an AT stretch is located at the 3 'end and if the last base is a T.
     A penalty is assigned only if there is a stretch of 5 or more similar bases.

- stability 3' penalty (thermodynamic stability of the 3' end)
   penalty = stability_3'_weight x 100 * (8.7 + DG) ^ 2,
     where DG is the hybridization free energy calculated with the Nearest Neighbor method, at 37 ° C of the last 5 bases at 3'.

c) Strutture (hairpin-loop e dimeri)
To identify the possible structures, a specific short sequence alignment software ('Short Promix Alignment', present in this same platform) is used. Subsequently, with the alignments obtained, any structures are predicted with the thermodynamic analysis of hybridization using the 'Nearest Neighbor' method with thermodynamic equilibrium. Only the structures whose fraction of the primer exceeds a certain threshold (definable by the user) are shown.

It should be noted that the Nearest-Neighbor method determines the thermodynamic parameters at equilibrium, while a PCR reaction is a dynamic process. For this reason, the software can display many structures, which may not affect the PCR reaction. However, the user can modify the thresholds set out in 'advance setting', thus obtaining fewer predictions.

Hot Start DNA polymerase
The structures are predicted at the annealing temperature (settable by the user). However, if the structures are interested in the 3 'terminations and if a' hot start 'DNA polymerase is not used, then the software also analyzes any structures at a lower temperature, selectable by the user (default 37 ° C) to predict possible elongations of primer even at low temperatures where the activity of DNA polymerase already exists (in this case, in the calculation, an efficiency of 10 times lower than normal is used)

DNA Polimerase proofReading
The use of a DNA polymerase 'proofreading', ie with 5'-3 'exonuclease activity, increases the danger of elongation of the primers at 3'. This type of enzyme is able to digest the non-complementary bases of the 3 'and then start the correct synthesis of the new filament. By selecting this option, the software will evaluate any lengthening of the 3 'of the primers even if the structures do not completely involve the terminations at 3'