MicroArray Design

Oligos are synthesised from the 3’ end and as such the primers will be 5’ truncated and this can make up as much as 30% of your oligo.

Sequences primed by the truncated molecule will be n-1 n-2 etc, but co-migrate with the correct product leading to multiple peaks for each base.

The best approach to overcome this is two-fold;
1.Purify all primers by PAGE.
2.Design sequencing primers featuring modified bases, particularly the 5’base to prevent n-1 primers being able to generate sequencing product.

Standard DNA sequencing primer 25mer Tm 68C- this interaction was modelled at 60C annealing, 3mM MgCl2, 50mM Nacl and no adjuncts were used-99.9% efficiency

Under the same conditions the n-1 primer will bind with a TM of 65C-99.9% Efficiency.

So when both are present we see that 93% of the sequence is the n primer and 7% is the n-1.

The problem arises if the longer primer degrades due to repeated freeze thaw, with the n-1 levels increasing to the point whereby they are visible in a sequencing trace.

The first solution is then to PAGE purify the primer to reduce the initial n-1 concentration to below 2%.

The better long-term solution is to correctly design PCR and sequencing reactions so that no such issues can arise.

The next two slides show the effect of SNPs on the reaction

First example shows a T>G at base 5- efficiency of reaction is still 96.5%.

First example shows a T>G at base 5- efficiency of reaction is still 96.5%.

This indicates that a single base mismatch under a primer cannot prevent a well designed primer from generating product.

We have never seen more than a 2.3C shift from a single A/T mismatch or more than 3C for G/C.

LNA based designs, verified by in silico PCR can really help with improving sequencing quality, along with reducing n-1 issues.

As LNA bases increase base stacking, an n-1 primer will feature greatly reduced stability if the modified molecule is missing.

A further advantage is the ability to be able to design shorter primers and that LNA modified sequencing primers have been shown to improve Phred scores.

The whole reaction can then be designed and modelled on your computer, so that your reactions will work first time.

As above,designed a modified sequencing primer to the same site-but this time it’s a 21mer with the 2 5’ bases modified by the substitution of LNA.

You can see that the n-1 primer would have a TM of 5C less than the correct primer and so it’s priming efficiency would be reduced to 4% while the correct product has 99% efficiency-the reduced cycles of a sequencing reaction mean that the n-1 product can now not be observed.

In summary

In a well designed assay, modelled in silico, allows the user to test their primers for SNP mismatch effects. A single SNP should not prevent a reaction from working.

N-1 can be improved by PAGE cleanup and the addition of LNA molecules to the primer.It is essential that one accurately model the effects of these substitutions-since mis-placement can adversely effect you reactions.

VisualOMP allows design and modelling of all hybridisation based assays including PCR, multiplex PCR, real-time, arrays, MLPA probes etc. The software will not allow any mis-priming or cross talk between molecules in an assay and so is invaluable in setting up multiplex assays.