Enhanced Diagnostic Tools
An effective and tried way to purify oligonucleotides is by denaturing PAGE; oligonucleotides analyzed by this method can be resolved by single nucleotide differences.
Principle: Each nucleotide (A,C,G and T) have a net charge that is different from each other. All nucleotides have a net negative charged and migrate towards the cathode (positive end of the electrophoresis device); however an oligomer that contains a higher ratio of C and A’s will run faster (have a higher net negative charge) than a same length oligo, of average base composition( 25% of each base) ; for example a 21mer made up only of C+A will run faster in a denaturing PAGE gel than a 21mer made up only of G+T.
Net Negative Charge: Adenosine and Cytosine nucleotides, have a higher net negative charge than Guanosine and Thymine nucleotides.
Mobility Difference: Furthermore, the difference in mobility is approximately one nucleotide for every 3 base difference in C+A or G+T composition; for example for a 21 mer with C+A only , this will run like a 17mer compared to a 21mer oligo that has only a total of 9 C+A; and it will run like a 14 mer compared to a 21mer with all G+T; this all G+T in turn will run like a 28mer, so the maximum difference in mobility can be seen in these two extreme cases: One 21mer(all C+A, the fast one) running like a 14mer and the other(all G+T, the slow one) running like a 28mer, all in all an approximate difference of 14 nucleotides for two oligos of the same length but with total different base composition.
Conclusion: One has to take into account the base composition, when judging the purities of oligos; when analyzing oligos of biased base compositions away from an equal ratio( 25% of each base ), one has to take into account the corresponding C+A content in order to avoid miscalling the correct size or purity level of a given oligo.
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