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Durante os ciclos de síntese, e em cada adição de base, cerca de aproximadamente 1% dos oligos não vão incorporar a base adequada. Isso leva à formação de mutantes de deleção. Isto pode acontecer em qualquer lugar dentro da sequência e leva a uma mistura de oligos de comprimento total, produtos truncados, e oligos com deleções internas.  Por esta razão, a sua escolha de purificação deve ser baseada em vários factores, incluindo onde será utilizado o seu oligo, o comprimento do oligo, e se serão adicionadas modificações ao oligo. Como regra geral, qualquer oligonucleotídeo com mais de 40 bases deve receber mais etapas de purificação, mas note que a purificação adicional resultará em uma diminuição no rendimento do oligo final.

  • Para algumas aplicações, tais como PCR ou sequenciação, a purificação por dessalinização padrão é bastante aconselhável porque os truncamentos e exclusões não afectarão os seus resultados consideravelmente.

 

  • Se tem um oligo grande sem modificações, deverá pedir a purificação cartdrige, uma vez que os fragmentos mais curtos e sais potenciais são eliminados.  O resultado é um oligonucleotídeo purificado livre de sais indesejáveis. De acordo com a nossa experiência, a purificação obtida com estes métodos está na faixa dos pequenos oligos não modificados (até 30 bases) bastante comparável à observada após purificação por HPLC.

 

  • Para outras aplicações como a clonagem e a mutagénese, o tamanho do produto final é de extrema importância, e a purificação PAGE deve ser fortemente considerada. A purificação PAGE resultará no mais alto nível de pureza em termos de produto de corpo inteiro, e seu oligos terão pelo menos 90% do tamanho total.  A purificação PAGE deve ser evitada para as seguintes modificações: Qualquer fluoróforo, Acrydite, modificadores Amino, Biotina, digoxigenina, eu Linker, espaçador 18 e modificadores de tiol. Em esses casos HPLC ou IE-HPLC seria o método de purificação a ser escolhido.

 

  • Se precisa de um produto limpo, mas também precisa de um rendimento mais alto, você deve optar por HPLC para oligos até 50nts e mais. Também deve ter os tipos de modificações em conta no seu oligo ao escolher um método de purificação. O processo de purificação é controlado e documentado por espectroscopia de UV. Isto permite que a concentração do produto puro seja feita atráves da escolha do pico desejado, ou "corte de qualidade", de modo a que qualquer outro pico não será selecionado. Para oligonucleótidos modificados o RP-HPLC também é o método de escolha.
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