PT1470252E - Método para montagem de um polinucleótdo de cadeia dupla - Google Patents

Método para montagem de um polinucleótdo de cadeia dupla Download PDF

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PT1470252E
PT1470252E PT02807744T PT02807744T PT1470252E PT 1470252 E PT1470252 E PT 1470252E PT 02807744 T PT02807744 T PT 02807744T PT 02807744 T PT02807744 T PT 02807744T PT 1470252 E PT1470252 E PT 1470252E
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oligonucleotide
polynucleotide
double
stranded
initiating
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Glen A Evans
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Centocor Inc
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Description

DESCRIÇÃO "MÉTODO PARA MONTAGEM DE UM POLINUCLEÓTDO DE CADEIA DUPLA"
ANTECEDENTES DA INVENÇÃO A presente invenção refere-se em geral à área da bioinformática e mais especificamente a métodos para montagem de polinucleótidos dirigida por computador.
Enzimas, anticorpos, receptores e ligandos são polipéptidos que evoluíram por pressão selectiva para realizar funções biológicas muito específicas no meio de um organismo vivo. A utilização de um polipéptido para aplicações tecnológicas específicas pode exigir que o polipéptido funcione em ambientes ou em substratos para os quais não foi evolutivamente seleccionado. Os polipéptidos isolados de microrganismos que prosperam em ambientes extremos proporcionam ampla evidência de que estas moléculas são, em geral, maleáveis no que respeita a estrutura e função. Contudo, o processo para isolar um polipéptido a partir do seu ambiente nativo é dispendioso e demorado. Deste modo, são desejados novos métodos para desenvolver sinteticamente o material genético que codifica um polipéptido possuindo uma actividade desejada.
Existem duas formas de obter material genético para manipulações de engenharia genética: (1) isolamento e purificação de um polinucleótido na forma de ADN ou ARN a partir de fontes naturais ou (2) a síntese de um polinucleótido utilizando várias abordagens químico-enzimáticas. A última 1

Claims (17)

  1. REIVINDICAÇÕES 1. Método de montagem de um polinucleótido de cadeia dupla compreendendo: (a) seleccionar um oligonucleótido de iniciação parcialmente de cadeia dupla, em que o referido oligonucleótido de iniciação compreende, pelo menos, uma parte saliente; (b) colocar em contacto o referido oligonucleótido de iniciação parcialmente de cadeia dupla com um oligonucleótido mais terminal seguinte de cadeia dupla, em que o referido oligonucleótido mais terminal seguinte de cadeia dupla é contíguo com o oligonucleótido de iniciação e compreende, pelo menos, uma parte saliente, e em que, pelo menos, uma parte saliente do referido oligonucleótido mais terminal seguinte de cadeia dupla é complementar a, pelo menos, uma parte saliente em do referido oligonucleótido de iniciação; (c) repetir (b) para adicionar sequencialmente o oligonucleótido mais terminal seguinte de cadeia dupla ao oligonucleótido de iniciação estendido, em que o referido polinucleótido de cadeia dupla é sintetizado, em que o referido oligonucleótido de iniciação no passo (a) tem uma parte saliente em 3' e uma parte saliente em 5', e/ou em que o referido oligonucleótido seguinte mais terminal tem uma parte saliente em 3' e uma parte saliente em 5'.
  2. 2. Método de montar um polinucleótido alvo compreendendo: (a) proporcionar uma sequência de polinucleótido alvo; (b) identificar, pelo menos, um oligonucleótido de iniciação parcialmente de cadeia dupla presente no referido polinucleótido alvo; em que o referido oligonucleótido de iniciação compreende uma parte saliente em 5' e uma parte saliente em 3'; (c) identificar um oligonucleótido mais terminal seguinte de cadeia dupla presente no referido polinucleótido alvo, em que o referido oligonucleótido mais terminal seguinte de cadeia dupla é contíguo com o oligonucleótido de iniciação e compreende uma parte saliente em 5' e uma parte saliente em 3', em que, pelo menos, uma parte saliente em do referido oligonucleótido mais terminal seguinte de cadeia dupla é complementar a, pelo menos, uma parte saliente em do referido oligonucleótido de iniciação; (d) colocar em contacto o referido oligonucleótido de iniciação com o referido oligonucleótido mais terminal seguinte de cadeia dupla em condições e durante um tempo adequados para o emparelhamento, em que a referida sequência de iniciação é estendida; e 2 (e) repetir (a) até (d) para adicionar sequencialmente o oligonucleótido mais terminal seguinte de cadeia dupla até ao oligonucleótido de iniciação estendido, em que um polinucleótido alvo é sintetizado.
  3. 3. Método da reivindicação 2, em que o referido oligonucleótido de iniciação é estendido de um modo bidireccional.
  4. 4. Método da reivindicação 2, em que o referido oligonucleótido de iniciação é estendido de um modo unidireccional.
  5. 5. Método da reivindicação 2, em que o referido oligonucleótido de iniciação é o oligonucleótido 5' mais terminal do referido polipéptido alvo.
  6. 6. Método da reivindicação 2, em que o referido oligonucleótido de iniciação é o oligonucleótido 3' mais terminal do referido polipéptido alvo.
  7. 7. Método da reivindicação 2, em que o polinucleótido alvo compreende uma sequência que codifica um polipéptido alvo.
  8. 8. Método da reivindicação 7, em que o referido polipéptido alvo é uma proteína.
  9. 9. Método da reivindicação 8, em que a referida proteína é uma enzima. 3
  10. 10. Método da reivindicação 2, em que o referido oligonucleótido de iniciação compreende uma sequência identificada por um programa de computador.
  11. 11. Método da reivindicação 10, em que o referido programa de computador compreende um algoritmo compreendendo os seguintes passos: (a) analisar uma sequência de proteína codão por codão; (b) proporcionar uma sequência de gene sintética utilizando os codões de E. coli de Tipo II; (c) analisar a sequência do passo (b) oligómero Q por oligómero Q em oligonucleótidos de comprimento Q com X sobreposição para o conjunto em sentido directo; (d) analisar a sequência em oligonucleótidos de comprimento Q da cadeia reversa sobrepondo-se em X nucleótidos; (e) determinar a sequência dos fragmentos "sufer" da extremidade da sequência REVERSA de comprimento Q/2; e (f) sintetizar a sequência utilizando sintetizadores de matrizes. 4
  12. 12. Método da reivindicação 2, em que a referida parte saliente em complementar do referido oligonucleótido mais terminal seguinte de cadeia dupla compreende cerca de cinquenta porcento do comprimento da cadeia possuindo a referida parte saliente em complementar.
  13. 13. Método da reivindicação 12, em que a referida cadeia possuindo a referida parte saliente em complementar tem cerca de 15 a 1000 nucleótidos de comprimento.
  14. 14. Método da reivindicação 13, em que a referida cadeia possuindo a referida parte saliente em complementar tem cerca de 20 a 500 nucleótidos de comprimento.
  15. 15. Método da reivindicação 14, em que a referida cadeia possuindo a referida parte saliente em complementar tem cerca de 25 a 100 nucleótidos de comprimento.
  16. 16. Método da reivindicação 2, em que o oligonucleótido de iniciação é ligado a um suporte sólido.
  17. 17. Método de montagem de um polinucleótido de cadeia dupla compreendendo: (a) sintetizar quimicamente a primeiro conjunto de oligonucleótidos compreendendo uma primeira cadeia de um polinucleótido de cadeia dupla; 5 (b) sintetizar quimicamente um segundo conjunto de oligonucleótidos compreendendo uma segunda cadeia complementar do referido polinucleótido de cadeia dupla, cada um dos referidos oligonucleótidos no referido segundo conjunto dos referidos oligonucleótidos sobrepondo-se com, pelo menos, um oligonucleótido no referido primeiro conjunto dos referidos oligonucleótidos/e (c) emparelhamento do referido primeiro e segundo conjuntos de oligonucleótidos para produzir de polinucleótido componente parcialmente de cadeia dupla; (d) repetir os passos (a) a (c) para preparar uma série polinucleótidos componentes parcialmente de cadeia dupla; (e) seleccionar, pelo menos, um polinucleótido componente parcialmente de cadeia dupla presente no referido polinucleótido alvo para servir como o polinucleótido de iniciação, em que o referido polinucleótido de iniciação compreende uma parte saliente em 5' e uma parte saliente em 3'; (f) adicionar o polinucleótido componente seguinte mais terminal, em que o referido polinucleótido componente seguinte mais terminal compreende, pelo menos, uma parte saliente em que é complementar a, pelo menos, uma parte saliente em do polinucleótido de iniciação; 6 (g) colocar em contacto o referido polinucleótido de iniciação com o referido polinucleótido componente seguinte mais terminal nas condições e durante um tempo adequado para o emparelhamento; e (h) repetir (e) até (g) para adicionar sequencialmente o referido polinucleótido componente seguinte mais terminal ao polinucleótido de iniciação estendido, em que um polinucleótido alvo é montado na ausência de polimerização enzimática. Lisboa, 25 de Janeiro de 2010 7
PT02807744T 2001-08-02 2002-08-02 Método para montagem de um polinucleótdo de cadeia dupla PT1470252E (pt)

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