HIGH THROUGHPUT METHOD FOR ASSEMBLY AND CLONING POLYNUCLEOTIDES COMPRISING HIGHLY SIMILAR POLYNUCLEOTIDIC MODULES

摘要

The present invention relates to a method for the assembly and cloning of polynucleotides comprising highly similar polynucleotidic modules, that is highly versatile, does not require intermediate amplification step and can be easily automated for high throughput production of customized polynucleotidic modules.

主权项

1) A method of generating and assembling polynucleotides comprising arrays of at least two highly similar polynucleotidic modules comprising the steps of:
a) generating at least one polynucleotidic building block comprising at least:
one polynucleotidic module;a single cleavage site for a first restriction enzyme A, placed on one side of the polynucleotidic module;a single cleavage site for a second restriction enzyme B, placed on the other side of the polynucleotidic module;wherein A and B can produce compatible cohesive ends;wherein cleavage of said polynucleotidic building blocks with restriction enzyme A results in a polynucleotide comprising a polynucleotidic module flanked on one side by a cohesive end that can be re-ligated with a polynucleotide building block cleaved by restriction enzyme B without restoring a sequence cleavable by restriction enzyme A and/or B;wherein cleavage of said polynucleotidic building blocks with restriction enzyme B results in a polynucleotide comprising a polynucleotidic module flanked on one side by a cohesive end that can be re-ligated with a polynucleotide building block cleaved by restriction enzyme A without restoring a sequence cleavable by restriction enzyme A and/or B; b) generating “n” polynucleotides linked to a solid phase comprising at least:
one polynucleotidic module; one end linked to a solid phase;a single cleavage site for a first restriction enzyme A, placed on the side of the polynucleotidic module that is linked to a solid phase;a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme A, and which ligation with a polynucleotidic building block cleaved by restriction enzyme A will not produce a sequence cleavable by restriction enzyme A and/or B; c) generating one C-terminal polynucleotidic building block comprising at least:
one polynucleotidic module;a single cleavage site for a first restriction enzyme A, placed on one side of the polynucleotidic module;a single cleavage site for a second restriction enzyme B, placed on the other side of the polynucleotidic module;wherein cleavage of said polynucleotidic building block with restriction enzyme B results in a polynucleotide comprising a polynucleotide module flanked on one side by a cohesive end that cannot be re-ligated with a polynucleotide building block cleaved by restriction enzyme A and/or B; d) cutting said one C-terminal polynucleotidic building block of c) with restriction enzyme A; e) ligating the resulting C-terminal polynucleotidic module with the free end of one polynucleotide of b) immobilized on a solid phase, thereby producing a new immobilized polynucleotide comprising one additional polynucleotidic module; f) cutting the resulting new immobilized polynucleotide with restriction enzyme A, thus producing a new polynucleotide having a free end compatible with the cohesive ends resulting from cleavage with restriction enzyme B; g) ligating the new polynucleotide with the free end of one polynucleotide of b) immobilized on a solid phase thus producing a new immobilized polynucleotide comprising one additional polynucleotidic module;