Oligonucleotide linkers comprising a variable cohesive portion and method for the preparation of polynucleotide libraries by using said linkers

摘要

The present invention relates to a linker or population of linkers that include an oligonucleotide fixed portion and an oligonucleotide variable portion represented by formula (N)n, wherein N is A, C, G, T or U, or their derivatives, and n is an integer equal to or higher than 1. A linker-polynucleotide or a population of linker-polynucleotides of the invention may be constituted by said linker or population of linkers and a target first strand polynucleotide bound to said linker. The invention also encompasses a method of preparing said linker or population of linkers and a method of preparing a linker-polynucleotide using said linker or population of linkers. The linkers or polynucleotide-linkers of the invention can be used in a method of preparing a cDNA library.

主权项

1. A method of preparing a double strand polynucleotide comprising:
i) mixing a population of linkers and a population of a single strand polynucleotides that are first strand cDNA to obtain a mixture comprising a population of linkers and a population of single strand polynucleotides, wherein the linkers comprise a first oligonucleotide strand comprising a fixed portion and a variable portion, and a second oligonucleotide strand comprising a fixed portion complementary to the fixed portion of the first oligonucleotide strand, and said first and second oligonucleotide strands are annealed together to form a double-stranded fixed portion so that the variable portion of said first oligonucleotide strand is overhanging, and wherein said double-stranded fixed portion of the linkers has a common sequence among all of the linkers of the population and comprises at least one restriction enzyme site, recombination site, RNA polymerase promoter site, marker or tag sequence, and the overhanging variable portion of said linkers is represented by the formula (G)m(N)n-m wherein N is A, C, G, T or U, n is an integer from 5 to 10, m is an integer from 1 to 3, the nucleotides (N) may be the same or different from each other, and nucleotides (G)m in the formula (G)m(N)n-m are located adjacent to the double-stranded fixed portion of the linkers, and wherein said population of linkers comprises linkers which have at least two different (N)n-m sequences, ii) annealing a variable portion of a first oligonucleotide strand of a linker to a target single strand polynucleotide that is contained in the population of single strand polynucleotides and that has a sequence complementary to said variable portion, iii) ligating the target single strand polynucleotide to a second strand of the linker, and iv) synthesizing a second single strand polynucleotide(s) complementary to said target single strand polynucleotide(s) by extending the first oligonucleotide strand of the linker to obtain the double strand polynucleotide.