END MODIFICATION TO PREVENT OVER-REPRESENTATION OF FRAGMENTS

摘要

The invention relates to a method of preparing a 5′ and 3′ modified library of template polynucleotides and also the use of the 5′ and 3′ modified library of templates in methods of solid-phase nucleic acid amplification. In particular, the invention relates to a method of preparing a 5′ and 3′ modified library of template polynucleotides which have common sequences at their 5′ ends and at their 3′ ends, wherein over-representation of “end” sequences of the primary polynucleotide molecules from whence the 5′ and 3′ modified library is generated is greatly reduced or prevented.

主权项

1. A method of generating a 5′ and 3′ modified library of template polynucleotide molecules from one or more primary polynucleotide molecules, wherein said primary polynucleotide molecules are modified primary polynucleotide molecules comprising a modification at or near each 5′-terminus that prevents ligation to their 5′-termini; said method comprising the step of:
a) Fragmenting the modified primary polynucleotide molecules to produce target polynucleotide duplexes, wherein the target polynucleotide duplexes comprise modified target polynucleotide duplexes comprising the modification at or near a 5′ terminus and unmodified target polynucleotide duplexes comprising two ligatable termini; b) ligating adapter polynucleotides to the two ligatable termini of the unmodified target polynucleotide duplexes to form one or more adapter-target-adapter constructs; c) carrying out an amplification reaction, wherein a primer oligonucleotide is annealed to both 5′-terminal adapter portions of each of the adapter-target-adapter constructs and extended by sequential addition of nucleotides to form extension products complementary to each strand of each of the adapter-target constructs, wherein the extension products have common sequences at their 5′ ends and common sequences at their 3′ ends and collectively provide a 5′ and 3′ modified library of template polynucleotide molecules;wherein said method prevents the over-representation of the sequences at either end of the primary polynucleotide molecules in the 5′ and 3′ modified library of template polynucleotide molecules.