Methods of preparation of gene-specific oligonucleotide libraries and uses thereof

A method of producing a target-specific library that comprises substantially all sequences of a pre-determined length or range of lengths that are comprised within a target polynucleotide sequence, the method comprising:

• digesting a double-stranded RNA copy of said target polynucleotide with a nuclease to generate fragments of from about 10 nucleotides to about 40 nucleotides in length;
• dephosphorylating said RNA fragments;
• ligating 2′-deoxyadenosine 3′-monophosphate (pdAp) to each end of said product of dephosphorylation;
• dephosphorylating the product of said ligation reaction;
• ligating product of said dephosphorylation reaction into a linearized vector having 3′-deoxythymidine overhangs;
• filling in gaps by using a DNA polymerase such as E. coli Pol l;
• amplifying the resulting vector in bacteria to replace RNA with DNA;
• wherein said resulting library of polynucleotides comprises substantially all sequences of a pre-determined length within said target polynucleotide sequence.

A method of producing a target-specific library that comprises substantially all sequences of a pre-determined range of lengths that are comprised within a target polynucleotide sequence, the method comprising:

• partially digesting a double-stranded DNA copy of said target polynucleotide with DNase I, and digestion is performed in the presence of Mn+2 to generate blunt-ended fragments of from about 10 nucleotides to about 40 nucleotides in length or a wider range that comprises the range 10 to 40 nucleotides;
• and
• ligating said DNA fragment to a first adapter;
• ligating the above product to a second DNA adapter.
• amplifying the product of the above reaction using primers complementary to said first and said second adapters.
• inserting said fragments into a vector or between fixed sequence segments of DNA.

A method of producing a target-specific library that comprises substantially all sequences of a pre-determined range of lengths that are comprised within a target polynucleotide sequence, the method comprising:

• hybridizing hemi-random probes to a ssDNA target, wherein said hemi-random probes comprise a fixed region comprising primer-binding sequences with encoded restriction enzyme recognition sites and a 10-nt randomized sequence located at the 5′ end in the case of one probe and at the 3′-end in the case of the other;
• ligating hybridized probes that hybridize to adjacent target sequences;
• amplifying the product of said ligating step;
• inserting the product of said amplification into a vector or between DNA sequences allowing expression of the inserted sequences.