Oligonucleotide-tethered nucleotides

A method for generating a library of nucleic acids from a sample comprising one or more nucleic acids, optionally wherein the sample comprises a plurality of cells, comprising:

• a. annealing a first primer which is at least partially complementary to the one or more nucleic acids,
• b. contacting the one or more nucleic acids with a nucleic acid polymerase, at least one nucleotide not tethered to an oligonucleotide, and at least one oligonucleotide-tethered dideoxynucleotide to form a plurality of nucleic acid strands comprising the oligonucleotide-tethered dideoxynucleotide at their 3′ end;
• c. annealing a second primer which is at least partially complementary to the tethered oligonucleotide, and
• d. allowing the polymerase to extend from a 3′ hydroxyl of the second primer annealed to the tethered oligonucleotide, thereby producing a library of nucleic acids.

A method for generating a library of nucleic acids from a sample comprising one or more nucleic acids, optionally wherein the sample comprises a plurality of cells or cell nuclei, comprising:

• a. annealing a first primer which is at least partially complementary to the one or more nucleic acids,
• b. contacting the one or more nucleic acids with a first nucleic acid polymerase, at least one nucleotide not tethered to an oligonucleotide, and at least one oligonucleotide-tethered dideoxynucleotide to form a plurality of first extension products comprising the oligonucleotide-tethered dideoxynucleotide at the 3′ end;
• c. annealing a splint oligonucleotide which is at least partially complementary to the tethered oligonucleotide of the first extension products, and
• d. contacting the first extension products with a nucleic acid polymerase and one or more nucleotides to allow the polymerase to extend across the annealed splint from the 3′ hydroxyl of the tethered oligonucleotide to produce a second extension product, thereby producing a library of nucleic acids.

An oligonucleotide-tethered nucleotide of Formula (A):

• or a salt thereof
• wherein NB is a nucleobase;
• Oligo is an oligonucleotide of 3 to 100 nucleotides;
• each of X and Q are independently chosen from, H, OH, N3, halo, alkyl, alkoxy, alkyl, alkenyl, alkynyl, acyl, cyano, amino, ester, and amido;
• each of Z and Y are independently chosen from a bond, amino, amido, alkyl, alkenyl, alkynyl, thioether, sulfonyl, sulfonamido, ether, ketone, carbonyl, anhydride, ester, imido, urea, urethane, and combinations thereof;
• and
• CXN is chosen from alkylene, alkenylene, alkynylene, ketone, carbonate, ester, ether, anhydride, amido, amino, aminoalkylene, imino, imido, diazo, carbamate ester, phosphodiester, sulfide, disulfide, sulfonyl, sulfonamido, and a heterocyclic group containing from one to four N, O, S atom(s) or a combination thereof where heterocyclic group is optionally substituted at carbon, nitrogen or sulfur atom(s).