TRANSITION-METAL CATALYST COMPOSITIONS AND METHODS FOR SEQUENCING BY SYNTHESIS
The present application relates to compositions and methods for sequencing by synthesis. A blocking group of a nucleotide may be removed by a transition metal catalyst, the transition metal catalyst activated by a non-reducing ligand and a reducing agent.
1 . A method for determining sequences of a plurality of target polynucleotides, comprising:
(a) contacting a solid support with sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides;
(b) contacting the solid support with a first aqueous solution comprising DNA polymerase and one or more of four different types of nucleotides under conditions suitable for DNA polymerase-mediated primer extension, wherein each of the nucleotides comprises a 3′ blocking group comprising an unsubstituted or substituted allyl group;
(c) incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides;
(d) performing one or more fluorescent measurements of the extended copy polynucleotides;
(e) removing the 3′ blocking group of the incorporated nucleotides in a second aqueous solution comprising a transition metal catalyst and one or more reducing agents, the transition metal catalyst is generated from a palladium or nickel complex with a water-soluble non-reducing phosphine or a N,N-bidentate non-phosphine ligand, and wherein the one or more reducing agents comprise a boron-containing reducing agent; and
(f) washing the solid support with a third aqueous solution after the removal of the 3′ blocking group of the incorporated nucleotides.
2 . The method of claim 1 , further comprising: repeating steps (b) through (f) until sequences of at least a portion of the target polynucleotides are determined.
3 . The method of claim 2 , wherein steps (b) through (f) are repeated at least 50 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, or at least 300 times.
4 . The method of claim 1 , wherein the transition metal catalyst is Pd(0) catalyst, and the Pd(0) catalyst is generated from the palladium complex with the water-soluble non-reducing phosphine.
5 . The method of claim 1 , wherein the water-soluble non-reducing phosphine comprises tris(substituted C 1 -C 6 alkyl)phosphine or tris(substituted C 6 -C 10 aryl)phosphine, or a combination thereof.
6 . The method of claim 5 , wherein the transition metal catalyst is a Pd(0) catalyst generated in situ from the palladium complex and the water-soluble non-reducing tris(substituted C 1 -C 6 alkyl)phosphine.
7 . The method of claim 6 , wherein the palladium complex comprises [Pd(Allyl)Cl] 2 , Na 2 PdCl 4 , K 2 PdCl 4 , Li 2 PdCl 4 , [Pd(Allyl)(THPP)]Cl, [Pd(Allyl)(THPP) 2 ]Cl, Pd(CH 3 CN) 2 Cl 2 , Pd(OAc) 2 , Pd(PPh 3 ) 4 , Pd(dba) 2 , Pd(Acac) 2 , PdCl 2 (COD), Pd(TFA) 2 , Na 2 PdBr 4 , K 2 PdBr 4 , PdCl 2 , PdBr 2 , or Pd(NO 3 ) 2 , or combinations thereof.
8 . The method of claim 7 , wherein the palladium complex comprises [Pd(Allyl)Cl] 2 or Na 2 PdCl 4 .
9 . The method of claim 5 , wherein the tris(substituted C 1 -C 6 alkyl)phosphine is a tris(carboxy substituted C 1 -C 6 alkyl)phosphine.
10 . The method of claim 9 , wherein the tris(substituted C 1 -C 6 alkyl)phosphine is
11 . The method of claim 5 , wherein the tris(substituted C 1 -C 6 alkyl)phosphine is:
12 . The method of claim 1 , wherein the molar ratio of the palladium complex and the water-soluble non-reducing phosphine is about 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5 or 1:10.
13 . The method of claim 1 , wherein the transition metal catalyst is Pd(0) catalyst, and the Pd(0) catalyst is generated from the palladium complex with the N,N-bidentate non-phosphine ligand.
14 . The method of claim 13 , wherein the N,N-bidentate ligand is selected from the group consisting of:
and salts thereof; wherein R 1 is independently —SO 3 H, —COOH, —PO 3 H 2 , or —NMe 3 + ; R 2 is —(CH 2 ) 1-3 COOH; and R 3 is —SO 3 H or —COOH.
15 . The method of claim 1 , wherein the boron-containing reducing agent is NH 3 BH 3 or B 2 (OH) 4 .
16 . The method of claim 1 , wherein the pH of the second aqueous solution is from about 7.0 to about 10, or from about 7.5 to about 9.5.
17 . The method of claim 1 , wherein the 3′ blocking group has the structure
attached to the 3′ oxygen of the nucleotide, wherein each of R a , R b , R c , R d and R e is independently H, halogen, unsubstituted or substituted C 1 -C 6 alkyl, or C 1 -C 6 haloalkyl.
18 . The method of claim 17 , wherein the 3′ blocking group of the nucleotide has the structure
attached to the 3′ oxygen of the nucleotide.
19 . The method of claim 1 , wherein the first aqueous solution or the third aqueous solution comprises at least one Pd(0) scavenger.
20 . The method of claim 19 , wherein the Pd(0) scavenger comprises one or more allyl moieties selected from the group consisting of —O-allyl, —S-allyl, —NR-allyl, and —N + RR′-allyl, and combinations thereof, wherein R is H, unsubstituted or substituted C 1 -C 6 alkyl, unsubstituted or substituted C 2 -C 6 alkenyl, unsubstituted or substituted C 2 -C 6 alkynyl, unsubstituted or substituted C 6 -C 10 aryl, unsubstituted or substituted 5 to 10 membered heteroaryl, unsubstituted or substituted C 3 -C 10 carbocyclyl, or unsubstituted or substituted 5 to 10 membered heterocyclyl; and R′is H, unsubstituted C 1 -C 6 alkyl or substituted C 1 -C 6 alkyl.
21 . The method of claim 20 , wherein the Pd(0) scavenger comprising one or more —O-allyl moieties is
(N-Boc tyrosine(allyl)-OH) or
(allyl-β-D-gluocopyranoside), or a salt thereof.
22 . The method of claim 20 , wherein the Pd(0) scavenger comprising one or more one or more —NR-allyl or —N + RR′-allyl moieties is
wherein Z − is Cl − or F − .
23 . The method of claim 19 , wherein the Pd(0) scavenger comprising one or more allyl moieties is in the first aqueous solution.
24 . The method of claim 1 , wherein the third aqueous solution further comprises at least one Pd(II) scavenger.
25 . The method of claim 24 , wherein the Pd(II) scavenger comprises L-cysteine or sodium thiosulfate.
26 .- 28 . (canceled)
29 . A kit for use with a sequencing apparatus, comprising:
an aqueous cleavage mixture comprising a transition metal catalyst and one or more reducing agents, wherein the transition metal catalyst is generated from a palladium or nickel complex with a water-soluble non-reducing phosphine or a N,N-bidentate non-phosphine ligand, and wherein the one or more reducing agents comprise a boron-containing reducing agent.
30 .- 51 . (canceled)