METHODS AND KITS FOR NUCLEIC ACID SAMPLE PREPARATION FOR SEQUENCING
The present disclosure relates to methods and kits for DNA library construction, particularly for consistent and reproducible DNA sequencing.
1 - 30 . (canceled)
31 . A method for nucleic acid library construction, the method comprising:
end repairing nucleic acid fragments in a first reaction mixture;
A-tailing the end-repaired nucleic acid fragments in a second reaction mixture; and
ligating nucleic acid adaptors to the A-tailed nucleic acid fragments in a third reaction mixture,
wherein one or more of the first reaction mixture, the second reaction mixture, and the third reaction mixture are rehydrated from a lyophilized form in a reaction volume comprising the nucleic acid fragments.
32 . The method of claim 31 , wherein at least the first reaction mixture is rehydrated from a lyophilized form.
33 . The method of claim 32 , wherein the lyophilized form is one or more lyophilized pellets, each lyophilized pellet having a diameter of from about 0.3 cm to about 1.0 cm.
34 . The method of claim 33 , wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume.
35 . The method of claim 31 , wherein the first reaction mixture comprises a polymerase and a polynucleotide kinase.
36 . The method of claim 35 , wherein the polymerase is a T4 DNA polymerase, and wherein the polynucleotide kinase is a T4 polynucleotide kinase.
37 . The method of claim 35 , wherein the first reaction mixture further comprises deoxynucleotide triphosphates, a source of magnesium, and a buffer.
38 . The method of claim 32 , wherein the nucleic acid fragments in the reaction volume are added to a single use reaction tube containing the lyophilized first reaction mixture.
39 . The method of claim 31 , wherein at least the second reaction mixture is rehydrated from a lyophilized form.
40 . The method of claim 39 , wherein the lyophilized form is one or more lyophilized pellets, each lyophilized pellet having a diameter of from about 0.3 cm to about 1.0 cm.
41 . The method of claim 40 , wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume.
42 . The method of claim 31 , wherein the second reaction mixture comprises a (3′→5′ exo-) polymerase.
43 . The method of claim 42 , wherein the (3′→′ exo-) polymerase is a Klenow exo-).
44 . The method of claim 42 , wherein the second reaction mixture further comprises a source of magnesium, a source of sodium, dATP, and a buffer.
45 . The method of claim 39 , wherein the end-repaired nucleic acid fragments in the reaction volume are added to a single use reaction tube containing the lyophilized second reaction mixture.
46 . The method of claim 31 , wherein at least the third reaction mixture is rehydrated from a lyophilized form.
47 . The method of claim 46 , wherein the lyophilized form is one or more lyophilized pellets, each lyophilized pellet having a diameter of from about 0.3 cm to about 1.0 cm.
48 . The method of claim 47 , wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume.
49 . The method of claim 31 , wherein the third reaction mixture comprises a nucleic acid ligase.
50 . The method of claim 49 , wherein the nucleic acid ligase is a T4 DNA ligase.
51 . The method of claim 49 , wherein the third reaction mixture further comprises a source of magnesium, ATP, and a buffer.
52 . The method of claim 46 , wherein the A-tailed nucleic acid fragments in the reaction volume are added to a single use reaction tube containing the lyophilized third reaction mixture.
53 . The method of claim 31 , wherein at least two of the first reaction mixture, the second reaction mixture, and the third reaction mixture are rehydrated from a lyophilized form.
54 . The method of claim 31 , wherein each of the first reaction mixture, the second reaction mixture, and the third reaction mixture is rehydrated from a lyophilized form.
55 . The method of claim 31 , wherein the nucleic acid fragments comprise DNA fragments.
56 . A method for nucleic acid library construction, the method comprising:
end repairing nucleic acid fragments in a first reaction mixture, wherein the first reaction mixture comprises a polymerase and a polynucleotide kinase;
A-tailing the end-repaired nucleic acid fragments in a second reaction mixture, wherein the second reaction mixture comprises a (3′→5′ exo-) polymerase; and
ligating nucleic acid adaptors to the A-tailed nucleic acid fragments in a third reaction mixture, wherein the third reaction mixture comprises a nucleic acid ligase,
wherein one or more of the first reaction mixture, the second reaction mixture, and the third reaction mixture are rehydrated from a lyophilized form in a reaction volume comprising the nucleic acid fragments.