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 . A method for DNA library construction, comprising:
modifying nucleic acid fragments; and
ligating nucleic acid adaptors to the modified nucleic acid fragments;
wherein one or more of the reactions are rehydrated from a lyophilized form.
2 . The method of claim 1 , comprising:
modifying the DNA fragments with a polymerase and a polynucleotide kinase in a first reaction mixture sufficient for end repair of the DNA fragments;
contacting the end repaired DNA fragments with (3′→5′ exo-) polymerase in a second reaction mixture sufficient for A-tailing the end-repaired DNA fragments;
ligating DNA adaptors to the A-tailed DNA in a third reaction mixture comprising 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.
3 . The method of claim 2 , comprising:
modifying DNA fragments with T4 DNA polymerase and T4 polynucleotide kinase in a first reaction mixture sufficient for end repair of the DNA fragments;
contacting the end repaired DNA fragments with Klenow (3′→5′ exo-) in a second reaction mixture sufficient for A-tailing the end-repaired DNA fragments;
ligating DNA adaptors to the A-tailed DNA in a third reaction mixture comprising T4 DNA 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.
4 . The method of claim 2 , wherein at least the first reaction mixture is rehydrated from a lyophilized form.
5 . The method of claim 4 , wherein the lyophilized form is one or more lyophilized pellets.
6 . The method of claim 4 , wherein the lyophilized form comprises T4 DNA polymerase, T4 DNA polynucleotide kinase, deoxynucleotide triphosphates, a source of magnesium, and a buffer.
7 . The method of claim 6 , wherein the rehydrated reaction mixture is from about 25 to about 150 μL volume.
8 . The method of claim 7 , wherein the rehydrated reaction mixture is about 50 μl or about 100 μl in volume.
9 . The method of claim 5 , wherein the lyophilized pellet has a diameter of from about 0.3 cm to about 1.0 cm.
10 . The method of claim 9 , wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume.
11 . The method of claim 4 , wherein the DNA fragments in the reaction volume are added to a single use reaction tube containing the lyophilized first reaction mixture.
12 . The method of claim 2 , wherein at least the second reaction mixture is rehydrated from a lyophilized form.
13 . The method of claim 12 , wherein the lyophilized form is one or more lyophilized pellets.
14 . The method of claim 12 , wherein the lyophilized form comprises Klenow (3′ →5′ exo-), a source of magnesium, a source of sodium, dATP, and a buffer.
15 . The method of claim 14 , wherein the rehydrated reaction mixture is from about 25 to about 200 μl volume.
16 . The method of claim 15 , wherein the rehydrated reaction mixture is about 50 or about 100 μl in volume.
17 . The method of claim 13 , wherein the lyophilized pellet has a diameter of from about 0.3 cm to about 1.0 cm.
18 . The method of claim 17 , wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume.
19 . The method of claim 12 , wherein the end repaired DNA fragments in the reaction volume are added to a single use reaction tube containing the lyophilized second reaction mixture.
20 . The method of claim 2 , wherein at least the third reaction mixture is rehydrated from lyophilized form.
21 . The method of claim 20 , wherein the lyophilized form is one or more lyophilized pellets.
22 . The method of claim 20 , wherein the lyophilized form comprises T4 DNA ligase, a source of magnesium, ATP, and a buffer.
23 . The method of claim 22 , wherein the rehydrated reaction mixture is from about 25 to about 200 μl volume.
24 . The method of claim 23 , wherein the rehydrated reaction mixture is from about 50 to about 100 μl in volume.
25 . The method of claim 21 , wherein the lyophilized pellet has a diameter of from about 0.3 cm to about 1.0 cm.
26 . The method of claim 25 , wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume.
27 . The method of claims 20 , wherein the A-tailed DNA in the reaction volume are added to a single use reaction tube containing the lyophilized third reaction mixture.
28 . The method of claim 2 , wherein at least two of the first reaction mixture, second reaction mixture, and third reaction mixture are rehydrated from lyophilized form.
29 . The method of claim 28 , wherein each of the first reaction mixture, the second reaction mixture, and the third reaction mixture are rehydrated from lyophilized form.
30 . A kit for DNA library construction and for performing the method of claim 1 , comprising:
a reaction tube containing first reaction components for end repair of DNA fragments to be added;
a reaction tube containing second reaction components for A-tailing of end-repaired DNA fragments;
a reaction tube contain third reaction components for ligating DNA adaptors to A-tailed DNA;
wherein one, two, or each of the first reaction components, the second reaction components, and the third reaction components are in lyophilized form.