Methods and kits for nucleic acid sample preparation for sequencing
Inventors
Finn, Patrick • Patton, Greg • Liu, Hongbo
Assignees
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Publication Number
US-10870847-B2
Publication Date
2020-12-22
Expiration Date
Abstract
The present disclosure relates to methods and kits for DNA library construction, particularly for consistent and reproducible DNA sequencing.
Core Innovation
The invention provides a method for DNA library construction that uses modifying nucleic acid fragments and ligating nucleic acid adaptors. At least one reaction is rehydrated from a lyophilized form in a reaction volume comprising the nucleic acid fragments, enabling DNA library construction reactions to be performed using lyophilized reaction mixtures.
The invention further specifies a DNA library construction sequence in which DNA fragments are modified with a polymerase and a polynucleotide kinase in a first reaction mixture sufficient for end repair. The end repaired DNA fragments are then contacted with a (3′→5′ exo−) polymerase in a second reaction mixture sufficient for A-tailing, and DNA adaptors are ligated to the A-tailed DNA fragments in a third reaction mixture comprising a nucleic acid ligase.
One or more of the first, second, and third reaction mixtures are rehydrated from a lyophilized form in a reaction volume comprising the DNA fragments. In representative embodiments, the end repair includes T4 DNA polymerase and T4 polynucleotide kinase, the A-tailing uses Klenow (3′→5′ exo−), and adaptor ligation uses T4 DNA ligase. The disclosed approach is described as room-temperature stable and supports end-to-end library construction using lyophilized, rehydratable reagents.
Claims Coverage
The partial content includes three independent claims. Across the independent claims, the core claim coverage centers on DNA library construction in which reaction mixtures for end repair, A-tailing, and adaptor ligation are rehydrated from a lyophilized form in a reaction volume containing DNA fragments.
Lyophilized rehydration for adaptor ligation in DNA library construction
A method 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 in a reaction volume comprising the nucleic acid fragments.
End repair, A-tailing, and adaptor ligation with lyophilized rehydration across reaction mixtures
A method comprising modifying DNA fragments with a polymerase and a polynucleotide kinase in a first reaction mixture sufficient for end repair; contacting the end repaired DNA fragments with a (3′→5′ exo−) polymerase in a second reaction mixture sufficient for A-tailing; ligating DNA adaptors to the A-tailed DNA fragments in a third reaction mixture comprising a nucleic acid ligase; wherein one or more of the first, second, and third reaction mixture are rehydrated from a lyophilized form in a reaction volume comprising the DNA fragments.
T4 end repair, Klenow A-tailing, and T4 ligase adaptor ligation with lyophilized rehydration
A method comprising modifying DNA fragments with T4 DNA polymerase and T4 polynucleotide kinase in a first reaction mixture sufficient for end repair; contacting the end repaired DNA fragments with Klenow (3′→5′ exo−) in a second reaction mixture sufficient for A-tailing; 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 in a reaction volume comprising the DNA fragments.
Across the independent claims, the inventive concept is grounded in DNA library construction where end repair, A-tailing, and adaptor ligation are carried out using one or more lyophilized reaction mixtures that are rehydrated in a reaction volume containing the DNA fragments.
Stated Advantages
Ambient shipping without dry/wet ice.
Stability due to enzyme immobilization upon lyophilization.
Reduced pipetting steps to lower contamination.
Consistent dosing across plates.
Single-use to avoid freeze-thaw variability.
Faster handling without thawing.
Documented Applications
Room-temperature stable end-to-end DNA library construction producing size-selected libraries.
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