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Methods and kits for nucleic acid sample preparation for sequencing

a technology of nucleic acid and kits, which is applied in the field of methods and kits for nucleic acid sample preparation, can solve the problems of non-homogenous thawing of products, increase the probability of cross contamination of stock liquid solutions, etc., and achieves the effects of reducing enzyme activity, simplifying shipping, and simplifying the storage of products

Inactive Publication Date: 2015-10-15
ARCHERDX LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent is about improving the reagents used for next-generation sequencing library construction. The invention involves using a lyophilized reaction mixture, which is a more stable and efficient enzyme formulation that can be shipped without the need for coolants. This simplifies shipping and reduces the risk of contamination. The lyophilized reaction mixture also requires fewer steps for sample preparation, resulting in consistent and reliable results. Overall, this invention provides a more efficient and consistent approach to sequencing library construction.

Problems solved by technology

Which if done incorrectly can yield non-homogenous thawing of the product and thus variation in efficiencies of each step in the sample preparation workflow.
Stock liquid solutions also increase the probability of cross contamination.

Method used

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  • Methods and kits for nucleic acid sample preparation for sequencing
  • Methods and kits for nucleic acid sample preparation for sequencing
  • Methods and kits for nucleic acid sample preparation for sequencing

Examples

Experimental program
Comparison scheme
Effect test

example 1

T4 Polynucleotide Kinase

[0030]FIG. 1 shows that T4 polynucleotide kinase successfully reactivates from its lyophilized format.

[0031]Three lyophilized pellet formulations of T4 polynucleotide kinase were treated in foil packs at 50° C. for 2 weeks. The assay measured radiolabeled γ-32P ATP incorporation into poly dT substrate, Activities of all three lyophilized pellet formulations track with a liquid formation stored at −20° C. Two weeks at 50° C. is equal to about 3 months at room temperature.

example 2

A-Tailing Formulation

[0032]FIG. 2 shows the results of an A-tailing formulation (comprising Klenow Fragment exo-)). DNA was treated with End Repair, and various amounts of Klenow (3′→5′ exo-), followed by ligation. Library fragments lacking a 3′ A from Klenow (Exo-) are expected to undergo blunt ended ligation. Input of Klenow from 15 Units to 1.9 Units has little effect on the A-Tailing step.

[0033]These data show that the lyophilized A-tailing formulation successfully reactivates and perform equivalently to the liquid formulation.

example 3

End Repair

[0034]As shown in FIG. 3, a lyophilized End Repair mix rehydrates and performs equivalently to the liquid reaction mixture. The assay involves treating with various amounts of End Repair and ligation.

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Abstract

The present disclosure relates to methods and kits for DNA library construction, particularly for consistent and reproducible DNA sequencing.

Description

PRIORITY[0001]This Application claims priority to U.S. Provisional Application No. 61 / 721,843, filed Nov. 2, 2012, which is heerebv incorporated by reference in its entiretyFIELD OF THE INVENTION[0002]The present disclosure relates to methods and kits for nucleic acid sample preparation, particularly for consistent and reproducible nucleic acid sequencing.BACKGROUND[0003]For nucleic acid sequencing, high throughput sample preparation products are generally shipped with significant quantities of dry ice (up to 50 lbs) to ensure that the reagents remain active upon receipt at the sequencing facility. Most of the enzymes required for sample preparation for next generation sequencing platforms are derived from mesophilic sources which are more sensitive to broad temperature fluctuations that may be observed during shipping. Even thermophilic sources, although they are more stable to temperature ranges, exhibit residual activities that may cause side reactions during shipping that negati...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/10C12Q1/68
CPCC12N15/1093C12Q1/6874C12Q1/6806
Inventor FINN, PATRICKPATTON, GREGLIU, HONGBO
Owner ARCHERDX LLC
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