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Massively parallel sequencing using unlabeled nucleotides

An unlabeled, deoxyribonucleotide technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, can solve the problems of signal reduction and low incorporation efficiency

Pending Publication Date: 2021-08-17
MGI TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, current SBS methods require expensive, reversibly terminated dNTPs (RTs), in which a label (e.g., a dye) on the base is attached to a cleavable linker, resulting in a) remaining on the incorporated base after cleavage of the label. Chemical scarring, b) less efficient incorporation, c) quenching, d) excited dye-induced termination of extension, and reduced signal in each sequencing cycle

Method used

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  • Massively parallel sequencing using unlabeled nucleotides
  • Massively parallel sequencing using unlabeled nucleotides
  • Massively parallel sequencing using unlabeled nucleotides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0450] Example 1: Rabbit anti-NLRT monoclonal antibody (mAb) and sequence

[0451] Generates 3'-azidomethyl-dA(N3A), 3'-azidomethyl-dC(N3C), 3'-azidomethyl-dG(N3G) or 3'-azide for KLH conjugation Rabbit monoclonal antibody to methyl-dT (N3T) (Yurogen Biosystems, Worcester, MA). Briefly, 8 rabbits were immunized with four different KLH-conjugated NLRTs, two rabbits for each of the four molecules. Bleeding analysis by ELISA was performed using each NLRT. On day 63 after immunization, rabbits were sacrificed and peripheral blood mononuclear cells (PBMC) or splenocytes were isolated. Rabbits were selected for cell sorting and culture of antibody-secreting B cells. Co-culture supernatants were screened using NLRT. Five or ten different anti-NLRT antibody clones (depending on target) were prepared for each of the four NLRTs, resulting in >30 mAb preparations.

[0452] Cloning of rabbit IgG genes from specific B cells identified by antigen screening. Obtain the heavy and ligh...

Embodiment 2

[0455] Example 2: Detection using a dN-azidomethyl-specific rabbit monoclonal antibody and a labeled goat anti-rabbit secondary antibody Measurement of NLRT incorporated in DNB arrays

[0456] Rabbit monoclonal antibodies N3A, N3T, N3G and N3C were used in this experiment. DNB arrays containing E. coli genomic DNA inserts were sensitized and primers extended using BG9 DNA polymerase (BGI, Shenzhen, China). Thirty antibody preparations were individually applied to separate lanes on the DNB array at 3 μg / mL or 25% culture supernatant as indicated and incubated at 35°C for 5 min (30 independent incubations). At the end of the incubation, unbound primary antibody was removed by washing the array at 35°C with antibody buffer (AbB) (Tris buffered saline pH 7.4 + 0.1% BSA and 0.05% Tween-20). The array was then incubated with Cy3-labeled goat anti-rabbit secondary antibody (Fab fragment) obtained from Jackson Immune Research (West Grove, PA, USA) at 35°C for 5 minutes. The arra...

Embodiment 3

[0462] Example 3: Sequencing by synthesis using labeled anti-NLRT monoclonal antibodies

[0463] E. coli genomic DNA libraries were prepared as previously described and arrayed on a BGISEQ-500 flow cell. Primers were added and sequence-by-synthesis was performed by primer extension using one target unlabeled nucleotide 3'-azidomethyl reversible terminator (dATP, dCTP, dGTP, dTTP) and three conventionally labeled reversible terminators in the ratio were: A-AF532 25% labeled, C-IF700 40% labeled, G-Cy5 35% labeled and T-ROX 35% labeled (in one experiment, two of the RTs (RT-A and RT -C) was routinely labeled, and two of the RTs (RT-G and RT-T) were detected by labeled monoclonal antibodies). The total concentration of 3′-blocked dNTPs was 1 μM for each nucleotide and incorporated using BG9 DNA at 55 °C and each cycle lasted 1 min. After incorporation and washing to remove unincorporated nucleotides, the array was incubated with a mixture of four directly labeled anti-3'-azid...

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Abstract

The invention provides compositions and methods for sequencing nucleic acids and other applications. In sequencing by synthesis, unlabeled reversible terminators are incorporated by a polymerase in each cycle, then labeled after incorporation by binding to the reversible terminator a directly or indirectly labeled antibody or other affinity reagent.

Description

technical field [0001] The present invention relates to nucleic acid sequencing and its use in medicine and biological sciences. [0002] sequence listing [0003] This application contains a Sequence Listing, which has been filed electronically in ASCII format, and is hereby incorporated by reference in its entirety. [0004] Cross References to Related Applications [0005] This application claims U.S. Provisional Patent Application 62 / 758,317, filed November 9, 2018, U.S. Provisional Patent Application 62 / 914,877, filed October 14, 2019, U.S. Provisional Patent Application 62 / 914,940, filed October 14, 2019 , and the priority of U.S. Provisional Patent Application 62 / 914,915, filed October 14, 2019, each of which is incorporated herein by reference. [0006] This application is related to United States Patent Publication US 2018 / 0223358 and International Patent Application PCT / US2018 / 012425 published as WO 2018 / 129214, both of which are incorporated herein by reference. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6874C12Q1/6876
CPCC12Q1/6874C12Q2525/186C12Q2563/131C12Q1/6876
Inventor 斯尼泽纳·德尔马纳茨李汉东马修·J·卡洛利昂·埃克哈特斯科特·加布伦茨拉多吉·德尔马纳茨周萍
Owner MGI TECH CO LTD
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