Single tube bead-based DNA co-barcoding for accurate and cost-effective sequencing, haplotyping, and assembly
A barcode and bead technology, applied in recombinant DNA technology, DNA preparation, combinatorial chemistry, etc., can solve problems such as limited use
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Embodiment 1
[0121] 2. Example 1: Methods and materials
[0122] 2.1. High molecular weight DNA separation
[0123] Follow RecoverEase TM A modified version of the DNA Isolation Kit (Agilent Technologies, La Jolla, CA) protocol was used to isolate long genomic DNA from cell lines (1).
[0124] Briefly, approximately one million cells were pelleted and lysed with 500ul of lysis buffer. After incubation at 4 °C for 10 min, add 20 μL of RNase-IT ribonuclease cocktail (cocktail) in 4 mL digestion buffer directly to the lysed cells and incubate on a heat block at 50 °C . After 5 minutes, add 4.5 mL proteinase K solution ( 1.1 mg / mL proteinase K, 0.56% SDS and 0.89X TE), and the mixture was incubated at 50°C for an additional 2 hours. Genomic DNA was then transferred to dialysis tubes with a molecular weight cut-off of 1000 kD (Spectrum Laboratories, Inc., Rancho Dominguez, CA) and dialyzed against 0.5X TE buffer overnight at room temperature.
[0125] 2.2 Barcoded bead construction
[...
Embodiment 2
[0159] Embodiment 2: detailed experimental scheme
[0160] 3.1 Materials
[0161] 1Kb Plus DNA Ladder (ThermoFisher, Cat. No. 10787018)
[0162] 100Kd MWCO Biotech CE dialysis tubing (Spectrum Labs, Cat. No. 131486)
[0163] 384-well Armadillo PCR plate (ThermoFisher, Cat. No. AB2384)
[0164] AMPure XP beads (Beckman Coulter, Cat. No. A63882 )
[0165] APE 1 (10000 units / mL) (New England Biolabs, Cat. No. M0282L)
[0166] ATP (100mM) (Teknova, Cat. No. A1210)
[0167] Barcoded bead construction oligonucleotides (IDT) (see notes)
[0168] Betaine (5M) (Sigma-Aldrich, Cat. No. B0300-5VL)
[0169] BSA (20mg / mL) (New England Biolabs, Cat. No. B9000S)
[0170] Universal Adapter Oligonucleotides (IDT)
[0171] DMF (~100%) (Sigma-Aldrich, Cat. No. D4551-250ML)
[0172] DMSO (100%) (Sigma-Aldrich, Cat. No. D9170-5VL)
[0173] dNTPs (25mM) (ThermoFisher, Cat. No. R1121)
[0174] Dialysis tubing (1000kD MWCO) (Spectrum Laboratories, Inc., Cat. No. 131486)
[0175] DTT (Si...
Embodiment 3
[0448] Example 3: 3' branch ligation, a new method for joining DNA to the 3'OH terminus of DNA or RNA and its application
[0449] 4.1 Introduction
[0450] This example generally describes a 3' branch connection. In the stLFR embodiments described herein, 3' branch ligation is used to add additional adapters (3' branch ligation adapters). See eg §1.1.2.
[0451] Ligase joins breaks in nucleic acids, which is critical for cell viability and viability. DNA ligases catalyze the formation of phosphodiester bonds between DNA ends and play key roles in DNA repair, recombination, and replication in vivo. RNA ligases add 5'-phosphoryl (5'PO4) and 3'-hydroxyl (3'OH) RNA ends via phosphodiester bonds and are involved in RNA repair, splicing, and editing. Organisms from all three kingdoms (bacteria, archaea, and eukaryotes) can be used in vitro as important molecular tools for applications such as cloning, ligase-based amplification or detection, and synthetic biology.
[0452] One...
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