Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library

<div><p>Chemical synthesis of oligonucleotides is a widely used tool in the field of biochemistry. Several methods for gene synthesis have been introduced in the growing area of genomics. In this paper, a novel method of constructing dsDNA is proposed. Short (28-mer) oligo fragments from a library were assembled through successive annealing and ligation processes, followed by PCR. First, two oligo fragments annealed to form a dsDNA molecule. The double-stranded oligo was immobilized onto magnetic beads (solid support) via streptavidin-biotin binding. Next, single-stranded oligo fragments were added successively through ligation to form the complete DNA molecule. The synthesized DNA was amplified through PCR and gel electrophoresis was used to characterize the product. Sanger sequencing showed that more than 97% of the nucleotides matched the expected sequence. Extending the length of the DNA molecule by adding single-stranded oligonucleotides from a basis set (library) via ligation enables a more convenient and rapid mechanism for the design and synthesis of oligonucleotides on the go. Coupled with an automated dispensing system and libraries of short oligo fragments, this novel DNA synthesis method would offer an efficient and cost-effective method for producing dsDNA.</p></div

Development of a Carbon Dot (C-Dot)-Linked Immunosorbent Assay for the Detection of Human α‑Fetoprotein

A sensitive, selective, environmentally friendly, high-throughput, well-plate-based immunosorbent assay was developed to detect human α-fetoprotein (AFP) using carbon dots (C-Dots). Highly fluorescent C-Dots were synthesized using a one-step hydrothermal reaction, with citric acid serving as the carbon source and ethylene diamine acting as the nitrogen source. The reaction conditions were optimized to obtain the desired surface functionality. Then, the C-Dots were used to label one member of the anti-AFP pair (Ab₂) via amine–amine coupling using glutaraldehyde. The capture anti-AFP (Ab₁) was coated onto polystyrene well plates and bovine serum albumin (BSA) was used to block unsaturated binding sites. AFP was incubated in Ab₁-coated wells; unbound AFP was then washed away with Tween-20. Next, the C-Dot-labeled Ab₂ was added to form a sandwich immunocomplex with the AFP bound to the Ab₁-coated wells. The fluorescence intensities detected from the C-Dots on these sandwich immunocomplexes were positively correlated to the concentrations of AFP antigen. A five-parameter logistic regression calibration curve was established between fluorescence and clinically important AFP concentrations (range: 0–350 ng/mL with a correlation coefficient of <i>R</i>² = 0.995). The results from the C-Dot-based immunoassay were in agreement with results from traditional immunoassays, which used horseradish peroxidase (HRP, <i>R</i>² = 0.964) and fluorescein isothiocyanate (FITC, <i>R</i>² = 0.973). These results indicated that C-Dots have great potential to be applied as biolabels for high-throughput well-plate-based immunoassays

Sequence of the oligo fragments used to form the complete dsDNA.

<p>Sequence of the oligo fragments used to form the complete dsDNA.</p

Schematic diagram for the proposed dsDNA synthesis.

<p>The overall procedure for dsDNA synthesis is composed of three processes: annealing, binding of streptavidin coated magnetic beads to biotinylated oligos, and ligation.</p

Agarose gel electrophoresis of annealed and ligation products.

<p>Lane 1 and 8, ladder; Lane 2, annealed product of N1b and N2b; Lane 3, annealed product of N3b and N4b; Lane 4, annealed product of N5b and N6b; Lane 5, “one-pot” ligation product of [(N1b-N2b)+(N3b-N4b)+(N5b-N6b)]; Lane 6, sequential ligation product of [(N1b-N2b)+(N3b-N4b)]+(N5b-N6b); Lane 7, ligation product of [(N1b-N2b)+(N5b-N6b)].</p

Schematic of the building blocks for DNA construction.

<p>Streptavidin-coated magnetic beads were used as solid support for dsDNA synthesis and oligo fragments were ligated to the building block one at a time.</p