This review describes the application of oligonucleotides, which are mainly obtained using DNA synthesizers of a new generation (microarray DNA synthesizers), for the enrichment of target genomic fragments. The methods of molecular hybridization, polymerase chain reaction, and CRISPR-Cas9 system for this purpose are considered. Examples of the practical use of the developed methods for research and diagnostic purposes are given.
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Chem Sci
December 2024
Department of Chemistry,, and Health Research Institute, Michigan Technological University Houghton Michigan 49931 USA
The longest oligos that can be chemically synthesized are considered to be 200-mers. Here, we report direct synthesis of an 800-mer green fluorescent protein gene and a 1728-mer 29 DNA polymerase gene on an automated synthesizer. Key innovations that enabled this breakthrough include conducting the synthesis on a smooth surface rather than within the pores of traditional supports, and the use of the powerful catching-by-polymerization (CBP) method for isolating the full-length oligos from a complex mixture.
View Article and Find Full Text PDFJ Am Chem Soc
December 2024
School of Applied and Interdisciplinary Sciences, Indian Association for the Cultivation of Science, Kolkata 700 032, India.
Phosphorodiamidate Morpholino Oligonucleotides (PMOs) have been well established in the milieu of FDA-approved oligonucleotide-based drugs in the past decade. Given their relevance in antisense therapeutics, a DNA/RNA synthesizer-compatible modular synthesis protocol of PMOs is long awaited to explore next-generation PMO chimeras with other therapeutically proven oligonucleotide backbones. Herein, we demonstrate a streamlined 5' → 3'phosphoramidite approach for the synthesis of PMOs using -butyl-protected 5'-morpholino phosphoramidites, which were synthesized from 5'-OH morpholino monomers derived from commercially available ribonucleosides.
View Article and Find Full Text PDFAppl Biosaf
September 2024
Built Biotechnologies Inc., Charlottesville, Virginia, USA.
Org Biomol Chem
September 2024
Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Methods Mol Biol
March 2024
MOE International Joint Research Laboratory on Synthetic Biology and Medicines, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, China.
Efficient preparation of DNA oligonucleotides containing unnatural nucleobases (UBs) that can pair with their cognates to form unnatural base pairs (UBPs) is an essential prerequisite for the application of UBPs in vitro and in vivo. Traditional preparation of oligonucleotides containing unnatural nucleobases largely relies on solid-phase synthesis, which needs to use unstable nucleoside phosphoramidites and a DNA synthesizer, and is environmentally unfriendly and limited in product length. To overcome these limitations of solid-phase synthesis, we developed enzymatic methods for daily laboratory preparation of DNA oligonucleotides containing unnatural nucleobase dNaM, dTPT3, or one of the functionalized dTPT3 derivatives, which can be used for orthogonal DNA labeling or the preparation of DNAs containing UBP dNaM-dTPT3, one of the most successful UBPs to date, based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT).
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