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How single mutations in genes can be repaired with single-strand DNA fragments

By Edited Jan 3, 2016 0 0

Short, single-strand nucleotides have emerged as a group of extremely useful and versatile molecules that can direct an array of changes in the eukaryotic cell. Sequence-specific strings of (less than fifty) nucleotides can serve as primers for the polymerase chain reaction and DNA sequencing or as antisense molecules that are complementary to mRNA regulatory regions and thereby block the translation of the desired protein. In less than a decade, the technology of RNAi has evolved from a little-known phenomenon in plants to a ubiquitous laboratory method for protein knockdown and the basis for numerous therapeutic trials. In addition, nucleotide strands can be used as a tool of sequence complementation; nucleic acids also exhibit unique structural properties that together with their simple and efficient custom synthesis can provide novel uses for structurally designed nucleotide molecules to act as aptameric drug compounds. Oligonucleotides have the capacity to join the growing field of small molecule-based therapies devised to enable translational control and selectively target the acquired oncogenic mutations of malignant cells.


Oligonucleotide-directed gene repair involves the direct correction of a mutation or mutations in the chromosomal copy of human genes, a process also known as targeted nucleotide exchange. Gene repair is initiated by the introduction of short single strands of naked DNA molecules that are designed to anneal with a complementary sequence in the target gene. A single base at the center of the oligonucleotide is designed to create a mismatch with the target; this mismatch can be resolved in several ways leading to the desired change in the targeted nucleotide. Such single base changes can be made to alter disease-causing mutations or to create stop codons in genes to truncate and disable the protein of interest. This technology can also be employed as a alternative means to introduce specific changes in the genes of plant and animal model systems.


The current goal of oligonucleotide-directed gene repair is to increase the frequency of successful repair events per treatment, in order to obtain a therapeutically beneficial level of correction. Beyond multiple transfections of oligonucleotide, a number of methods can by employed to increase the success of gene repair. These techniques include increasing the accessibility of the DNA target by relaxing chromatin, synchronizing the cell cycle of target cells in order to deliver the oligonucleotides at the most opportune time, increasing the amount of DNA repair proteins that are required for facilitation of gene repair, and prolonging the half-life of the oligonucleotide after transfection.

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