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Open Reading Frame Cloning
Open reading frame (ORF) cloning is the cloning of only the coding sequence of a gene, no introns, from the start to stop site. ORF cloning usually begins with polymerase chain reaction (PCR) amplification of the desired ORF from complementary DNA (cDNA). cDNA is created from single-stranded messenger RNA (mRNA) so it only contains the coding regions and untranslated 5’ and 3’ ends of genes. The ORF is then inserted into a vector, which is a DNA molecule for transport of DNA into a host cell, to create a circularized piece of DNA called a plasmid. The plasmid is added to host cells, usually bacteria, where it is replicated and then purified out of the cells. ORF cloning is especially useful for the production of a specific protein product for a gene of interest. Genes can code for several proteins by alternative splicing of mRNA to create ORFs with different coding sequences. By cloning from the mRNA with the desired ORF, only one protein product will be translated. Sequencing of the cloned ORF is suggested to ensure expression of the desired protein product. The applications for ORF cloning include recombinant protein expression, the creation of tagged fusion proteins, protein localization studies, and functional assays. (Credit: Brooke Anderson-White)
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