Molecular Biology Problem Solver 51. Sách được nhiều nhà khoa học có uy tín, nhiều kinh nghiệm trong nghiên cứu thực nghiệm trình bày những vấn đề thường hay phát sinh trong phòng thí nghiệm. Do vậy mà sách không trình bày các protocol hay quy trình như các sách khác, thay vào đó các tác giả sẽ trình bày các vấn đề nhằm giúp giúp đọc giả: Tự nâng cao khả năng chẩn đoán nguyên nhân khi gặp các vấn để về kỹ thuật, quy trình, hóa chất, thuốc thử trong quá trình thực nghiệm trong phòng thí. | transmembrane receptor or anchored to the surface . through a glycosyl phosphatidylinositol phosphate GPI linkage . Fortunately we usually have the luxury of working with genes that are at least partially characterized by their biological properties. But what about the genes of unknown origin or function In this new age of genomics many of the genes we obtain are like genes belonging to large families of related genes that share only a minimal percentage of homology with a known gene. Despite these similarities there is often no way to know whether the same expression and purification methods used for one orthologue or homologue will be effective for another. Thus one is immediately faced with the challenging prospect of having to consider multiple expression strategies in order to get the protein expressed and purified to sufficient levels in an active form in addition to not knowing what activity to look for. Can You Obtain the cDNA Before embarking on an expression project you will need to locate a cDNA copy of the gene of interest. It is also possible in theory to express genomic DNA containing introns provided that the expression host will recognize the proper splice junctions. In practice however this is not often the most efficient route to expression because it is not usually known how the introns will affect expression levels or whether the desired splice variant will be expressed. Furthermore most mammalian genes are interrupted by multiple intron sequences that can span many kilobases in length. This can make subcloning of genomic DNA considerably more difficult than for the corresponding cDNA. The three most common ways to obtain a known gene of interest include purchase from a distributor of clones from the Integrated Molecular Analysis of Genomes and their Expression IMAGE consortium http requests from a published source such as an academic lab or RT-PCR cloning from RNA derived from a cell or tissue source. IMAGE clones can be found
