TAILIEUCHUNG - analysis of genes and genomes phần 3

Khái niệm cơ bản của Nhân bản Các khả năng để phá vỡ và tái tham gia các phân tử DNA gần như sẽ dẫn đến những thí nghiệm đầu tiên trong nhân bản DNA vào năm 1972 (Jackson, Symons và Berg, 1972). | 78 BASIC TECHNIQUES IN GENE ANALYSIS 2 a O O Lys-NH2 DNA ligase ATP O -O-M- P-O-P- I I I O- O- O- complex b 5 I C A T A O G T A T 3 I 1- O-P-O- CAT HO-I 13 GTA 1 3y G T A 5 1 . r CATA 5 I 1 3 C A T A G T A O . gtat c a t 3 I zb-O-P- 1 1 I 5 Figure . The mechanism of DNA joining by DNA ligase. See the text for details. This figure is adapted from Doherty et al. 1996 The Basics of Cloning The ability to break and rejoin DNA molecules almost at will led to the first experiments in DNA cloning in 1972 Jackson Symons and Berg 1972 . 79 5 3 Insert BamHI EcoRI G G A T C C G A A T T C C C T A G G C T T A A G THE BASICS OF CLONING Cut with BamHI and EcoRI 5 G A T C C 3 ế cttaa 5- DNA ligase 3 G Figure . Breaking and joining DNA using restriction enzymes and DNA ligase. Linear DNA insert and a closed-circular plasmid DNA vector each contain the recognition site for BamHI and EcoRI. Mixing the DNA fragments with compatible ends together in the presence of DNA ligase can result in the formation of vector-insert hybrid DNA molecules For the first time it was possible to extract a fragment of DNA from one source and insert or clone it into the DNA from another source. Perhaps the most common type of cloning experiment involves the insertion of a foreign piece of DNA into a suitable vector so that the foreign DNA may be propagated in E. coli. In Chapter 3 we will discuss the various different 80 BASIC TECHNIQUES IN GENE ANALYSIS 2 types of vector that are available but at this stage we could consider the vector as a closed-circular double-stranded plasmid DNA molecule. If we wish to insert foreign DNA sequences into this vector we need to cut it to produce a linear DNA onto which we can attach other DNA sequences using DNA ligase. Let us first consider the insertion of DNA into the vector using two different restriction enzymes Figure . Treatment of both a vector and insert DNA sequences with the restriction enzymes will generate a number of DNA fragments. In .

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