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Biotechnology & Genetic Engineering Lecture ( 10 ) Assi. Prof. Rajaa Al –Anbaqi Cloning :What is Cloning ? The terms recombinant DNA technology ,DNA cloning ,molecular cloning ,or gene cloning all refer to the same process ; the transfer of a DNA fragment of interest from one organism to a self –replicating genetic element such as a bacterial plasmid ( cloning vector ) . The DNA of interest can then be propagated in a foreign host cell . This technology has been around since the 1970 s , and it has become a common practice in molecular biology labs today . Cloning is frequently employed to amplify DNA fragmentes containing genes , an essential step in their subsequent analysis . How does molecular cloning work ? Cloning of any DNA sequence involves the introduction of a foreign piece of DNA into an extra chromosomal element ( cloning vector ) of an organism which then produces copies of the vector as it replicates itself ,thereby amplifying the DNA of interest . The whole process can be summarized in the following steps : fragmentation , ligation ,transfection , screening / selection , and conformation of ins 1 The first recombinant DNA molecules were generated at Stanford University in 1972, utilising the cleavage properties of restriction enzymes (scissors) and the ability of DNA ligase to join DNA strands together (glue). The importance of these first tentative experiments cannot be overestimated. Scientists could now join different DNA molecules together and could link the DNA of one organism to that of a completely different organism. The methodology was extended in 1973 by joining DNA fragments to the plasmid pSC101,which is an extra chromosomal element isolated from the bacterium Escherichia coli. These recombinant molecules behaved as replicons; that is, they could replicate when introduced into E. coli cells (host cells). Thus, by creating recombinant molecules in vitro, and placing the construct in a bacterial cell where it could replicate in vivo, specific fragments of DNA could be isolated from bacterial colonies that formed clones (colonies formed from a single cell, in which all cells are identical) when grown on agar plates. This development marked the emergence of the technology that became known as gene cloning (Fig. 1.3) Or another way for explain it: The mainstay of genetic manipulation is the ability to isolate aGene cloning enables isolation and identification of individual genes. Single DNA sequence from the genome. This is the essence of gene cloning and can be considered as a series of four steps (Fig. 1.1) A useful analogy is to consider gene cloning as a form of molecular agriculture, enabling the production of large amounts (in genetic engineering this means micrograms or milligrams) of a particular this ability to isolate a particular gene sequence is still a major aspect of gene manipulation. 2 1- The first step requires basic step it generation of DNA fragments (see map1) by Preparation of DNA fragments for sequencing is largely dependent on the scale of the sequencing project. If the aim is to sequence part of a gene that has already been isolated and identified, the process is relatively straightforward and usually requires that the fragment is of a suitable length and in a suitable form for the sequencing procedure in use If, however, the aim is to sequence a much larger piece of DNA (such as an entire chromosome), the problem is much greater. In this case the sequencing strategy is important, and there are two approaches to this. The first is an ordered sequencing strategy. The second is the so-called shotgun sequencing method, where the fragments are generated and processed at random. Assembly of the sequence is then carried out by searching for sequence overlaps using a computer. The two strategies are shown in 3.7 3 Generation and preparation of DNA fragments is fairly simple on a purely technical level. The fragments are often cloned sequences that are presented for sequencing in a suitable vector; with careful attention to detail where the fragment is within the genome; there are basically two approaches. The first is an ordered sequencing strategy. The second is the so-called shotgun sequencing method , The sequencing protocol is essentially a technical procedure. There are two main methods for sequencing DNA. The first is a chemicals and the second is Enzymatic method . but the most widely used techniques are based on the enzymatic method .Whatever the method , the desired result is to generate a set of overlapping fragments that terminate at different bases and differ in length by one nucleotide. This is known as a set of nested fragments. The final stage Analysis of fragments is similar for both methods (chemicals and enzymatic methods)) it usually involves separation of the fragments on a polyacrylamide gel (electrophoresis) and autoradiography which are dentified as they pass a detector. When the generation of 4 fragments (for enzymatic sequencing or chemical sequencing ) and usually involves subcloning into different vectors. 2- Joining to a vector or carrier molecule Gene cloning is achieved by using a vector (carrier) to propagate the desired sequence in a host cell. Choosing the right vector/host combination is one of the critical stages of a cloning procedur. Many plasmid vectors are now available and some types can be used directly for DNA sequencing experiments. Another method is to clone the DNA into a vector such as the bacteriophage M13, which produces single-stranded DNA during infection. This provides a suitable substrate for the sequencing reaction .The various types of host cell and vector systems and methods for getting DNA into cell .We need now focus on A- the vectors characters , B-types of vector systems and C- methods for getting DNA into cell . A- There are certain essential features that vectors must posses:-Ideally 1- They should be fairly small DNA molecules, to facilitate isolation and handling. 2- There must be an origin of replication, so that their DNA can be copied and thus maintained in the cell population as the host organism grows and divides. 3 - It is desirable to have some sort of selectable marker that will enable the vector to be detected. 4- The vector must have at least one unique restriction endonuclease recognition site to enable DNA to be inserted during the production of recombinants. Plasmids have these features and are extensively used as vectors in cloning experiments. B- Types of vector systems:- There are two main types :1- Plasmids :- Many types of plasmids are found in nature, in bacteria and some yeasts. They are circular DNA molecules, relatively small when compared to the host cell chromosome and have been Specially constructed. Plasmids are extrachromosomal genetic elements that are not essential for bacteria to survive but often confer advantageous traits (such as antibiotic resistance) on the host cell. The antibiotic resistance genes encoded by plasmid DNA (pDNA) are often used in the construction of vectors for genetic engineering, as they provide a convenient means of selecting cells containing the plasmid. When plated on growth medium that contains the appropriate antibiotic, only the plasmid-containing cells will survive. This is a very simple and powerful selection method asmid found in the host cell, known as the copy number. ALow-copy-number plasmids . B- High copy- number plasmids 2- Phages :- The term ‘bacteriophage’ is often shortened to ‘phage’ and can be used to describe either one or many particles of the same type. Thus, we might say that a test tube contained one ^ phage or 2 × 106 _ phage particles. The plural term ‘phages’ is used when different types of phage are being considered; we therefore talk of T4, M13, and ^as being phages Bacteriophages are essentially bacterial viruses and usually consist of a DNA genome enclosed in a protein head (capsid). As with other viruses, they depend on the host cell for their propagation and do not exist as free-living organisms. Structurally, phages fall into three main groups: (1) tailless (2) Bacteriophages are essentially bacterial viruses and usually consist of a DNA genome enclosed in a protein head (capsid). As 5 with other viruses, they depend on the host cell for their propagation and do not exist as free-living organisms. head with tail, and (3) filamentous. The genetic material may be single or double-stranded DNA or RNA, with double-stranded DNA (dsDNA) found most often. In tailless and tailed phages the genome is encapsulated in an icosahedral protein shell called a capsid (sometimes known as a phage coat or head). The genome of phage ^ is 48.5 kb in length, and encodes some 46 genes (Fig. 5.4). The entire genome has been sequenced (this was the first major sequencing project to be completed, and represents one of the milestones of molecular genetics), and all the regulatory sites are known. At the ends of the linear genome there are short (12 bp) single-stranded regions that are complementary. These act as cohesive or ‘sticky ends, which enable circularisation of the genome following infection. The region of the genome that is generated by the association of the cohesive ends is known as the cos site . The utility of phage ^ as a cloning vector depends on the fact that not all of the ^ genome is essential for the phage to function. Thus, there is scope for the introduction of exogenous DNA, although certain requirements have had to be met during the development of cloning vectors based on phage^ . First, the arrangement of genes on the ^ genome will determine which parts can be removed or replaced for the addition of exogenous DNA. It is fortunate that the central region of the ^ genome (between positions 20 and 35 on the map shown in Fig. 5.4) is largely dispensable, so no complex rearrangement of the genome in vitro is required. The central region controls mainly the lysogenic properties of the phage, and much of this region can be deleted without impairing the functions required for the lytic infection cycle. Second, wild-type^ phage will generally have multiple recognition sites for the restriction enzymes commonly used in cloning procedures. This can be a major problem, as it limits the choice of sites for the insertion of DNA. In practice, it is relatively easy to select for phage that have reduced numbers of sites for particular restriction enzymes, and the technique of mutagenesis in vitro may be used to modify remaining sites that are not required. Thus, it 6 is possible to construct phage that have the desired combination of restriction enzyme recognition sites. Life cycle of bacteriophago . 3- Other vectors :- Cosmids and phagemids incorporate some of the characteristics of both plasmid and phage-based vectors. Cosmids , This has been exploited in the construction of vectors that are made up of plasmid sequences joined to the cos sites of phage^.Hybrid plasmid/phage vectors in which the phage functions are expressed and utilised in some way are known as Phagemids . 7 3-introduction into a host cell for amplification :- back to map 5 8