Direct And Indirect Mutation Analysis III - Recombinant DNA Technology PDF 2020

Direct and indirect mutation analysis III: Fundamentals of recombinant DNA technology (restriction endonucleases and vectors) Research Assist. Merdiye Mavis  Recombinant DNA Recombinant DNA: a piece of DNA that has been created by insertion of a DNA fragment...

Direct and indirect mutation analysis III: Fundamentals of recombinant DNA technology (restriction endonucleases and vectors) Research Assist. Merdiye Mavis  Recombinant DNA Recombinant DNA: a piece of DNA that has been created by insertion of a DNA fragment from one cell or organism into the replicating DNA of another cell or organism. Combination of at least two DNA molecules.  Recombinant DNA Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources. These sequences that are created by genetic engineering technologies would not be found in the genome naturally. DNA sequences used in the construction of rDNA molecules >> can originate from any species. Using recombinant DNA technology and synthetic DNA – literally any DNA sequence may be created – and introduced into any of a very wide range of living organisms Recombinant DNA technology or genetic engineering have at their core the process of gene cloning.  Gene Cloning Introduction of a foreign DNA molecule into a replicating cell permits the cloning or amplification of that DNA. Many identical copies of the DNA of interest can be produced. Alternative to gene cloning is PCR.  Basics Steps of Gene Cloning 1. A fragment of DNA, containing the gene to be cloned, is inserted into a circular DNA molecule called a vector, >> to produce a recombinant DNA molecule. 2. The vector transports the gene into a host cell, which is usually a bacterium, although other types of living cell can be used. 3. Within the host cell the vector multiplies, producing numerous identical copies, not only of itself but also of the gene that it carries.  Basics Steps of Gene Cloning 4. When the host cell divides, copies of the recombinant DNA molecule are passed to the progeny and further vector replication takes place. 5. After a large number of cell divisions, a colony, or clone, of identical host cells is produced. – Each cell in the clone contains one or more copies of the recombinant DNA molecule the gene carried by the recombinant molecule is now said to be cloned.  Vectors for Gene Cloning A vector is a molecule of DNA to which the fragment of DNA to be cloned is joined. Commonly used vectors include plasmids and viruses.  Essential properties of a vector include: Must be able to replicate within the host cells – numerous copies of the recombinant DNA molecule can be produced and passed to the daughter cells Must be capable of autonomous replication within a host cell Needs to be relatively small, ideally less than 10 kb in size – Large molecules tend to break down during purification – Large molecules are hard to manipulate It must contain at least one specific nucleotide sequence recognized by a restriction endonuclease. It must carry at least one gene that confers the ability to select for the vector, such as an antibiotic resistance gene.  Plasmids Plasmids are circular and double-stranded molecules of DNA that lead an independent existence in the bacterial cell. Most plasmids possess at least one DNA sequence that can act as an origin of replication, so they are able to multiply within the cell independently of the main bacterial chromosomes. Plasmids almost always carry one or more genes. Often these genes are responsible for a useful characteristic displayed by the host bacterium. For example, the ability to survive in normally toxic concentrations of antibiotics such as chloramphenicol or ampicillin is often due to the presence in the bacterium of a plasmid carrying antibiotic resistance genes.  Contains selective marker: for selection of cells containing a plasmid In the laboratory, antibiotic resistance is often used as a selectable marker to ensure that bacteria in a culture contain a particular plasmid. Size and Copy Number of Plasmids Desirable size: less than 10 kb Copy number: – the number of molecules of an individual plasmid that are normally found in a single bacterial cell. – High copy number is desired. – Needs to be present in the cell in multiple copies so that large quantities of the recombinant DNA molecule can be obtained. – However low-copy-number plasmids may be preferably used in certain circumstances, for example, when the protein from the cloned gene is toxic to the cells  Bacteriophages Bacteriophages, or phages are viruses that specifically infect bacteria. The bacteriophages used for cloning are the phage λ and M13 phage. Like all viruses, phages are very simple in structure, consisting merely of a DNA molecule carrying a number of genes. These genes include several for replication of the phage, surrounded by a protective coat or capsid made up of protein molecules. Useful for cloning large DNA fragments (10-23 kb) Bacteriophage Lambda as a Cloning Vector The general pattern of infection of a bacterial cell by a bacteriophage  Other vectors: Naturally occurring viruses that infect mammalian cells (retroviruses,for example) Artificial constructs such as cosmids - useful for cloning very large DNA fragments Bacterial or yeast artificial chromosomes (BACs or YACs, respectively)  Enzymes for Cutting DNA - Restriction Endonucleases DNA molecules should be cut in a very precise and reproducible fashion. Each vector molecule must be cleaved at a single position – to open up the circle – so that new DNA can be inserted Each vector molecule must be cut at exactly the same position on the circle.  It is also necessary to cleave the DNA that is to be cloned. 2 reasons for this: – If the aim is to clone a single gene, which may consist of only 2 or 3 kb of DNA, then that gene will have to be cut out of the large (often greater than 80 kb) DNA molecules. – Large DNA molecules may have to be broken down to produce fragments that are small enough to be carried by the vector.  Type II restriction endonucleases cut DNA at specific nucleotide sequences Type II restriction endonucleases >> each enzyme has a specific recognition sequence at which it cuts a DNA molecule and nowhere else. The recognition sequences are palindromes. They have twofold symmetry. A Palindrome  Type II restriction endonucleases - examples PvuI cuts DNA only at the hexanucleotide CGATCG. Many restriction endonucleases recognize hexanucleotide target sites, but others cut at four, five, eight, or even longer nucleotide sequences. There are also examples of restriction endonucleases with degenerate recognition sequences >> meaning that they cut DNA at any one of a family of related sites. HinfI recognizes GANTC, so cuts at GAATC, GATTC, GAGTC, and GACTC. The recognition sequences for some of the most frequently used restriction endonucleases.  Blunt ends and Sticky ends Blunt Ends Many restriction endonucleases make a simple double-stranded cut in the middle of the recognition sequence resulting in a blunt end or flush end. PvuII and AluI are examples of blunt end cutters.  Sticky ends Some enzymes do not cut the two DNA strands at exactly the same position. Instead the cleavage is staggered, usually by two or four nucleotides, so that the resulting DNA fragments have short single-stranded overhangs at each end. These are called sticky or cohesive ends, as base pairing between them can stick the DNA molecule back together again. Restriction endonucleases with different recognition sequences may produce the same sticky ends.  Formation of recombinant DNA from restriction fragments with “sticky” ends.  Ligation – joining DNA molecules together Final step in construction of a recombinant DNA molecule >> joining together of the vector molecule and the DNA to be cloned. This process is referred to as ligation, and the enzyme that catalyzes the reaction is called DNA ligase. In the test tube, purified DNA ligases can join together individual DNA molecules or the two ends of the same molecule. The chemical reaction involved in ligating two molecules is that two phosphodiester bonds must be made, one for each strand. Ligation of blunt-ended and sticky- ended molecules  Sticky ends increase the efficiency of ligation Two blunt-ended fragments can be joined together. This reaction can be carried out in the test tube but it is not very efficient. This is because the ligase is unable to “catch hold” of the molecule to be ligated, and has to wait for chance associations to bring the ends together. In contrast, ligation of complementary sticky ends is much more efficient. This is because compatible sticky ends can base pair with one another by hydrogen bonding a relatively stable structure for the enzyme to work on. Introduction of DNA into Living Cells  Transformation—the uptake of DNA by bacterial cells Not all species of bacteria are equally efficient at DNA uptake. Most species of bacteria, including E. coli, take up only limited amounts of DNA under normal circumstances. In order to transform these species efficiently, the bacteria have to undergo some form of physical and/or chemical treatment that enhances their ability to take up DNA. Cells that have undergone this treatment are said to be competent.  Selection for transformed cells Transformation of competent cells is an inefficient procedure. Some way must be found to distinguish a cell that has taken up a plasmid from the many thousands that have not been transformed.  For example, E. coli cells are normally sensitive to the growth inhibitory effects of the antibiotics ampicillin and tetracycline. Cells that contain the plasmid pBR322 are resistant to these antibiotics. This is because pBR322 carries two sets of genes, one gene that codes for a b-lactamase enzyme that modifies ampicillin into a form that is non-toxic to the bacterium, and a second set of genes that code for enzymes that detoxify tetracycline. After a transformation experiment with pBR322, only those E. coli cells that have taken up a plasmid are ampRtetR and able to form colonies on an agar medium that contains ampicillin or tetracycline. Non-transformants, which are still ampStetS, do not produce colonies on the selective medium. Transformants and non-transformants are therefore easily distinguished.  Identification of recombinants Which of the transformed colonies comprise cells that contain recombinant DNA molecules, and which contain self ligated vector molecules?  Identification of recombinants With most cloning vectors, – insertion of a DNA fragment into the plasmid destroys the integrity of one of the genes present on the molecule. Recombinants can therefore be identified because the characteristic coded by the inactivated gene is no longer displayed by the host cells. Insertional inactivation  Recombinant selection with pBR322—insertional inactivation of an antibiotic resistance gene BamHI, for example, cuts pBR322 at just one position, within the cluster of genes that code for resistance to tetracycline. A recombinant pBR322 molecule, one that carries an extra piece of DNA in the BamHI site, is no longer able to confer tetracycline resistance on its host, as one of the necessary genes is now disrupted by the inserted DNA. Cells containing this recombinant pBR322 molecule are still resistant to ampicillin, but sensitive to tetracycline(ampRtetS ).  After transformation, – the cells are plated onto ampicillin medium – incubated until colonies appear All of these colonies are transformants  untransformed cells are ampS and so do not produce colonies on the selective medium BUT only a few contain recombinant pBR322 molecules Most contain the normal, self-ligated plasmid.  To identify the recombinants – the colonies are replica plated onto agar medium that contains tetracycline – Incubation – some of the original colonies regrow, but others do not. Those that do grow >> consist of cells that carry the normal pBR322 The colonies that do not grow >> are recombinants (ampRtetS) Samples for further study can be recovered from the original ampicillin agar plate.  Insertional inactivation does not always involve antibiotic resistance – Lac Selection/Blue-White Screen pUC8 – carries ampicillin resistance gene – lacZ′ gene -- which codes for part of the enzyme b-galactosidase.  B-galactosidase is normally coded by the gene lacZ, which resides on the E. coli chromosome. Some strains of E. coli have a modified lacZ gene, one that lacks the segment referred to as lacZ′ and coding for the a- peptide portion of b-galactosidase. These mutants can synthesize the enzyme only when they harbor a plasmid, such as pUC8, that carries the missing lacZ′ segment of the gene.  A cloning experiment with pUC8 involves selection of transformants on ampicillin agar followed by screening for b-galactosidase activity to identify recombinants. Cells that harbor a normal pUC8 plasmid are ampR and able to synthesize b- galactosidase Recombinants are also ampR but unable to make b-galactosidase. Screening for b-galactosidase presence or absence: There is a lactose analog called X-gal which is broken down by b-galactosidase to a product that is colored deep blue. If X-gal (plus an inducer of the enzyme such as isopropylthiogalactoside, IPTG) is added to the agar, along with ampicillin – then non-recombinant colonies, the cells of which synthesize b-galactosidase, will be colored blue, – recombinants with a disrupted lacZ′ gene and unable to make b-galactosidase, will be white. This system, which is called Lac selection.  Transfection Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. Transfection Methods Cloning serves two main purposes: 1. It allows a large number of recombinant DNA molecules to be produced from a limited amount of starting material. – At the outset only a few nanograms of recombinant DNA may be available, but each bacterium that takes up a plasmid subsequently divides numerous times to produce a colony, each cell of which contains multiple copies of the molecule. 2. Purification - Can provide a pure sample of an individual gene, separated from the other genes in the cell.