Lecture_3-Vectors.pdf
- 1. Recombinant DNA Technology (content # 2) Cloning Vectors Dr. Amir Hameed
- 2. • The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector. • A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes. • Most vectors are genetically engineered. • The cloning vector is chosen according to the size and type of DNA to be cloned. Cloning Vector
- 3. • The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together. • After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use. • A DNA molecule needs to display several features to be able to act as a vector for gene cloning. Cloning Vector
- 4. • Cloning vector is used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and expressed. • It is used to amplify a single molecule of DNA into many copes. • Cloning vectors are DNA molecules that are used to "transport" cloned sequences between biological hosts and the test tube. • Without Cloning Vector, Molecular Gene Cloning is totally impossible. Why Cloning Vector?
- 6. • Scientists (Herbert Boyer, Keiichi Itakura and Arthur Riggs) working in Boyer’s lab (University of California) recognized a general cloning vector with unique restriction sites for cloning a foreign DNA and the expression of antibiotic resistance genes for selection of transformed bacteria. • In 1977, they described the first vector designed for cloning purposes, pBR322 – a plasmid. • This vector was small, ~4 kb in size, and had two antibiotic resistance genes for selection. History
- 8. All commonly used cloning vectors have some essential features: • Origin of replication (ori): – This makes autonomous replication in vector. – ori is a specific sequence of nucleotide from where replication starts by DNA polymerase of host. – When foreign DNA is linked to the sequence along with vector replication, foreign (desirable) DNA also starts replicating within host cell. • Cloning Site: – Cloning site is a place where the vector DNA can be digested and desired DNA can be inserted by the same restriction enzyme. – It is a point of entry or analysis for genetic engineering work. – Recently recombinant plasmids contain a multiple cloning site (MCS) which have many (up to ~20) restriction sites. Features of A CloningVector
- 9. • Selectable Marker – Selectable marker is a gene that confers resistance to particular antibiotics or selective agent that would normally kill the host cell or prevent its growth. – A cloning vector contains a selectable marker, which confer on the host cell an ability to survive and proliferate in a selective growth medium containing the particular antibiotics. • Reporter Gene or MarkerGene – Reporter genes are used in cloning vectors to facilitate the screening of successful clones by using features of these genes that allow successful clone to be easily identified. – Such feature present in cloning vectors is used in blue- white selection.
- 10. • Additional Properties of Vectors: ─ It should be short, small. ─ Compatible with host cell. ─ Incompatible with other vector. ─ Should become high in copy number. ─ It should able to express itself utilizing the host machinery. ─ It should be able to move under two system (Prokaryote and Eukaryote system).
- 12. • Plasmid • Bacteriophage • Cosmid • Bacterial Artificial Chromosome(BAC) • Yeast Artificial Chromosome (BAC) • Human Artificial Chromosome(HAC) • Retroviral Vectors Types of Cloning Vectors
- 14. • Plasmid is an autonomously replicating circular double stranded extra- chromosomal DNA which is physically separated from a chromosomal DNA andcan replicate independently. • They are most commonly found in bacteria, sometimes they are present in archaea and eukaryotic organisms. • The size of the plasmid varies from 1 to over 250 kb. • Most general plasmids may be used to clone DNA insert of up to 10 kb in size. • Many plasmids (Relaxed) have high copy number (50) and high copy number is useful as it produces greater yield of recombinant plasmid for subsequent manipulation • However (Stringent) low copy number (1 or 2) plasmids may be preferably used in certain circumstances, for example, when the protein from the cloned gene is toxic to the cells. Example: pBR322, pUC18, F plasmid, Col Plasmid etc. Plasmid
- 16. • The name ‘pBR322’ conforms with the standard vector nomenclature. • ‘p’ indicates that this is indeed a plasmid • ‘BR’ identified the laboratory where it was originally constructed (BR stands for Bolivar and Rodriguez the two researchers who developed it. • ‘322’ distinguishes this plasmid from the same laboratory (there are also p pBR327 etc.) The Nomenclature of Plasmid Cloning Vector
- 17. Types of Plasmids Plasmids fall into two groups: conjugative and non-conjugative. Conjugative plasmids are characterized by the ability to promote sexual conjugation between bacterial cells. Conjugation and plasmid transfer are controlled by a set of transfer or tra genes, which are present on conjugative plasmids but absent from the non-conjugative type. A non-conjugative plasmid may, under some circumstances, be co-transferred along with a conjugative plasmid when both are present in the same cell. In fact, cells of E. coli have been known to contain up to seven different plasmids at once. To be able to coexist in the same cell, different plasmids must be compatible. Different types of plasmid can therefore be assigned to different incompatibility groups on the basis of whether or not they can coexist.
- 18. Plasmid classification (based on plasmid genes) • Fertility or F plasmids carry only tra genes and have no characteristic beyond the ability to promote conjugal transfer of plasmids. A well-known example is the F plasmid of E. coli. • Resistance or R plasmids carry genes conferring resistance to one or more antibacterial agents, such as chloramphenicol, ampicillin, and mercury. R plasmids are very important in clinical microbiology as their spread through natural populations can have profound consequences in the treatment of bacterial infections. An example is RP4, which is commonly found in Pseudomonas, but also occurs in many other bacteria. • Col plasmids code for colicins, proteins that kill other bacteria. An example is ColE1 of E. coli. • Degradative plasmids allow the host bacterium to metabolize unusual molecules such as toluene and salicylic acid, an example being TOL of Pseudomonas putida. • Virulence plasmids confer pathogenicity on the host bacterium; these include the Ti plasmids of Agrobacterium tumefaciens, which induce crown gall disease on dicotyledonous plants.
- 19. Why Plasmids are Good Cloning Vectors: • Small size (easy to manipulate and isolate). • Circular (more stable). • Replication independent of host cell. • Several copies may be present (facilitates replication). • Frequently have antibiotic resistance (detection easy). Disadvantages Using Plasmids: • Cannot accept large fragments. • Sizes range from 0 – 10kb. • Standard methods of transformation are inefficient.
- 20. • Viruses that specifically infect bacteria. • The bacteriophages used for cloning are the phage λ and M13 phage. • There is an upper limit on the amount of DNA that can be packed into a phage (a maximum of 53 kb). • There is also a lower size limit for DNA that can be packed into a phage, and vector DNA that is too small cannot be properly packaged into the phage. Bacteriophages
- 21. Bacteriophages
- 22. • Phage lambda is a bacteriophage or phage, i.e. bacterial virus, that uses E. coli as host. • Its structure is that of a typical phage: head, tail, tail fibres. • Lambda viral genome: 49kb DNA with a 12 base ssDNA "sticky end" at bothends; these ends are complementary in sequence and can hybridize to each other (this is the cos site: cohesive ends). • Infection: lambda tail fibres adsorb to a cell surface receptor, the tail contracts, and the DNA is injected. • The DNA circularizes and lambda begins its life cycle in the E. colihost. • There are two kinds of λ phage vectors - insertion vector and replacement vector. – Insertion vectors contain a unique cleavage site whereby foreign DNA with size of 5–11 kb may be inserted. – In replacement vectors, the cleavage sites flank a region containing genes not essential for the lytic cycle may be deleted and replaced by the DNA insert in the cloning process. Phage Lambda
- 27. • M13 is an example of a filamentous phage. • M13 genome is only 6.4 kb and unusually ssDNA. • M13 vectors are used for obtaining ss copies of DNA which are suited for DNA sequencing and in vitro mutagenesis. • Injection of an M13 DNA molecule into an E. coli cell occurs via the pilus. • Replicates in bacteria until over 100 copies are present in the cell. • Examples M13mp18 & M13mp19 M13 Phage Vector M13 vectors are also used in phage display, a technique for identifying pairs of genes whose protein products interact with one another.
- 30. • A phagemid is a plasmid that contains an f1 origin of replication from an f1 bacteriophage • It can be used as a type of cloning vector in combination with helper filamentous phage M13. • A phagemid can be replicated as a plasmid, and also be packaged as single stranded DNA in viral particles. • pEMBL18 & pEMBL19 Phagemid
- 31. Phage Vectors Present TwoAdvantages Over PlasmidVectors- 1. They are more efficient than plasmids for cloning of large DNA fragments; the largest cloned insert in lambda phage is 24 kb, while for plasmid vector it is less than 15 kb. 2. It is easier to screen a large number of phage plaques than bacterial colonies for identification of recombinant vectors.
- 32. • Cosmids are plasmids that incorporate a segment of bacteriophage λ DNA that has the cohesive end site (cos) which contains elements required for packaging DNA into λ particles. • It is normally used to clone large DNAfragments between 25-45 kb (genomic libraries). • They can replicate as plasmids if they have a suitable origin of replication. ColE1 ori for ds DNA replication, or f1 ori for ss DNA replication in prokaryotes • They can also be packaged in phage capsids, which allows the foreign genes to be transferred into cells by transduction. Advantages : • High transformation efficiency. • The cosmid vector can carry up to 45 kb whereas plasmid and Lambda phage vectors are limited to 25 kb. Cosmid
- 33. Scheme of DNA cloning in a cosmid vector
- 34. • Yeast artificial chromosomes (YACs) provide the largest insert capacity of any cloning system (100–1000 kb of transgene). • YACs are genetically engineered chromosomes derived from the DNA of the yeast, Saccharomyces cerevisiae. • The YAC vector itself provides the essential elements for propagation of DNA as a chromosome in the yeast Saccharomyces cerevisiae. • YACs typically contain a yeast centromere, two functional telomeres, and auxotrophic markers for selection of the YAC in an appropriate yeast host. • YAC ligated with a bacterial plasmid can be used both in yeast and bacteria. • YAC carry the largest inserts of all and are used extensively in cloning large genomes such as the human genome and in expression libraries. Yeast Artificial Chromosome (YAC)
- 35. • The centromere, which is required for the chromosome to be distributed correctly to daughter cells during cell division. • Two telomeres, the structures at the ends of a chromosome, which are needed in order for the ends to be replicated correctly and which also prevent the chromosome from being nibbled away by exonucleases. • The origins of replication, which are the positions along the chromosome at which DNA replication initiates, similar to the origin of replication of a plasmid. Yeast Artificial Chromosome (YAC)
- 37. • BAC vectors are similar to standard E. coli plasmid vectors. • Contain the origin and genes encoding the ori binding proteins required for plasmid replication. • Derived from a naturally occurring large plasmid, the F ʹ-plasmid. • Low copy number (1-2 copies per cell) • The bacterial artificial chromosome's usual insert size is 150-350 kb. • BACs are preferred for different kind of genetic studies of inherited or infectious diseases because they accommodate much larger sequences without the risk of rearrangement, and are therefore more stable than other types of cloning vectors. Bacterial Artificial Chromosome (BAC)
- 39. • PAC was developed by Nat Sternberg (1990s). The constructed vector incorporates features of both P1 bacteriophage and F ʹ plasmid and can be transformed into E.coli by electroporation. In a PAC vector, inserts of size 100-300 kb can be cloned. It is devoid of problem such as instability of cloned DNA. Advantages of BACs compared to YACs • Stable • Ease to transformation • Speed of growth of E. coli host • Simpler to purify • More user friendly • They are helpful in the development of vaccines P1-Derived Artificial Chromosome (PAC)
- 40. • Human artificial chromosome (HAC) is a microchromosome that can act as a new chromosome in a population of human cells e.g. (human HT1080 cells). • roughly 6–10 megabases (Mb) in size instead of 50–250 Mb for natural chromosomes, and able to carry new genes introduced by human researchers. • It is a tool for expression studies and determining human chromosome function and can carry very large DNA fragments. • HACs differ in this regard, as they are entirely separate chromosomes. This separation from existing genetic material assumes that no insertional mutants would arise Human Artificial Chromosome (HAC)
- 42. • Most plants are subject to viral infection, so much more convenient to use • Viruses transformation can be achieved simply by rubbing the virus. • One problem majority of plant viruses have genomes not of DNA but of RNA. Only two classes of DNA virus are– the caulimoviruses and geminiviruses • Caulimovirus vectors: • in 1984, used a caulimovirus vector to clone a new gene into turnip plants • the virus genome the capacity for carrying inserted DNA is still very limited. • adopting a helper virus strategy • cauliflower mosaic virus (CaMV) vector + wildtype CaMV • Narrow host range (turnips, cabbages and cauliflowers) • highly active promoters that function in all plants. Plant viruses as cloning vectors
- 43. • Geminivirus vectors: particularly interesting natural hosts include maize and wheat, Cotton, Vegetables. • during the infection cycle the genomes of some geminiviruses undergo rearrangements and deletions, which would scramble up any transgene. • virus-induced gene silencing (VIGS) • a technique used to investigate the functions of individual plant genes. natural defence mechanisms RNA silencing, results in the degradation of viral mRNAs along with homologous host mRNA trnascripts Plant viruses as cloning vectors
- 44. • methods for cloning in humans for gene therapy. • important progress has also been made with insects. a novel type of vector • The fruit fly, Drosophila melanogaster, the most important model organisms • Gene mapping in insects, animals. the discovery of homeotic selector genes • model for the study of human developmental processes. • cloning in • Drosophila makes use of a transposon called the P element. • Transposons, common in all types of organism, are short pieces of DNA • (usually <10 kb ) that can move from one position to another in the cell. • P elements, one of several types of transposon in Drosophila, 2.9 kb contain three genes flanked by short, inverted repeat sequences at either end. • transposase, the enzyme that carries out the transposition process, and the inverted repeats form the recognition sequences that enable the enzyme to identify the two ends of the inserted transposon. Animal viruses as cloning vectors
- 45. Animal viruses as cloning vectors
- 46. • Cloning in mammals: • At present, gene cloning in mammals is carried out for one of three reasons: • To achieve a gene knockout, which is an important technique used to help • determine the function of an unidentified gene. These experiments are • usually carried out with rodents, such as mice. • For the production of recombinant protein in a mammalian cell culture, and in the related technique of pharming. the genetic engineering of a farm animal so that it synthesizes an important protein in its milk. • In gene therapy, in which human cells are engineered in order to treat a disease. Animal viruses as cloning vectors
- 47. Animal viruses as cloning vectors
- 48. Animal viruses as cloning vectors Adenoviruses, which enable DNA fragments of up to 8 kb to be cloned, longer than is possible with an SV40 vector, though adenoviruses are more difficult to handle because their genomes are bigger. Papillomaviruses, which also have a relatively high capacity for insert. Bovine papillomavirus (BPV), which causes warts on cattle, is particularly attractive unusual infection cycle in mouse cells, taking the form of a multicopy plasmid with about 100 molecules present per cell. It does not cause death of the mouse cell, and BPV molecules are passed to daughter cells on cell division, giving rise to a permanently transformed cell line. Shuttle vectors consisting of BPV and E. coli sequences, and capable of replication in both mouse and bacterial cells, have been used for the production of recombinant proteins in mouse cell lines.
- 49. Animal viruses as cloning vectors Adeno-associated virus (AAV), which is unrelated to adenovirus but often found in the same infected tissues, mainly because AAV utilizes some of the proteins synthesized by adenovirus in order to complete its replication cycle. In the absence of this helper virus, the AAV genome inserts into its host’s DNA. With most integrative viruses this is a random event, but AAV has the unusual property of always inserting at the same position, within human chromosome 19. Knowing exactly where the cloned gene will be in the host genome is important if the outcome of the cloning experiment must be checked rigorously, as is the case for applications such as gene therapy. AAV vectors are therefore considered to have major potential in this area. Retroviruses, which are the most commonly used vectors for gene therapy. Although they insert at random positions, the resulting integrants are very stable, which means that the therapeutic effects of the cloned gene will persist for some time.
- 51. • Insert size • Vector size • Restriction size • Cloning efficiency What Determines Choice of Vector? Vector Insert size (kb) Plasmid <10 kb Bacteriophage 9 – 15 kb Cosmids 23 – 45 kb BACs ≤ 300 kb PACs 100 – 300 kb YACs 100 – 3000 kb