1. This is a modern biotechnological advance , in which a desired gene fragment can be inserted in to a cloning vector and the resulting DNA (Recombinant DNA) can be amplified in suitable host. 2. A vector can be a plasmid, cosmid,bacterophage,retroviruses, animal and plant viruses or artificial chromosomes like YAC, BAC,or HAC.(Yeast artificial chromosome, bacterial……..) 3. The rec. DNA produced can be amplified or cloned in a suitable vector like bacteria for plamids, cosmids or bacterophages, plant and animal cells for viruses .
Involves five steps:
1. Enzyme restriction digest of DNA sample.
2. Enzyme restriction digest of DNA plasmid vector (same Res.Enzyme). 3. Ligation of DNA sample products and plasmid vector.
4. Transformation with the ligation products.
5. Growth on agar plates with selection for antibiotic resistance. [pic]
← APPLICATION OF RECOMBINANT DNA technology
← Gene therapy
← Recombinant Vaccines
← Genetically modified crops
← Monoclonal antibodies
← Cell/tissue culture
← Production of next generation antibiotics
← Bioterrorism detection
DNA molecules that are used to “transport” cloned sequences between biological hosts and the test tube.
Cloning vectors share four common properties: 1. Ability to promote autonomous replication.
2. Contain a genetic marker (usually dominant) for selection. 3. Unique restriction sites to facilitate cloning of insert DNA. 4. Minimum amount of nonessential DNA to optimize cloning. Main types of cloning vectors
Plasmid, bacteriophage, cosmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), HAC, retrovirus, baculovirus vector etc.
← Bacterial cells may contain extra-chromosomal DNA called plasmids. ← Plasmids are usually represented by small, circular DNA. ← Some plasmids are present in multiple copies in the cell ← F –plasmid= capable of conjugation
← R-plamid= Encode antibiotic resistance
← Plasmid vectors are ≈1.2–3kb and can clone up to 12 kb of insert DNA IT CONTAINS,
1. replication origin (ORI) sequence : DNA segment recognized by the cellular DNA-replication enzymes. 2. a gene that permits selection, antibiotic resistance like ampr;tetr etc ampr encodes the enzyme beta-lactamase, which inactivates ampicillin. Selective marker is required for maintenance of plasmid in the cell. 3. Multiple cloning site(mcs) :Exogenous DNA can be inserted in mcs .It is a DNA segment with several unique sites for restriction endo- nucleases located next to each other.Restriction sites of the polylinker are not present anywhere else in the plasmid.Cutting plasmids with one of the restriction enzymes that recognize a site in the polylinker does not disrupt any of the essential features of the vector. 4. Plasmid vectors are used to clone DNA ranging in size from several base pairs to several thousands of base pairs (100bp -10kb).
5. ColE1 based, pUC vehicles commercially available ones, eg pGEM3, pBlueScript
6. Some are expression vectors and have sequences that allow RNA
polymerase to transcribe genes (promoters, Terminators and ribosome binding sites)
7. DNA sequencing primers
CLONING IN PLASMID VECTOR
← Examples of plasmid vectors
← First artificial cloning vector developed in 1977from E.Coli (By Boliver and Rodrigues). ← Contain 4361 base pairs
← Two selectable markers (Ampicillin and Tetracycline resistance genes) ← Several unique restriction sites scattered throughout plasmid 0 restriction sites for Hinlll,Eco RV,BamH1 etc. ← Some retriction sites lie within antibiotic resistance genes = means of screening for inserts ← ColE1 ORIGIN(Ori)
← Found ~20 copies per bacterial cell
← Derivative of pBR322
← Advantages over pBR322:
← Smaller 2 kbp– so can accommodate larger DNA fragments during cloning (5-10kbp) ← Higher copy # per cell (500 per cell = 5-10x more than pBR322) ← Multiple cloning sites clustered in same location = “polylinker” ← Interruptable gene encoding for enzyme beta galactosidase (lacZ) ← Polylinker resides in the middle
← Beta galactosidase enzyme activity can be used as marker for gene insertion,which can convert X-gal ubtartes to blue coloured substrate. ← Disrupted gene = nonfunctional ; blue colonies, self ligated vector has intact lacZ gene,gives blue colonies. ← Amp resistance gene still present, Tetr. resistance gene- absent BLUE WHITE SCREENING
The Major Limitation of Cloning in Plasmids
← Upper limit for clone DNA size is 12 kb, only small insert can be inserted ← Requires the preparation of “competent” host cells ← Preference for smaller clones to be transformed
← If it is an expression vector there are often limitations regarding eukaryotic protein expression- lack Post transltional modification in bacterial system Agrobacterium tumefaciens -Ti plasmid -to introduce genes into plants
virus that infects bacteria are bacterePohages can incorporate desired DNA up to 23 kb size. ← 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 and spikes. ← Infection: lambda tail fibres adsorb to a cell surface receptor, the tail contracts, and the DNA is injected. ← The DNA circularizes at the cos site, and lambda begins its life cycle in the E. coli host Prophage : phage incorporated in genome , replicate along with host genome. Stages of viral replication
Lambda genome is approximately 49 kb in length.Only 30 kb is required for lytic growth.Thus, one could clone 19 kb of “foreign” DNA.Packaging efficiency 78%-100% of the lambda genome. 49 kb linear DNA with a 12 base ssDNA “sticky end” at both ends; these ends are complementary in sequence and can hybridize to each other (this is the cos site: cohesive ends). [pic] [pic]
2 types of lambda vectros
1. Lambda replacement vector : phage DNA parts are removed and insert foreign genes 2. Lambda insertionvector: No phage DNA is removed,so only lower size fragments can be inserted M 1 phage vectors
Filamentous bacteriophage of E.Coli with 7.kb long ss circular DNA;Large
inerts can be inerted VIRAL SELECTION
Selection is by plaques or viral infected regeons (clear areas) formed in bacterial lawn. COSMIDS
← Hybrid vectors: plasmids that contain bacteriophage lambda cos sites (plamid + phage cos site) . Clone large inserts of DNA: size ~ 48 kb ← DNA (~ 33-48 kb) cloned into restriction site, the cosmid packaged into viral particles and these phages used to infect E.coli ← Cosmid can replicate in bacterial cell, so infected cells grow into normal colonies ← Insert DNA limited by the amount of DNA that can fit into phage capsule ← Presence of the Cos site permits in vitro packaging of cosmid DNA into Lambda particles Animal viruses AND Plant viruses : (write abt,insert gene& lytic cycle) ADVANTAGES OF VIRAL VECTORS
1. They can introduce genes by direct infection of host cells 2. they contain promoters for gene expression
3. They can replicate in large numbers inside a host cell 4. Retroviruses can integrate in to host genome ,
so that they can propagate along with host cell genome EX. Of animal viral vector: SV-40 (Simian Virus 40)-mammalian virus;Ds Circular gene. 5.2kb EX. Of plant viral vector
CaMV (Cauliflower mosaic virus – Ds DNA virus)
TMV-Tobacco Mosaic Virus-RNA virus
Gemini virus – ss DNA virus
RETROVIRUSES(draw diagrams if needed- lytic cycle)
← Retroviral vectors are used to introduce new or altered genes into the genomes of human and animal cells. ← Retroviruses are RNA viruses.
← The viral RNA is converted into DNA by the viral reverse transcriptase and then is efficiently integrated into the host genome ← Any foreign or mutated host gene introduced into the retroviral genome will be integrated into the host chromosome and can reside there practically indefinitely. ← Retroviral vectors are widely used to study oncogenes and other human genes. BAC/YAC/HAC
← Cloning vehicles that propogate in eukaryotic cell hosts as eukaryotic Chromosomes BACs (Bacterial artificial chromosomes)
← Large low copy number plasmids (have ori and selectable marker) ← Can be electroporated into E. coli
← Useful for sequencing genomes, because insert size 100 – 300kb ← YAC (Yeast Artificial Chromosome)
← Can be grown in E.coli and Yeast
← Miniature chromosome (contains ori, selectable markers, two telomeres, and a centromere ← useful for sequencing
← Clone very large inserts of DNA: 100 kb – 10 Mb
← Features:Final chimeric DNA is a linear DNA molecule with telomeric ends: Artificial Chromosome[pic]
Cite this Recombinant DNA Technology
Recombinant DNA Technology. (2016, Oct 30). Retrieved from https://graduateway.com/recombinant-dna-technology/