5 Recombinant DNA Technology.ppt
- 1. Recombinant DNA Technology Dr Shivaraj Gowda Associate Professor. Dept of Biochemistry, J. N. Medical College, Belgaum
- 2. Molecular Cloning The process of inserting a piece of DNA molecular of interest into a DNA carrier (vector) and making multiple copies of the DNA of interest in a host cell such as bacteria Purposes of molecular cloning • Isolation of a gene from a pool of genetic materials • Amplification of modified forms of genetic materials • Manipulation of a piece of DNA for further experiments Vector (DNA carrier) modified forms of genetic materials • Plasmids • Cosmids • YAC • Bateriophage • Virus
- 3. Overview • Part A • rDNA Objectives • Steps in rDNA Processing • Part B • Cloning Vectors – Plasmids – Phage – BACs and YACs
- 4. • Genetic engineering plays a very important role, not only in scientific research, but also in the diagnosis and treatment of disease. • Recombinant DNA is a tool in understanding the structure, function, and regulation of genes and their products PART A
- 5. • Genetic engineering produces proteins that offer advantages over proteins isolated from other biological sources. • These advantages include: – High purity – High specific activity – Steady supply – Batch-to-batch consistency
- 6. • The objectives of Recombinant DNA technology include: – Identifying genes – Isolating genes – Modifying genes – Re-expressing genes in other hosts or organisms
- 7. • These steps permit scientists and clinicians to: – Identify new genes and the proteins they encode – To correct endogenous genetic defects – To manufacture large quantities of specific gene products such as hormones, vaccines, and other biological agents of medical interest
- 9. The fundamentals of current DNA technology are very largely based on two quite different approaches to studying specific DNA sequences within a complex DNA population DNA cloning. The desired fragment must be selectively amplified so that it is purified essentially to homogeneity. Thereafter, its structure and function can be comprehensively studied, for example by DNA sequencing, in vitro expression studies, etc., and various manipulations can be achieved to change its structure by in vitro mutagenesis. Molecular hybridization. The fragment of interest is not amplified, but instead is specifically detected within a complex mixture of many different sequences. Its chromosomal location can be determined in this way and some information can be gained regarding its structure. If expressed, the sequence of interest can be detected within a complex RNA or cDNA population from specific cells, enabling comprehensive analysis of its expression patterns
- 10. Essentially two different DNA cloning approaches are used: Cell-based DNA cloning. This was the first form of DNA cloning to be developed, and is an in vivo cloning method. The first step in this approach involves attaching foreign DNA fragments in vitro to DNA sequences which are capable of independent replication. The recombinant DNA fragments are then transferred into suitable host cells where they can be propagated selectively. Cell-free DNA cloning. The polymerase chain reaction (PCR) is a newer form of DNA cloning which is enzyme mediated and is conducted entirely in vitro.
- 11. The essence of cell-based DNA cloning involves four steps Construction of recombinant DNA molecules by in vitro covalent attachment (ligation) of the desired DNA fragments (target DNA) to a replicon (any sequence capable of independent DNA replication). This step is facilitated by cutting the target DNA and replicon molecules with specific restriction endonucleases before joining the different DNA fragments using the enzyme DNA ligase. Transformation. The recombinant DNA molecules are transferred into host cells (often bacterial or yeast cells) in which the chosen replicon can undergo DNA replication independently of the host cell chromosome(s). Selective propagation of cell clones involves two stages. Initially the transformed cells are plated out by spreading on an agar surface in order to encourage the growth of well-separated cell colonies. These are cell clones (populations of identical cells all descended from a single cell). Subsequently, individual colonies can be picked from a plate and the cells can be further expanded in liquid culture. Isolation of recombinant DNA clones by harvesting expanded cell cultures and selectively isolating the recombinant DNA.
- 12. Two basic types of extrachromosomal replicons are found in bacterial cells: Plasmids are small circular double-stranded DNA molecules which individually contain very few genes. Their existence is intracellular, being vertically distributed to daughter cells following host cell division, but they can be transferred horizontally to neighboring cells during bacterial conjugation. Natural examples include plasmids which carry the sex factor (F) and those which carry drug-resistance genes. Bacteriophages are viruses which infect bacterial cells. DNA-containing bacteriophages often have genomes containing double-stranded DNA which may be circular or linear. Unlike plasmids, they can exist extracellularly. The mature virus particle (virion) has its genome encased in a protein coat so as to facilitate adsorption and entry into a new host cell.
- 13. Vectors -- the DNA carriers Must have a origin of replication Allow the vector as well as the foreign DNA to amplify in the host cell 1) Plasmids 2) Phages Origin of replication Antibiotic-resistant genes Allow the host to grow on selective media Can selectively amplify this specific vector in the host cell Multiple cloning sites Allow insertion of foreign DNA
- 14. Plasmid Cloning Vectors • Plasmids are circular, double-stranded DNA molecules that exist in bacteria and in the nuclei of some eukaryotic cells. • They can replicate independently of the host cell. The size of plasmids ranges from a few kb to near 100 kb • Can hold up to 10 kb fragments • Plasmids have an origin of replication, antibiotic resistance genes as markers, and several unique restriction sites. • After culture growth, the clone fragment can be recovered easily. The cells are lysed and the DNA is isolated and purified. • A DNA fragment can be kept indefinitely if mixed with glycerol in a –70 degrees C freezer.
- 15. Phage Cloning Vectors • Fragments up to 23 kb can be may be accommodated by a phage vector • Lambda is most common phage • 60% of the genome is needed for lytic pathway. • Segments of the Lambda DNA is removed and a stuffer fragment is put in. • The stuffer fragment keeps the vector at a correct size and carries marker genes that are removed when foreign DNA is inserted into the vector. • Example: Charon 4A Lambda • When Charon 4A Lambda is intact, beta-galactosidase reacts with X-gal and the colonies turn blue. • When the DNA segment replaces the stuffer region, the lac5 gene is missing, which codes for beta-galactosidase, no beta-galactosidase is formed, and the colonies are white.
- 16. Cosmid Cloning Vectors • Fragments from 30 to 46 kb can be accommodated by a cosmid vector. • Cosmids combine essential elements of a plasmid and Lambda systems. • Cosmids are extracted from bacteria and mixed with restriction endonucleases. • Cleaved cosmids are mixed with foreign DNA that has been cleaved with the same endonuclease. • Recombinant cosmids are packaged into lambda caspids • Recombinant cosmid is injected into the bacterial cell where the rcosmid arranges into a circle and replicates as a plasmid. It can be maintained and recovered just as plasmids. Shown above is a 50,000 base-pair long DNA molecule bound with six EcoRI molecules, and imaged using the atomic force microscope. This image clearly indicates the six EcoRI "sites" and allows an accurate restriction enzyme map of the cosmid to be generated. http://homer.ornl.gov/cbps/afmimaging.htm
- 17. Bacterial Artificial Chromosomes(BACs) and Yeast Artificial Chromosomes(YACs) • BACs can hold up to 300 kbs. • The F factor of E.coli is capable of handling large segments of DNA. • Recombinant BACs are introduced into E.coli by electroportation ( a brief high- voltage current). Once in the cell, the rBAC replicates like an F factor. • Example: pBAC108L • Has a set of regulatory genes, OriS, and repE which control F-factor replication, and parA and parB which limit the number of copies to one or two. • A chloramphenicol resistance gene, and a cloning segment. • YACs can hold up to 500 kbs. • YACs are designed to replicate as plasmids in bacteria when no foreign DNA is present. Once a fragment is inserted, YACs are transferred to cells, they then replicate as eukaryotic chromosomes. • YACs contain: a yeast centromere, two yeast telomeres, a bacterial origin of replication, and bacterial selectable markers. • YAC plasmidYeast chromosome • DNA is inserted to a unique restriction site, and cleaves the plasmid with another restriction endonuclease that removes a fragment of DNA and causes the YAC to become linear. Once in the cell, the rYAC replicates as a chromosome, also replicating the foreign DNA.
- 18. Recombinant DNA Technology. A plasmid and the gene of interest are both cut with the same restriction endonuclease. The plasmid and gene now have complementary "sticky ends." They are incubated with DNA ligase, which reforms the two pieces as recombinant DNA. Recombinant DNA is allowed to transform a bacterial culture, which is then exposed to antibiotics. All the cells except those which have been encoded by the plasmid DNA recombinant are killed, leaving a cell culture containing the desired recombinant DNA. DNA cloning allows a copy of any specific part of a DNA (or RNA) sequence to be selected among many others and produced in an unlimited amount. This technique is the first stage of most of the genetic engineering experiments: production of DNA libraries, PCR, DNA sequencing, et al.
- 19. Making Recombinant DNA (rDNA): An Overview