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recombinant DNA with subtopics
- 3. RECOMBINANT DNA Presented by: Syeda Saba kazmi Dated:2/9/2015
- 4. OUTLINE rDNA • rDNA technology •Basic techniques •Restriction enzymes •Vectors •applications Site directed mutagenesis • About random mutagenesis • Description of site directed mutagenesis DNA sequencing • Definition • Sanger method • Applications • references
- 5. Genetic engineering & Biotechnology •The modification of DNA of an organism to produce new genes with new characters Genetic Engineering •Use of living organisms to perform practical tasksBiotechnology
- 7. Recombinant DNA Recombinant DNA RE=again Combinant=binding/combining DEFINITION: Recombinant DNA (rDNA) molecules are DNA molecules formed by bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms
- 9. Carry genetic information. Components of DNA are universal (Nucleotides), only the difference exists in arrangement of bases rDNA is artificial new DNA that can be synthesized in vivo or vitro through biological techniques e.g. molecular cloning DNA rDNA
- 10. History of rDNA Idea: Peter Lobban, Student of Biochemistry Department at Stanford University Medical School in 1972 -1973 Insulin (First drug for human), developed by Eli Lilly and Company 1980 Stanley N. Cohen and Herbert W. Boyer
- 11. Stanley N. Cohen (1935–) (top) and Herbert Boyer (1936–) (bottom), who constructed the first recombinant DNA using bacterial DNA and plasmids. Stanley N. Cohen , who received the Nobel Prize in Medicine in 1986 for his work on discoveries of growth factors.
- 12. Creating recombinant DNA Molecular cloning • Molecular cloning is the laboratory process used to create recombinant DNA molecular cloning involves replication of the DNA within a living cell. Polymerase chain reaction(PCR) • PCR is the laboratory process used to create recombinant DNA that replicates DNA in the test tube, free of living cells.
- 13. Recombinant DNA Technology DEFINITION Intentional modification of organisms’ genomes for practical purposes Three goals Eliminate undesirable phenotypic traits Combine beneficial traits of two or more organisms Create organisms that synthesize products humans need
- 14. How is Recombinant DNA made? There are three different methods by which Recombinant DNA is made. Transformation Phage Introduction Non-Bacterial Transformation
- 16. Transformation(Steps) *select a piece of DNA to be inserted into a vector. *cut that piece of DNA with a restriction enzyme *ligate the DNA insert into the vector with DNA Ligase *The vector is inserted into a host cell One example of a possible host cell is E. Coli.
- 18. Phage Introduction Phage introduction is the process equivalent to transformation, except a phage is used instead of bacteria. E.g. lambda or MI3 phages to produce phage plaques which contain recombinants.
- 20. Non-Bacterial Transformation very similar to Transformation But does not use bacteria such as E. Coli for the host. In microinjection, the DNA is injected directly into the nucleus of the cell being transformed. or the host cells are bombarded with high velocity micro projectiles, such as particles of gold or tungsten that have been coated with DNA.
- 22. Applications of rDNA technology Better Crops (drought & heat resistance) Recombinant Vaccines (Hepatitis B) Prevention and cure of sickle cell anemia Prevention and cure of cystic fibrosis Production of clotting factors Production of insulin Production of recombinant pharmaceuticals Plants that produce their own insecticides Germ line and somatic gene therapy
- 23. Insect-resistant tomato plants The plant on the left contains a gene that encodes a bacterial protein that is toxic to certain insects that feed on tomato plants. The plant on the right is a wild-type plant. Only the plant on the left is able to grow when exposed to the insects.
- 24. Transgenic animals Green fluorescence Red fluorescence
- 26. A transgenic mouse Mouse on right is normal; mouse on left is transgenic animal expressing rat growth hormone
- 27. Cloned gene Retrovirus capsid Bone marrow cell from patient Inject engineered cells into patient. Insert RNA version of normal allele into retrovirus. Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into chromosome. Bone marrow Somatic cells Only! Not for reproductive cells !!
- 29. just a joke
- 30. Tools of Recombinant DNA Technology •Bacterial enzymes that cut DNA molecules only at restriction sites RESTRICTION ENZYMES •Two groups based on type of cut •Cuts with sticky ends •Cuts with blunt ends
- 32. The Tools of Recombinant DNA Technology • Nucleic acid molecules that deliver a gene into a cell • Include viral genomes, transposons, and plasmids Vectors • Small enough to manipulate in a lab • Survive inside cells • Contain recognizable genetic marker • Ensure genetic expression of geneare 4 different type of vectors: Plasmid vectors Lamda (λ) phage vectors Cosmids Expression vectors
- 35. The Tools of Recombinant DNA Technology • A collection of bacterial or phage clones • Each clone in library often contains one gene of an organism’s genome Gene Libraries • Library may contain all genes of a single chromosome • Library may contain set of cDNA complementary to mRNA Cont ….
- 37. Multiplying DNA in vitro: • Large number of identical molecules of DNA produced in vitro • Critical to amplify DNA in variety of situations in vivo The Polymerase Chain Reaction (PCR) • Epidemiologists use to amplify genome of unknown pathogen Cont ….
- 38. Multiplying DNA in vitro: • Separates molecules based on electrical charge, size, and shape • Allows scientists to isolate DNA of interest Gel Electrophoresis and the Southern Blot • Negatively charged DNA drawn toward positive electrode • Smaller fragments migrate faster than larger ones Cont ….
- 41. What Is a Mutation? Genetic information is encoded by the sequence of the nucleotide bases in DNA of the gene. The four nucleotides are: adenine (A), thymine (T), guanine (G), and cytosine (C), a mutation is a change in the order of these nucleotides. A change in the order can cause the gene to encode for wrong proteins and inhibit the function of the gene or cause the gene to be virtually inactive.
- 42. Random mutagenesis • based on process of natural evolution • NO structural information required • NO understanding of the mechanism required Random Mutagenesis •Generation of genetic diversity • Screening and natural selection Cont …..
- 43. Site-directed Mutagenesis Or Site Directed Mutagenesis is a powerful technique where site specific changes in DNA sequence are produced in vitro-for instance to change an amino acid residue into another by changing the codon sequence within the gene sequence Site Directed Mutagenesis is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule known as a plasmid. Wild-type gene sequence must be known.
- 44. CAG GTC CAG + primer CAG primer GCC + polymerase CAG GCC replication CAG GTC Wild type translation Val Mutant Wild type protein CGG GCC translation Thr Mutant protein Only one amino acid changed Val → Thr (1) (2) (3) (4) (5) (6) Smith (1993)
- 45. Site directed Vs Random mutagenesis -> site-directed mutagenesis -> point mutations in particular known area -> random mutagenesis -> point mutations in all areas within DNA of interest
- 46. INVENTION Site Directed Mutagenesis using oligonucleotide was first described in 1978 by Michael Smith & shared Nobel Prize in chemistry in October 1993 with Kary B. Mullis who developed the PCR technique. Site –directed mutagenesis Requirements: -> Knowledge of sequence and preferable Structure (active site,….) -> Understanding of mechanism (knowledge about structure – function relationship)
- 47. Site Directed Mutagenesis Cassette mutagenesis Oligonucleotide directed mutagenesis Using M13 DNA Using Plasmid DNA 3. PCR amplified Oligonucleotide directed mutagenesis 4. Random mutagenesis With Degenerate Oligonucleotide primers or Using Nucleotide Analogues
- 48. CASSETTE MUTAGENESIS Cleavage by a Restriction Enzyme (RE) at a particular site in the plasmid. Ligation of an Oligonucleotide containing Mutation in the gene of interest to the plasmid. RE that cuts at the plasmid and Oligonucleotide is same, permitting sticky ends of the plasmid & inserts to ligate to one another.
- 49. Site-directed mutagenesis – Oligonucleotide - directed method Synthetic single-stranded fragments of DNA used for the mutated clones. In order to work, the primers must meet the following criteria: -must contain desired mutation. -mutation should be in the middle of the primer. -the GC content should be at a minimum of 40% and should terminate in one or more of C or G bases. Oligonucleotides are…
- 50. Oligonucleotide directed mutagenesis Basic Methods 1. Base pair substitution: Change of one nucleotide (A-> C) 2. Insertion: Gaining one additional nucleotide 3. Deletion: Loss of one nucleotide
- 53. 1. Synthesize an Oligonucleotide containing the changed sequence. Ex.---ATT---Wild type sequence (Codon for ILe) ---CTT---Desired Change (Leu) ---GAA---Mutagenic Oligonucleotide (MO) 2.Hybridize MO ss form of gene cloned into M13. 3.Synthesize second strand of DNA with KLENOW fragment &dNTPs. 4.Seal nick in new strand with T4 DNA ligase. 5.Introduce into E. Coli. 6.ss + phage isolated from plaques & screened by hybridization. Oligonucleotide directed mutagenesis with M13 DNA:
- 54. Oligonucleotide Directed Mutagenesis Using Plasmids More common Increases the chances of obtaining the desired mutation in the target gene Antibiotic resistance gene restored and a second one eliminated
- 55. Uses of Site Directed Mutagenesis 1.Site Directed Mutagenesis is also used to ‘engineer’ commercially important Proteins for many different purposes, for example •Improve stability •Change specificity •Reduce toxicity
- 56. Site Directed Mutagenesis enabled new approaches to drug designing – particularly in order to improve FUNCTION. Random Mutagenesis is used to construct large & diverse clone libraries of mutated DNA fragments.
- 57. DNA sequencing DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of DNA.
- 58. Applications of DNA sequencing Knowledge of DNA sequences has became valuable for basic biological research virology medical diagnosis biological systematics forensic biology
- 59. DNA sequencing helps to Molecular biology: to study genomes and the proteins they encode. allows researchers to identify changes in genes identify potential drug targets. Evolutionary biology: to study how different organisms are related and how they evolved.
- 60. DNA sequencing helps to Metagenomics: The field of metagenomics identification of organisms present in a body of water, sewage, dirt, debris filtered from the air, or swab samples from organisms. Sequencing enables researchers to determine which types of microbes may be present in a microbiome.
- 61. Sanger Sequencing/ chain termination method The most popular method for doing this is called the dideoxy method or Sanger method (named after its inventor, Frederick Sanger, who was awarded Nobel prize in chemistry in 1980[his second] for this achievment).
- 63. The Procedure The DNA to be sequenced is prepared as a single strand. This template DNA is supplied with a mixture of all four normal (deoxy) nucleotides in ample quantities dATP ,dGTP ,dCTP ,dTTP. a mixture of all four dideoxynucleotides, each present in limiting quantities and each labeled with a "tag" that fluoresces a different color: ddATP ,ddGTP ,ddCTP ,ddTTP
- 64. DNA polymerase I Because all four normal nucleotides are present, chain elongation proceeds normally until, by chance, DNA polymerase inserts a dideoxy nucleotide instead of the normal deoxynucleotide. At the end of the incubation period, the fragments are separated by length from longest to shortest. The resolution is so good that a difference of one nucleotide is enough to separate that strand from the next shorter and next longer strand. Each of the four dideoxynucleotides fluoresces a different color when illuminated by a laser beam and an automatic scanner provides a printout of the sequence.
- 66. Other methods • Maxam-Gilbert sequencing • Advanced methods and de novo sequencing • Shotgun sequencing • Bridge PCR