Published on February 27, 2014
ENNADA…….RASCALA…MIND IT..!!!!!! 2
WILL THERE BE ANOTHER YOU??????............ 3
Fifteen years ago, scientists in Edinburgh announced to the world an incredible breakthrough: the creation of the first cloned animal–a sheep who originated from a cell taken from an adult mammal. Dolly’s birth sparked a vigorous debate about the controversial technique and its potential application to humans. 5
DOLLY…. a female domestic sheep, and the first mammal to be cloned from an adult somatic cell (5 July 1996 – 14 February 2003) 6
Copycat….!!!!!!!!!!!!! The world's first cloned kitten, named Cc. It was created by scientists in Texas using a cell taken from an adult tortoise shell. The photo, taken on December 22 2001 when the kitten was seven weeks old, was made public 7 in February 2002.
Genetic Engineering OR Recombinant DNA hhhgenetic Technology
Introduction Genetic engineering is a tool of biotechnology Sophisticated and most advanced. Genetic Engineering includes techniques of DNA analysis to manipulate DNA change DNA sequence and bring about a desirable genetic expression. 9
Applications of Genetic engineering fields of medicine, agriculture, animal farming, ecology, paleontology, etc. Medical applications of DNA technology 1. Basic research - understanding of structure and functions of DNA & proteins. 2. Diagnosis of diseases - genetic and microbial. 10
Medical applications ………contd 3. Forensic applications 4. Production of proteins for Replacement therapy Disease prevention (e.g. insulin) (e.g. vaccines) Diagnostic tests (e.g. monoclonal antibodies). 5. Treatment of genetic diseases (gene therapy) 11
Applications in agriculture PLANTS 1. disease-resistant and insect-resistant, high yielding crops 2. Hardier fruit 3. 70-75% of food in supermarket is genetically modified.
Applications in animal farming Genetically modified organisms are called transgenic organisms. 1. Mice – used to study human immune system 2. Chickens – more resistant to infections 3. Cows – increase milk supply and leaner meat 4. Goats, sheep and pigs – produce human proteins in their milk.
Transgenic Goat Human DNA in a Goat Cell . This goat contains a human gene that codes for a blood clotting agent. The blood clotting agent can be harvested in the goat’s milk.
Applications in ecology Recombinant Bacteria- bacteria which can be engineered to “eat” oil spills.
Some Important Tools of Genetic Engineering 1) Enzymes Restriction Endonucleases (REs): DNA ligase DNA Polymerases Reverse transcriptases 2)Vectors Plasmid Bacteriophage, Cosmid Yeast
Some Important Tools of Genetic Engineering Enzymes Restriction Endonucleases (REs): used as scissors to cut DNA -DNA scissors at specific DNA sequences to generate a set of smaller fragments. Genomic DNA DNA fragments 17
DNA ligase Joins two DNA molecules or fragments. 18
DNA Polymerases Synthesis of DNA using DNA template and dNTPs Reverse transcriptase Enzyme found in retroviruses that makes DNA copy, using RNA as template RNA cDNA dsDNA 19
Vectors Into the DNA of the vector a foreign DNA can be inserted, integrated/incorporated. Use : For amplification by cloning and for gene therapy. Examples : Plasmid Bacteriophage, Cosmid Yeast 20
Plasmid A small, circular, dsDNA present in bacteria Confer antibiotics resistance against the bacteria many copies of plasmid in a bacterium replicate independent of the bacterial DNA. 21
Bacteriophage is a virus that can infect bacteria Cosmid plasmid + Cos sites for binding to bacteriophages can carry larger DNA fragments 22
Restriction Endonucleases (REs) •recognize specific DNA sequences- called “palindrome” (restriction sites) • Example : EcoR I (E. coli RY 13) recognises sequence 5’ GAATTC 3’. •cuts the phosphodiester bonds of the DNA on both the strands. 23
Action of EcoRI Cuts both strand of the DNA 24
Action of RE Plasmid RE DNA RE Plasmid with a cut DNA fragment 25
Restriction enzyme nomenclature Why the funny names? • • • • • • • • EcoRI – BamHI – DpnI – HindIII – BglII – PstI – Sau3AI – KpnI – Escherichia coli strain R, 1st enzyme Bacillus amyloliquefaciens strain H, 1st enzyme Diplococcus pneumoniae, 1st enzyme Haemophilus influenzae, strain D, 3rd enzyme Bacillus globigii, 2nd enzyme Providencia stuartii 164, 1st enzyme Staphylococcus aureus strain 3A, 1st enzyme Klebsiella pneumoniae, 1st enzyme
Restriction Endonucleases (REs) •Examples: EcoRI; Hpa I; BamHI; Taq I. REs are isolated from bacteria. Biological function of RE in bacteria : is to recognize and cleave foreign DNA (e.g. DNA of an infecting virus). 27
Applications of REs in Genetic Engineering 1) sequencing of DNA 2) cloning of DNA 3) 4) antenatal diagnosis of inherited disorders ( RFLP analysis) DNA finger printing (having forensic applications) 5) for Southern blot technique (for detecting the presence of a particular base sequence in the sample DNA). 28
Some Important Techniques in DNA Analysis and Genetic Engineering: DNA Amplification: production of many identical copies of a DNA fragment of interest. Uses 1) further DNA analysis or 2) for large-scale genetic expression (protein production). 29
Types of DNA amplification Cloning Polymerase Chain Reaction (PCR) in vivo method using bacteria an in vitro method using DNA polymerase used to amplify longer segments of DNA shorter segments of DNA can be amplified suitable for large-scale shorter time for amplifying DNA fragments protein production 30
Cloning Production of an identical copy of either DNA or a cell or an organism is called cloning. -2 Types. 1)Molecular cloning -production of identical DNA molecules (i.e., identical in base-sequence) 2)Somatic cloning -production of cells or organisms with identical genetic makeup. 31
DNA Cloning Recombinant DNA Technology- Cloning a DNA Fragment Two principal steps : Constructing a recombinant DNA molecule -gene of one species is transferred to another living organism. -usually, a human gene is transferred to a bacteria. Amplifying the recombinant DNA molecule in a bacterial host 32
Constructing recombinant DNA molecule Chimeric DNA / DNA chimera 33
Amplifying the recombinant DNA molecule in a bacterial host 1. Transfection / transformation 2. Amplify in a suitable culture medium 34
Selection Isolation Amplification 35
2. Selection, Isolation and Amplification of Recombinant DNA: by specific techniques (eg. by antibiotic sensitivity technique) and allowed to multiply in a suitable culture. 3. Release of the Cloned DNA Molecules from the Bacteria: by using the same RE as used for cleaving of DNA 36
Applications of recombinant DNA Technology Used in the fields of Medicine, Agriculture, Animal Farming, Ecology, Paleontology, etc. 37
Medical applications of Recombinant DNA Technology 1.Production of proteins for Replacement therapy Disease prevention (e.g. insulin) (e.g. vaccines) Diagnostic tests (e.g. monoclonal antibodies). 2.Treatment of genetic diseases (gene therapy) 38
Production of Proteins Using Recombinant DNA Technique : proteins, especially human proteins produce large amounts of proteins provide human proteins, which are not antigenic when administered to humans. 39
Proteins produced are used for: Replacement therapy and other treatments (e.g. insulin, growth hormone, interleukins, antihemophilic factor, interferon, etc.). Disease prevention (e.g. vaccines, such as hepatitis B antigen) Diagnostic tests (e.g. monoclonal antibodies). 40
Human insulin is produced using Recombinant DNA Technique : Recombinant insulin (Humulin) Recombinant Human Growth Hormone
Vaccines production • Vaccines like hepatitis B vaccine, are produced using Recombinant DNA Technique
Polymerase Chain Reaction (PCR) in vitro method for DNA amplification much faster more sensitive method than cloning. very little DNA sample is sufficient can only amplify short segments of DNA cannot be used for amplifying genes and for production of proteins 43
Use : To amplify a short sequence of DNA Procedure : (1) A mixture of DNA sample + dNTP’s + Primers + Enzyme : Taq DNA polymerase (2) Treatment of the mixture : 1 cycle 94 - 95 C Denaturation of DNA 30 – 60 sec 52 - 54 C Annealing of primers 30 – 60 sec 72 C Extension of the DNA 1 min 44
Test DNA sample Separation DNA strands Primers anneal 1 cycle Extension by DNAP No. of cycles : 30 - 45 Product : Every cycle the DNA doubles 45
Advantages of PCR 1. Very little DNA sample is required 2. Amplification time is very short. 3. Amplification rate is high. Applications of PCR Useful : when insufficient DNA molecules are present in test samples for DNA analytical techniques. 46
Uses of PCR 1. Diagnostic uses used to quickly detect microbial infections, when the number of microbes is less in the sample. Examples :Diagnosis of Tuberculosis (TB) Mycobacterium tuberculi AIDS HIV 47
2. Prenatal diagnosis of genetic disorders Sections of genes, having particular mutations known to cause a disease are Amplified Sequenced Diagnosis Example : Detection of Sickle cell anemia (HbS) 48
3. Forensic Uses: Samples used : Blood, saliva, semen, hair Obtained from : a victim or suspect Volume of the sample : is insufficient Sample PCR Amplified DNA Amplification of DNA DNA analytical techniques i.e., DNA fingerprinting 49
Gene therapy : treatment of Genetic disorders . •Severe Combined Immuno Deficiency (SCID) Adenosine deaminase • Cystic Fibrosis Chloride channel • Familial Hypercholesterolemia Receptor for LDL • Hemophilia Clotting factor (factor VIII or IX) 50
Gene therapy • Gene therapy is aimed at treating genetic disorders • involves introduction of normal foreign gene into somatic cells of the patient having the genetic disease to compensate for the defective protein, which is the product of the mutant gene. 51
Gene therapy-procedure • The procedure involves, 1) isolation of the healthy gene 2) incorporation of this gene into a carrier or vector and 3) delivering the vector into the target cells. 52
Gene therapy-proceedure • isolation of the healthy gene -done by isolating its mRNA first -using this mRNA as a template, cDNA is synthesised, using reverse transcriptase. -from thic cDNA, double strande DNA is synthesised using DNAP. 53
Isolation Of The Healthy Gene 54
Gene therapy-proceedure.. 2) incorporation of this gene into a carrier or vector -Vectors used are retroviruses, adenoviruses and plasmid-liposome complexes. -this is done by recombinant DNA technology, as described earlier. 55
Gene therapy-proceedure…. • delivering the vector into the target cells. • The vector with normal gene is now introduced to the patient. •The cells of this patient will start producing the normal protein which was deficient earlier. • Disease is cured. 56
Replacement of mutant protein in genetic disorders 57
Major vectors used for transfer of the gene in gene therapy Retroviruses Adenoviruses Plasmid-liposome complexes. 58
Reverse Transcriptase (RT) RNA cDNA dsDNA stored in DNA library DNA library a collection of DNA fragments of one organism, each carried by a plasmid / virus. 59
Uses of DNA library 1. For Protein expression o Incorporation into a vector - DNA Chimera o Cloned Suitable bacteria o Protein expressed 2. As a probe for analytical techniques o Detecting specific nucleotide sequence in test samples. o As in Southern and Northern blot techniques. 60
DNA Probes are Single stranded, fragments / pieces of DNA Contain nucleotide sequence complimentary to the target sequence Radiolabeled with radioisotopes (usually 32P) to visualize on an X-ray film Use : for detecting a target sequence in Southern and Northern blot techniques 61
Examples for probes Synthetic oligonucleotides RNA Antibodies (protein) -as a probe for protein molecule -in Western blot technique. 62
Blot techniques : Analytical techniques used in Recombinant technology Done on test samples Types Detection of Southern DNA Northern RNA Western Protein 63
Applications of Blot techniques : in • Research • Diagnosis of diseases (microbial and genetic) • Forensic medicine. Southern Blot Technique Process : 6 steps 1) Extraction of DNA from the test sample/cells 2) Digestion by a suitable RE – Product DNA fragments 64
3) Electrophoresis of the digest - Separation of fragments 4) Denaturation of DNA and blotting onto a membrane (nitrocellulose membrane) 5) Adding a radiolabeled DNA probe 6) Autoradiography : Visualization on X-ray film . DNA fragments hybridized with the radiolabeled DNA probes. 65
Blood stain DNA extracted Transfer to membrane RE treated Nitrocellulose membrane DNA Probes (radiolabeled) to the membrane Exposure to X-ray film DNA fragments separated by gel electrophoresis DNA denaturation with alkali DNA pattern 66
The pattern observed on Southern blot analysis depends on : • the specific RE used • location of the restriction site in the DNA sample • the probe used. 67
Restriction Fragment Length Polymorphism (RFLP) Analysis Extraction of Human chromosome Digestion with one or more REs Southern blot Visualization Application : molecular analysis of genes involved in disease. 68
Medical Applications of Genetic Engineering 1. Basic research for understanding structure and functions of DNA and proteins. Recombinant DNA technology has made possible : • Complete sequencing of the human genome (Human Genome Project) • Gene localizing and • defining the map of the human genome. 69
• Isolation and detailed molecular analysis of genes involved in disease (using RFLP analysis). 2. Diagnosis of diseases - genetic and microbial. Techniques used : PCR, Southern blot & RFLP Test sample : Amniotic fluid Time of Test : Prenatal diagnosis 70
Sample PCR Amplified DNA Amplification of DNA DNA analytical techniques 3. Forensic Uses: For identifying dead bodies Settling parental disputes. Identifying criminals. 71
Samples used : Blood, saliva, semen, hair Obtained from : a victim or suspect Volume of the sample : is insufficient Sample PCR Amplified DNA Amplification of DNA DNA analytical techniques i.e., DNA fingerprinting 72
4. Production of Proteins Using Recombinant DNA Technique : proteins, especially human proteins produce large amounts of proteins provide human proteins, which are not antigenic when administered to humans. 73
Proteins produced are used for: Replacement therapy and other treatments (e.g. insulin, growth hormone, interleukins, antihemophilic factor, interferon, etc.). Disease prevention (e.g. vaccines, such as hepatitis B antigen) Diagnostic tests (e.g. monoclonal antibodies). 74
5. Treatment of genetic diseases : Example : Gene therapy Involves Introduction of normal foreign gene Into somatic cells of the patient having the genetic disease To compensate for the defective protein, Which is the product of the mutant gene. 75
Genetic disorders treated by Gene therapy (attempt) : •Severe Combined Immuno Deficiency (SCID) Adenosine deaminase • Cystic Fibrosis Chloride channel • Familial Hypercholesterolemia Receptor for LDL • Hemophilia Clotting factor (factor VIII or IX) 76
1. 2. 3. 4. What are restriction Endonucleases? Give two examples. (3) What is reverse transcriptase? What is its significance? (3) Reverse transcriptase. (3) What is plasmid? What are its applications in recombinant DNA technology? (4) 5. Discuss in detail recombinant DNA technology and its clinical application. (5) 6. What is “Recombinant DNA”? Mention applications of genetic engineering. (1+3 =4 ) 7. Describe the clinical applications of recombinant DNA technology. (4 ) 8. Give two applications of recombinant DNA technology. (3) 9. What is Polymerase Chain Reaction (PCR)? Mention application of PCR. (3) 10.What is polymerase chain reaction? Mention its applications (3) 11.PCR (4 ) 12.Polymerase chain reaction (3) 13.Gene therapy (4) 14.What is gene therapy? Name vectors used for gene therapy. (3) 77
MULTIPLE CHOICE QUESTIONS 1. DNA Scissors is______________. a) DNA Polymerase b) 3’→5’ Exonuclease c) Restriction endonuclease d) RNase H. 2. Two fragments of DNA are joined by ______. a) DNA Polymerase b) DNA Ligase c) Topoisomerase d) Reverse transcriptase. 78
3. An example for an RNA dependant DNA polymerase is ____________. a) DNA Polymerase b) RNA Polymerase c) Primase d ) Reverse transcriptase. 4. ____________ confer antibiotic resistance to bacteria. a) Genomic DNA b) Mitochondria c) Cell wall d ) Plasmids. 79
5. ____________ is an example for a recombinant protein used in disease prevention. a) HB antigen b) Interleukins c) Interferons d ) Insulin. . ___________ is an example for a recombinant 6. protein used in replacement therapy. a) HB antigen b) Antibodies c) Oral polio vaccine d ) Insulin. 80
7. Applications of REs in genetic engineering include these except_____________. a) Cloning of DNA b) Antenatal diagnosis of inherited disorders c) Radiolabeling d) DNA finger printing. . 8. The function of polymerase chain reaction is to _____________. a) amplify DNA b) destroy DNA c) synthesize proteins d) confer antibiotic resistance. 81
9. These can be used as a vector in DNA cloning except a) Plasmid b) Cosmid c) Oligonucleotides d) Bacteriophage. 10. Inserting DNA fragment of interest into the DNA of. a vector produces a molecule which is called by all these names except a) Recombinant DNA b) Recombinant protein c) DNA chimera d) Chimeric DNA. 82
11. Ideally, for cloning, both the vector and the DNA of interest should be cleaved with the same a) Endonuclease b) Exonuclease c) Restricted endonuclease d) RNase H. 12. These are advantages of PCR technique except a) High rate of amplification b) Less time required c) Small amounts of test sample is needed d) High rate of errors. 83
RNA cDNA dsDNA 84
Calcification Inhibitors in CKD and Dialysis Patients
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