This presentation is all about biotechnology. It is about the basic aspects of Biotechnology and covers a lot of topics under biotechnology, recombinant DNA technology. This is specifically for the HSC students of Mumbai. I hope that it helps.
The cell or organism from which the required gene is taken is called donor.Molecular scissors are enzymes that cut the DNA strands at desired sites.
A cosmid, first described by Collins and Hohn in 1978, is a type of hybrid plasmid (often used as a cloning vector) that contains aLambda phage cos sequence. Cosmids' (cos sites + plasmid = cosmid) DNA sequences are originally from the Lambda phage. Cosmids can be used to build genomic libraries.Cosmids are able to contain 37 to 52 kb of DNA, while normal plasmids are able to carry only 1–20 kb. They can replicate as plasmids if they have a suitable origin of replication:
Kary received noble award in 1993 for this.
Ethical--pertaining to or dealing with morals or the principles of morality; pertaining to right and wrong in conduct
Biotechnology: Process and Application
Definition• Biotechnology is the integration of natural science and organisms, cells, parts thereof and molecular analogues for products and services.
Application of fermentation inproduction of wine and other alcoholic beverages is also a biotechnological technique
But with time biotechnology gradually became more sophisticated.
DNA manipulation Protein Tissueengineering culture BiotechnologyImmobilized Protoplast enzymes fusion Cell catalysis
Biotechnology led to production of many products andprovides many services for human welfare.
Dragon Fly The biotechnology industry has mushroomed since 1992, with U.S. health care biotech revenues from publicly traded companies rising from $8 billion in 1992 to $58.8 billion in 2006.There were 180,000 people employed by U.S. biotechnology companies in2006. There are more than 400 biotech drug products and vaccines currently in clinical trials targeting more than 200 diseases, including various cancers, Alzheimer’s disease, heart disease, diabetes, multiple sclerosis, AIDS and arthritis.
In-Vitro Fertilization Also called as Test tube baby
The recombinant DNA technique was first proposed by Peter Lobann A. Dale Kaiser
The present day rDNA technology flourished after the work of
Gene coding for Plasmid antibiotic resistance Vector CloningSalmonella typhimuriumThey successfully linked a genecoding for antibiotic resistance witha native plasmid of Salmonellatyphimurium with the vector plasmidand then cloning it in E.coli. E. coli
What is recombinant DNA ?• Technique of manipulating the genome of a cell or organism so as to change the phenotype desirably. Seedless guava Calorie free sugar
Introducing in Host Isolating Culturing the genomic cells DNA Insertion of DNA in a vector TransformationFragmenting of host cell this DNA Screening the fragments
Isolating Isolating genomic genomic DNA DNA from the donor. FragmentingFragmenting this DNA using this DNA molecular scissors.
Screening theScreening the fragments for afragments “desired gene”. Inserting theInsertion of fragments with the DNA in a desired gene in a vector „cloning vector‟.
Introducing the recombinantIntroducing vector into a competent host in Host cell Culturing these cells to obtainCulturing multiple copies or clones ofthe cells desired DNA fragments Using these copies to transform suitable host cellsTransformation so as to express the desired of host cell gene.
Tools used in recombinantDNA technology• Enzymes• Vectors
Tools used in recombinant DNA technology• Enzymes Act as biological scissors. Most commonly used are: Restriction endonuclease DNA ligase DNA polymerase Alkaline phosphatases
Tools used in recombinant DNA technology• Vectors Low molecular weight DNA molecules. Transfer genetic material into another cell. Capable of multiplying independently.
Vector Bacteriophage DNA Artificial Plasmid Vector DNA Cosmid
Insertion of vector in targetcell is called• Bacterial cells – Transformation• Eukaryotic cells – Transfection• Viruses - Transduction
Insertion of vector in targetcellVectors used:• Bacteria- plasmids, cosmid, lambda phage• Insects- baculoviruses• Plants- Ti plasmid• Yeast cells- YAC (yeast artificial chromosome)
HOST DONOR DNA DNA Fragmented by Restriction Endonuclease DNA strands with sticky endsSticky ends base pair with complementary sticky ends DNA ligase links them to form rDNA Cloned In vitro In vivoPolymerase chain Prokaryotic or eukaryotic cell,reaction (PCR) mammalian tissue culture cell
Some examples of therapeutic products made by recombinant DNA techniques¶ Blood Proteins: Erythropoietin, Factors VII, VIII, IX; Tissue plasminogen activator; Urokinase.¶ Human Hormones: Epidermal growth factor; Follicle stimulating hormone, Insulin.¶ Immune Modulators: α Interferon, β Interferon; Colony stimulating hormone; Lysozyme; Tumor Necrosis factor.¶ Vaccines: Cytomegalovirus; Hepatitis B; Measles; Rabies
Transposons• Transposons are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell.• Also called „Jumping genes‟.
• 1st transposons were discovered by Barbara McClintockin Zea mays (maize)
Types of transposons• According to their mechanism they are classified as:
Retrotransposons• Follows method of “Copy and Paste”.• Copy in two stages. DNA RNA DNA Transcription Reverse Transcription
DNA transposons• Follows the method of “Cut and Paste”.• Do not involve RNA intermediate. Enzyme Transposase Cuts out transposon Ligates in new position
Plasmid• Plasmids are small, extra chromosomal, double stranded, circular forms of DNA that replicate autonomously.• The term was introduced by in 1952. Joshua Lederberg
Plasmid• Found in bacterial, yeast and occasionally in plants and animal cells.• Transferable genetic elements or ‘Replicons’.• Size- 1 to 1000 kilo bp.• Related to metabolic activity.• Allows bacteria to reproduce under unfavorable conditions.
PlasmidNomenclatureLower case P (p)First letters of researchers name or place where it was discovered.Numerical numbers given by workers.
Plasmid Eg. Plasmid pBR 322BR is for Bolivar and Rodriguez, who designated it as 322
PlasmidEg. Plasmid pUC 19 UC stands for University of California
Plasmid- Cosmids• Cosmids are plasmids with cos sequence.• They are able to accommodate long DNA fragments that plasmids can’t.
A bacteriophage is a virus that infects bacteria. Virulent portion is deleted. Genetic material can be ssRNA, dsRNA, ssDNA, dsDNA.
For Single genes- Plasmids are used For Large pieces of DNA- Bacteriophages
48.5 kb in length. Cos sites of 12 bp at the ends. Cohesive ends allow circularizing DNA in host.
(1) Phage attaches to a specific host bacterium.(2) Injects its DNA,(3) Disrupting the bacterial genome and killing the bacterium, and(4) Taking over the bacterial DNA and protein synthesis machinery to make phage parts.(5) The process culminates with the assembly of new phage, and(6) The lysis of the bacterial cell wall to release a hundred new copies of the input phage into the environment.
RESTRICTION FRAGMENTSA restriction fragment is a DNA fragment resulting from the cutting of a DNA strand by the restriction enzyme. Process is called restriction.
RESTRICTION FRAGMENTS Steward Linn along with Werner Arber in 1963 isolated two enzymes. One of them is Restriction Endonuclease. Restriction Endonuclease can cut DNA. Restriction Endonuclease are basic requirement for gene cloning or rDNA technology.
RESTRICTION FRAGMENTS NucleasesThey remove They make cutsnucleotides at specific Exonuclease Endonuclease positions withinfrom the endsof the DNA the DNA
TYPES OF REN REN Type I Type II Type III Mostly used in rDNA technology. More than 350 types of type II endonucleases with recognition sites are known. Can be used to identify and cleave within
NOMENCLATURE OF REN First letter- genus name of bacteria (in italics). Next- first two letters of the species name (in italics). Next- strain of the organism. Roman number- order of discovery.
NOMENCLATURE OF REN Eg. - EcoR IE- Escherichia, co- coli, R-strain Ry 13,I- first endonuclease to be discovered. Eg.- Hind IIIH- Haemophilus, in- influenzae, d- strain Rd,III- third endonuclease to be discovered.
RECOGNITION SEQUENCE (RESTRICTION SITES) It is the site/ sequence where REN cuts the DNA. Sequence of 4-8 nucleotides. Most restriction sites are Palindromes.
In DNA, palindrome is a sequence of base pairs that reads the same on the two strands when orientation of reading is kept same.
CLEAVAGE PATTERNS OF REN REN recognizes the restriction site. Cleave the DNA by hydrolyzing Phosphodiester bonds. Isolate a particular gene. Single stranded ends called sticky ends.
These sticky ends can form hydrogen bonded base pairs with complementary sticky ends or any other cleaved DNA.
Gel Restriction fragments electrophoresis yield a band pattern characteristic of the original DNA molecule & restriction enzyme used.Bands
PREPARING AND CLONING A DNA LIBRARY Collection of DNA fragments from a particular species that is stored and propagated in a population of micro organisms through molecular cloning.
GENOMIC LIBRARY Collection of all clones of DNA fragments of complete genome of an organism. All DNA fragments are cloned and stored as location of desired gene is not known. Screening of DNA fragments can be done by Complementation Or by using Probes.
Construction of Genomic Library.Entire genome isolated Cut into fragments by REN Fragments inserted in Vector Recombinant vectors are transferred into suitable organism Transferred organisms are cultured and stored
CDNA LIBRARY cDNA is Complementary DNA. Produced using Teminism i.e. Reverse Transcriptase. Constructed for eukaryotes.
cDNA is made from mRNA AAAAAAA Mature mRNAStart Stop TTTTTTT Add polyT primer, nucleotides, and Reverse Transcriptase AAAAAAA DNA/RNA TTTTTTT RNA removed (by NaOH) and second strand synthesized TTTTTTT Complementary DNA cDNA
Gene Amplification (PCR) It is obtaining multiple copies of a known DNA sequences that contain a gene. Done artificially by using PCR (Polymerase Chain Reaction)
PCR (Polymerase Chain Reaction) Developed by in 1983. Kary Mullis In Vitro technique. Scientific technique to generate billions of copies of a particular DNA sequence in a short time.
Requirements for PCR technique Primers-forward andA DNA segment DNA segment Primers reverse, are100-35,000 bp in syntheticlength to be oligonucleotides andamplified. complementary to PCR the desired DNA segmentFour types of Thermostabledeoxyribonucleotid dNTPs DNA Enzyme that canes i.e. dCTP, dGTP, polymerase withstand upto 94°dTTP, dATP C.
Steps of PCR technique The double strand melts open to single stranded DNA, all enzymatic reactions stop (for example : the extension from a previous cycle). Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore. The bases (complementary to the template) are coupled to the primer on the 3 side (the polymerase adds dNTPs from 5 to 3, reading the template from 3 to 5 side, bases are added complementary to the template)
The exponential amplification of the gene inPCR.
Application of biotechnology inagriculture- Bt crops
Bacillus thuringiensis • Soil bacterium. • Produces a protein that has insecticidal properties. • Traditionally used as spray.
Mechanism of Bt Bt (in inactive form) sprayed on Crops• Bt produces Bt toxins which are inactive protoxins. Eaten by insect• When an insect ingests it, inactive protoxin gets Toxin gets activated by alkaline pH of insect’s gut converted into active form due to alkaline pH of the insect’s gut. Swelling of gut of insect• This led to swelling of gut and ultimately death of insect Death of insect
Crop plants are now engineered to express Bt toxin.• Cry gene in Bt produces inactive protoxins.
Agrobacterium tumefaciens• Soil bacterium.• Causes crown gall tumors in dicotyledonous plants.• T DNA (gall producing gene) occurs in Ti plasmid.• Ti plasmid is used as vector for Mechanism higher plants.• Many genetically modified plants are produced using A. tumifaciens. Tumor
• Desirable genes such as Cry gene an Nif gene is cloned inside A. tumifaciens and then transferred into another plant.Nif Geneisolated fromRhizobium
Examples1. Flavr savr tomato 2. Golden Rice Longer shelf life. Greater pro vitamin A content. Antisense DNA is introduced that Genetically engineered. retards ripening
Bio-Safety Issues Biosafety issues Impact on human health Genetically Impact on and modified Agriculture environment organisms Ethical issues
Genetic modification of organismscan lead to Contamination of gene pools. Consumption may lead to allergies. Hazardous microbes may escape laboratory Therefore manipulation of organisms needed regulation
Genetic Engineering ApprovalCommitteeIn India, GEAC takes decision regarding validity of GMresearch and introduction of GM products.
Biopiracy The patenting of plants, genes, and other biological products that are indigenous to another country Developed countries patent the knowledge and resources of underdeveloped countries and enjoy immense profits.
BiopatentA patent is grantedby the governmentto the inventor forbiological entities,processes andproducts.
Case Study Texmati was derived by crossing Indian Basmati rice with a semi dwarf variety. A Texas based company got patent on rights of basmati.Indian Basmati rice Texmati rice