Bacterial Genetics

Learning the Basics
Dr.T.V.Rao MD

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Genetics Guide Life

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Genes are Eternal Run In Progeny

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Understanding Genetics
We resemble and differ because of Genetic configurations Parents - Son - Daughter, how they resemble each other. They breed true from Generation to Generation

But vary in small proportions in progeny.
Bacteria too obey the laws of Genetics
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Watson - Crick Discovery of DNA

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Beginning of Bacterial Genetics
• The principles of Genetics were applied to bacteria and viruses • Advances in Genetic process also of lead to fundamental advances in Biology and Biochemistry. A Birth of New Branch of Science

Molecular Biology
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DNA A Complex Structure Makes Life

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DNA ( Deoxyribonucleic Acid )
• DNA is composed of Many Units of Adenine – Thymine A – T Guanine – Cytosine G - C A+ T G+C proportion differ for each species DNA replicates first unwinding at one end to form a fork Each strand of fork acting as template for the synthesis of complementary strand
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Structure of DNA

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DNA
• A DNA molecule is composed of two chains of Nucleotides wound together in the form of a Double Helix • Each chain has back bone of Deoxyribose and Phosphates residues arranged alternatively Dr.T.V.Rao MD

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Structure of DNA
• Attached to each Deoxyribose and phosphate residues arranged alternatively • Attached to each Deoxyribose are of four nitrogen bases • Purines - Adenine, Guanine • Pyramidine Thymidine and Cytosine
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How RNA differs from DNA
• RNA contains - Sugar Ribose instead of Deoxyribose • Uracil is present instead of Thymine • Types of RNA Messenger RNA mRNA Ribosomal RNA rRNA Transfer RNA tRNA
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DNA - RNA

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Knowledge on DNA lead to advances in Molecular Biology
• Central dogma of Life – Deoxyribonucleic acid • DNA carries the Genetic information • DNA is transcribed to RNA – Polypeptides Cell Function depends upon specific polypeptides – Proteins – Enzymes DNA is a store house of Protein synthesis DNA acts a Template for synthesis of mRNA Virus differs from other as they contains either DNA or RNA
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What is a Code in Genetics
• Code is a unit consists of sequence of three Bases • Code is triplet A-T- C • A code can make single Amino acid • More than one code present for making similar sequence of Amino acid • AGA make Arginine • AGC, CGU, CGG, also code for similar Amino acid • Some Codons UAA Dr.T.V.Rao MD don't code for any Amino acid called as Nonsense codon

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What is a Gene
• Gene is a sequence of DNA carrying codons specifying for particular polypeptide. • DNA contains many Genes( A combinations of hundreds and thousands of Nucleotides )
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Bacterial Chromosome
• Contains a Double stranded molecules of DNA arranged in circular form. • Length 1,ooo microns. • Bacterial DNA contains about 4,000kilobases • I kb = 1000 base pairs ( A-T ) ( G-C) • Humans have about 3,000 kb pairs.
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How bacterial Genome differs from Higher forms of Life
• Several stretches of DNA don't appear to function as codons, occurs between the coding sequences of Gene. called as INTRONS. • Coded are called as EXONS • In transcription introns are excised when form RNA before translated by ribosomal proteins.
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Extra chromosomal Genetic Elements
Bacteria posses Extra chromosomal genetic elements Not Essential for survival of Bacteria But makes the Bacteria Resistant to antibiotics, and makes them survive Able to produce toxins
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Plasmids
• Plasmids are circular DNA molecules present in the cytoplasm of the Bacteria • Capable of Autonomous replication • Can transfer genes from one cell to other • Act as vectors in Genetic engineering. • Can also present in Yeasts
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Plasmid ( Blue )

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Plasmids
• Plasmid seem to be ubiquitous in bacteria, May encode genetic information for properties

1 Resistance to Antibiotics 2 Bacteriocins production 3 Enterotoxin production 4 Enhanced pathogen city 5 Reduced Sensitivity to mutagens
6 Degrade complex organic molecules
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R plasmid

R: drug resistance

RTF: transfer of R plasmid

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Plasmids
• Can be integrated with Chromosomal DNA • Episomes Integrated form of plasmid with DNA

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Potentials of Plasmids
• Plasmids can be self transmissible and Non transmissible • Transfers the Sex and Drug resistance with the help of restriction end nucleases

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Classification of Plasmids
• Incompatibility typing • Don't accommodate others which are similar • Other methods of Classification Centrifugation Electrophoresis Genetic methods
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Genotypic and Phenotypic variation
• Genome – Sum total of Gene that make up the genetic apparatus of cell established as Genotype. • Hereditary constitution of cell this transmitted to its progeny • Phenotype – is the physical expression in a environment. Change according to environment.
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What is Phenotypic expression
• • • • Exhibit – different phenotypes Appearance differs in different situations. Eg Typhoid bacilli flagellated normally But grown in Phenol agar don't grow flagella So flagella are lost physical variation • Lactose fermentation in E.coli dependent on Beta Galactosidase When lactose present - test is positive When lactose is absent - test turns negative
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Different Enzymes Guided by Genomic configurations
• Inducer enzyme acts in the presence of substrate • Constitutive enzyme acts irrespective of presence or absence of enzyme. • Phenotypic variations influenced by environment limited in range by genes • Genotypic variations are stable not influenced by environment.
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Principles of Genotypic variations
• Mutations • Genotypic by transfer of genes Transformation Transduction (Lysogenic conversion) Conjugation
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Replica Plating, pt. 2

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Mutations in Bacteria
• Bacteria Multiply by asexual binary fission • Altered Nucleotide sequence in expresses new or altered characteristics • Selective value to the organism • Evolutionary value • Acquires Antibiotic resistance grows in body without inhibition • Become a prominent organism • Phenotypic variation occurs when genes changes in response to the environment but reversible.
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Mutations
• Mutation is a Random, Undirected, Heritable variation • Caused by alteration in the Nucleotide sequence at some point of DNA which can occur due to Addition Deletion Substitution of one or more bases
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Mutation Type
Frameshift (deletion)
(leu) (ser) (arg)

Normal AAT AGT GCC
(leu) (val) (pro)

Mutant AAT AGT GCC A

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Mutation Type
Frameshift (insertion)
(leu) (ser) (arg)

Normal AAT AGT GCC
(leu) (glut) (cyst)

Mutant AAT CAGT GCC

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Mutations can occur in any sequence,inveitable, useful for Survival

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Multiple Mutations
• Causes extensive chromosomal rearrangement • Missense mutation -Triplet code is acted so as to specify an Aminoacid different from that normally located at particular position in the protein • Nonsense mutation - Deletion of nucleotide within a gene may cause premature polypeptide chain termination by nonsense codon • Tran version is Substitution of purine for pyramidine or vice versa in the base pairing
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Mutations
• Suppressor Mutation is reversal of mutant phenotype by another mutation at a point of DNA distant from that of original mutation.
• All genes are susceptible for mutations, but all mutations are not expressed • Lethal mutation is harmful destroy the vital functions
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Mutations
• Conditional Lethal mutant may be Live under certain conditions • Common example is temperature ( its ) mutant • Temp sensitive ( ts) mutant lives at 350c but not at 390c • Each gene undergoes mutation at a fixed frequency. Bacteria undergo mutations at 10-4 - to 10-10 • Tutomerisim T – A is replaced by G - A
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Mutagenic Agents
• U V rays • Alkyl ting agents • Arcidine Dyes

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GENE TRANSFER Occurs by Complex Mechanisms

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The three bacterial sexual processes
– 1.

Conjugation: direct transfer of DNA from one bacterial cell to another. –2. Transduction: use of a Bacteriophages (bacterial virus) to transfer DNA between cells. –3. Transformation: naked DNA is taken up from the environment by bacterial cells.
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Transformation of Genetic material ( Gene Transfer )

• Different Mechanisms Transformation Transduction Conjugation
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Gene Transfer Processes for Bacteria and Their Viruses
1. Conjugation
2. Transformation

3. Transduction 4. Infection with bacteriophage

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What is Transformation
• Transformation is defined as transfer of Genetic information through the activity of DNA • Griffith experiment Mice injected with Live non capsulated ( R ) Pneumococci with heat killed capsulated (S) Pneumococci Lead to death of Mice with isolation of Live capsulated Pneumococci It means that some factor from Dead pneumococci transferred to live non pathogenic Pneumococci
Dr.T.V.Rao MD Avery, McCleod, Mc Cartny in 1944 identified to be DNA 47

Griffith Phenomenon

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Demonstration of transformation
Avery, MacLeod, and McCarty (1944)

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Transduction
• Generalized involve any segment of DNA

• Restricted when specific Bacteriophages traduces only a particular genetic trait. • Transduction effects Plasmids ,and Episomes • Plasmid transfer induces Penicillin resistance in Staphylococcus • Helps Genetic mapping, also in eukaryotic cell • Helps Genetic Engineering
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Transduction
• Transduction is defined as transfer of portion of DNA from one bacteria to another by Bacteriophages, is known as Transduction

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DNA transfer through Bacteriophages
• When the Phage particle infects another bacteria DNA transfer is effected and the recipient cell acquires new characters coded by donor DNA
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Bacteriophages
• Are viruses that parasitize bacteria and consists of Nucleic acid core and a protein coat • A phage particle may have at its core besides its own nucleic acid and a segment of the Host Dr.T.V.Rao MD DNA

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Transduction Types
• Two types of Transduction • 1 Lytic and 2 Lysogenic • 1 Virulent or Lytic cycle after large number of progeny are built up inside the host bacterium ruptures and phages are released
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Lysogenicity creates new characters • Eg - Lysogenic conversion in Diphtheria bacilli which acquires toxigenicity by lysogenization with phage beta • Elimination of phage for toxigenic strain renders nontoxigenic
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Conjugation Lederberg - Tatum
• A process by which a Donor cell or male cell makes contact with another cell, the recipient or Female cell.

• DNA is directly transferable
• Plasmid Carry genetic information necessary for conjugation to occur. • Only cell that contain such plasmids can act as donor. the cell lacking a corresponding plasmid act as recipient. • Requires direct contact between donor and Dr.T.V.Rao MD 56 recipient

Conjugation
• The ability to conjugate is conferred by the F plasmid. A plasmid is a small circle of DNA that replicates independently of the chromosome. Bacterial cells that contain an F plasmid are called “F+”. Bacteria that don’t have an F plasmid are called “
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Conjugation - Transferring genes with plasmids
• Plasmids mediating conjugation carry genes coding for properties, of 1-2 microns long protein appendage termed Pilus on the Donor cell
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Mechanism of Transfer I: Conjugation

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Conjugation

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Simple Conjugation

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Conjugation

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Pilus helps Conjugation
• Different types of Pilus are specified by different types of plasmids and can help in aid of plasmid classification. • Only one strand of circular DNA of the plasmid nicked upon at a specific site and passed into a recipient. • Spread to all other cells.
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F factor
• Transfer factor that contains the genetic information necessary for synthesis of Sex Pilus and for self transfer without any other identifiable genetic materials such as drug resistance
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F factor helps transformation
• F+ called as Donor bacteria can transform F- into F+ cell Can be Episomes able to exist in some cells in the integrated state in the donor cell chromosome Can transform chromosomal genes to recruitment with high frequency are known as Hfr cells Conversion of F+ cells into Hfr state is reversible. F factor incorporates some chromosomal genes and is called as F’ Sexduction The process of transfer of host genes through F’ factor
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DNA transfer through Bacteriophages
• When the Phage particle infects another bacteria DNA transfer is effected and the recipient cell acquires new characters coded by donor DNA
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Types of DNA transfer through Bacteriophages

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Colicinogenic ( Col ) Factor
• Coli form Bacteria produce Colicins • Colicins are lethal to other Enterobacteriaceae • Pyocins produce by Pseudomonas • Diptherocins produced by C.diptheria
• Plasmid transmits col factor leads to self transfer of chromosomal segments
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Resistance Transfer Factor RTF
• Plasmids – helps to spread multiple drug resistance • Discovered in 1959 Japan • Infections caused due to Shigella spread resistance to following Antibiotics

Sulphonamides Streptomycin Chloramphenicol, Tetracycline
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RTF
• Shigella + E.coli excreted in the stool resistant to several drugs in vivo and vitro • Plasmid mediated – transmitted by Conjugation • Episomes spread the resistance
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Bacterial Conjugation: High Frequency Transfer (Her) Cells

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Hfr Conjugation

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Sequence of RTF transmission

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Hfr cell conjugating a Normal cell

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Composition of RTF
• Plasmid consists of two components • A transfer factor RT, helps conjugational transfer and resistant determinants ( r ) to each of the several drugs • RTF + r determinants are known as R factor
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R factor
• R factor can contain several determinants as many as 8 or > 8 drugs • Guide the cell for production of Enterotoxins too • But R factors can be inhibited by Bile salts R factors can be transferred to animals
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Genesis of R factors
• In discriminate use of Antibiotics in vet nary Medicine has increased the spread of R factors to Human • Addition of Antibiotics to Animal feeds to be prohibited.
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Genetic Mechanisms of Drug Resistance
• Bacteria acquire drug resistance through several Mechanisms • Mutations • Genetic transfer Transformation, Transduction Conjugation Several Biochemical Mechanisms Decreasing permeability of drugs, Attaining alternative pathways Produce enzymes and inactivate drugs
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Genetic Mechanisms in Bacteria helps to Spread the Infectious diseases

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Mutations
• Mutilations can be 1 Stepwise mutation as in Penicillin use 2 One step mutation Streptomycin use May show low resistance or High resistance If tuberculosis is treated with sole drug as of Only Streptomycin some resistant mutants appear and replaces sensitive bacteria in due course so the occurrence of MDR - TB
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Other Mechanisms
• Use of Penicillin created resistant Staphylococcus by transduction • R factors created resistance to several drugs, caused increased virulence • Spread to several humans and animals

Best option- To restrict use of Antibiotics
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Transposable Genetic Elements
Structurally / Genetically – Discrete sequence of DNA – Move around in a cut and paste manner between Chromosomal and Extra chromosomal DNA molecules within cells. Called as Transposons _ Jumping Genes Genetic transfer due to Transposition Small Transposons 1 – 2 Kb Not self replicating and depend on Plasmid or Chromosome for replication. A chunk of DNA is added by Transposons.
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Transposons and R factor
• R forms may have evolved as a collection of Transposons • Each carrying Genes that confers resistance to one or several Antibiotics • Seen in Plasmids, Microorganisms Animals Laboratory Manipulations are called as Genetic Engineering
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Molecular Genetics
• Analysis and manipulation of DNA using Biochemical and Microbiological techniques
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Genetic Engineering
• Under standing Molecular genetics in Biochemistry fuels genetic Engineering • Recombinant DNA (renal) techniques changed the ideals of Medicine

• Genetic Engineering await many surprises?
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Genetic Engineering
Genetic Engineering Was Born from

Genetic Recombination
•Genetic engineering involves changing the genetic material in an organism to alter its traits or products •A recombinant DNA molecule contains DNA fragments spliced together from 2 or more organisms
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Modern applications
• Pharmaceutical production
–Insulin, interferon, hormones, vaccines etc.

• Genetically engineered plants • Animal gene alterations • Gene probes • DNA fingerprinting • The human genome initiative
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Genetic Engineering
• Isolation of Genes coding for any desired protein from Microorganism or from cell of higher life forms including human beings and their introduction into a suitable microorganism in which genes would function directing the production of specific proteins
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Genetic Engineering changing the Diagnostic and Therapeutic Protocols in MEDICINE

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Research on Gene transfer shapes the future of Science

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Genetically Engineered Products
• Can prepare desired protein in pure form in economic way Somatostatin • Commercial preparations pdf Cloned Human Insulin Interferons

Hepatitis B vaccine

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Restriction Endonucleases
• A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA. The enzyme makes two incisions, one through each of the sugar-phosphate backbones (i.e., each strand) of the double helix without damaging the nitrogenous bases They work with cutting up foreign DNA, a process called
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Restriction Endonucleases Made the advances in Genetic Engineering

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DNA Probes
• There are Radioactive Biotinylated otherwise labeled copies united single stranded DNA Contains 20 -25 nucleotides Helps detection of Homology DNA by Hybridization. Helps Diagnosis of Infectious Diseases Minute quantities of DNA can be detected.
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Blotting Techniques
• Drug fragments obtained by restriction enzyme digestion on separation Gel can be transferred to Nitrocellulose or nylon membranes • Several methods 1 Southern blotting 2 Northern Blotting 3 Western blotting
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western blot
• The western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions)
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Western Blotting
• In Western Blot Protein ( Antigen ) mixture is separated by SDS ( Sodium dodecyl sulfate – polyacrylamide gel electrophoresis ) Blotted on to Nitro cellulose strips and identified by radio labeled or enzyme labeled antibodies as probes
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Western Blot

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• Western Blot testing is confirmatory test for diagnosis of HIV/AIDS • Identifies antibodies directed against different antigens in pathogen Surface, Core Dr.T.V.Rao MD RT antigen

Western Blot to confirm HIV Infections made land mark Diagnostic tool

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Polymerase chain reaction Kary B Mullis 1983
• Rapid • Automatic amplification of specific DNA sequences • Nobel prize winning Technology 1993

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PCR -Sequences
• PCR consists of several cycles of sequential DNA replication where the products of first cycle becomes the template for the Next • It makes available abundant quantities of specific DNA sequences starting
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Genetic Mapping
• Genetic sequences for Bacteriophages and virus • Genetic mapping is done most of the Human Genes

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Newer Understanding on GENES

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Human Genome Project

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Human Genome Project
• Completed in 2003, the Human Genome Project (HGP) was a 13-year project coordinated by the U.S. Department of Energy and the National Institutes of Health. During the early years of the HGP, the Welcome Trust (U.K.) became a major partner; additional contributions came from Japan, France, Germany, China, and others
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Genetics are Complex - Leading the birth of BIOINFORMATICS

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Genes Evolved and made us Men What NEXT ?

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Are We Playing with Genes in the Right Direction ?

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Understanding of human Genome is Changing the Future of Medicine

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• Programmed Created by Dr.T.V.Rao MD for Undergraduate Medical Students in the Developing World • Email • doctortvrao@gmail.com

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