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8.Microbialgenetics.pptx.....................................
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- 2. BASIC PRINCIPLES OF MOLECULARBIOLOGY DNA – Essential material of heredity Possesses all the information for protein synthesis Exceptions are RNA viruses DNA carries genetic information Transcribed on RNA – translated (polypeptide)
- 3. STRUCTURE OF DNA Doublehelix Backbone deoxyribose and phosphate residues Purines: Adenine (A) and guanine (G) Pyrimidines: Thymine (T) and cytosine (C) Hydrogen bond- between strands Bacteria grouped based on GC content
- 4. STRUCTURE OF DNA Aschematic drawing of the Watson–Crick’s structure of DNA showing helical sugar–phosphate backbone of the two strands held together by hydrogen bonding between the bases The chemical nature of a segment of double-stranded DNA
- 5. STRUCTURE OF RNA Similar toDNA except • Contains ribose sugar instead of deoxyribose • Uracil instead of thymidine DNA acts as template for RNA synthesis Types of RNA • Messenger RNA (mRNA) • Ribosomal RNA (rRNA) • Transfer RNA (tRNA)
- 6. TERMS RELATED TO GENETICS Gene:A segment of DNA carrying specific codons for the synthesis of a particular polypeptide Genome: Sum of an organism’s genetic material Extrachromosomal Genetic Elements • In addition to chromosomal DNA • Not essential for the normal life and functioning of the host bacterium • Confer drug resistance and toxigenicity on the bacteria • Gives survival advantage
- 7. EXTRACHROMOSOMAL GENETIC ELEMENTS Plasmids CircularDNA in cytoplasm Replicate independently Transferred from one cell to another Important tool in genetic engineering Conjugative and non-conjugative plasmids Episomes Plasmid integrated with chromosomal DNA
- 8. Transposable genetic elements • Transposons(‘jumping genes’) • Structurally and genetically discrete DNA segments • Move around in a ‘cut-and-paste’ manner between chromosomal and extrachromosomal DNA molecules within cells. • this mode of genetic transfer: transposition • Small transposons: ‘Insertion sequences’ or IS • Transposon insertion acquisition of new characteristics
- 9. PHENOTYPE • Physical expressionof genotype in a given environment GENOTYPE • All the genes present in a cell (the genome) collectively constitute its genotype • Transmitted to its progeny GENETIC VARIATIONS • Due to alterations in the genome • Stable and heritable • Occur by mutation or by one of the mechanisms of genetic transfer
- 10. MUTATIONS Heritable changesin DNA Alteration in nucleotide sequence Substitution of a nucleotide Deletion of a nucleotide Addition of nucleotides Two types o Spontaneous o Induced mutation by mutagens – UV rays
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- 12. BACTERIAL RECOMBINATION A piece ofDNA (containing specific genes) is created artificially from two or more sources Types of recombination • Homologous: Between similar DNA molecules • Non-homologous: Between non-similar pieces of DNA • Site-specific: Between short sequences present in dissimilar molecules • Replicative recombination: Generates a new copy of a segment of the DNA
- 13. NUCLEIC ACID PROBES • DNAprobes • Hybridise with DNA or RNA • Radioactively labelled • Used to diagnose infectious diseases • High degree of specificity • Detects minute quantity • Detect microbes impossible to culture
- 14. BLOTTING TECHNIQU ES • Southern blotting -Identifying DNA fragment - DNA-DNA hybridisation - Detects genetic disorders and cancers • Northern blotting - Identifies RNA - Detects mRNA in a sample for gene expression
- 15. • Western blotting: -Identify proteins (antigens) - Detect HIV antibodies • Eastern blotting: - Analyse protein post-translational modification (PTM) - Used to detect carbohydrate epitope
- 16. DNA AMPLIFICATION TECHNIQUE Polymerase chainreaction • Amplification of specific DNA sequence • Diagnosis of infectious diseases • Genetic of neoplastic diseases • Forensic investigations • Used in phylogenetic evolution Reverse transcriptase PCR — RNA viruses Real-time PCR (Quantitative) — Viral load Nested PCR — Two primers used Multiplex PCR — More than one primers used
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- 18. (a) Thermocycler (conventional PCR)to amplify the desired DNA and (b) gel documentation of amplified DNA bands of specific base pairs compared to a molecular weight ladder (Source: Department of Microbiology, Pondicherry Institute of Medical Sciences, Puducherry)
- 19. DNA AMPLIFICATION TECHNIQUE ConventionalRT-PCR (Source: Molecular division, Dept of Microbiology, Pondicherry institute of medical sciences, Puducherry) Quantitative PCR(qPCR) (Source: Pushpagiri Institute of Medical sciences and research centre, Thiruvalla, Kerala)
- 20. NON- AMPLIFICATIO N TEST LAMP(loop-mediatedisothermal assay) Constant temperature Does not require thermocycler DNA microarrays Many DNA spots on a solid surface Molecular epidemiology Plasmid profile analysis Genomic fingerprinting Typing of microorganisms
- 21. NON- AMPLIFICATION TEST Genetic mapping • Sequenceseveral DNA fragments simultaneously • Subsequently perform bioinformatics analyses on these fragments • Differentiate microorganisms at the genetic level Uses • Rapid detection of pathogens in clinical material • Detecting molecular markers • Tracing the source of infection in an outbreak • Identifying new microorganisms or mutants • Detection of genes responsible for drug resistance
- 22. NON- AMPLIFICATIO N TEST Sequence-based assays Many DNA sequenced in parallel Analysed by bioinformatics Advantages Highly sensitive and specific Relatively rapid compared to culture Can detect several characteristics of the microorganism, e.g., antibiotic resistance and virulence Can be used for epidemiological purposes
- 23. APPLICATION OF GENETIC ENGINEERING IN MICROBIOLO GY Recombinant DNA(rDNA ) Technology Involves cloning Specific gene product obtained in large amounts Desired proteins obtained in pure form Cloned human insulin and growth hormone Safer vaccines: Hepatitis B vaccine and rabies vaccine production Test kits in diagnosis of infectious diseases