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Questions and Answers
Which of the following are types of DNA mutations?
Spontaneous mutations can only affect non-vital functions.
False
What are mutagens?
Agents that produce DNA alterations
______ is a technique that allows for precise localization of a specific segment of nucleic acid within a histological section.
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Match the DNA amplification method with its description:
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Which of the following are types of RNA? (Select all that apply)
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The observable properties in an organism are referred to as __________.
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Exons are coding sequences of genes, while Introns are functional genes.
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What is the purpose of Transformation in gene transfer?
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Match the mechanism of gene transfer with the correct description:
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What is the central dogma of molecular biology?
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Which of the following describes the DNA expression of information?
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Which code terminates the messages for the synthesis of a polypeptide?
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Which of the following classes of microbial adhesins measure virulence as a quantitative measure of pathogenicity?
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Which of the following is included in exotoxins?
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Study Notes
Bacterial Genetics and Molecular Diagnosis of Infectious Diseases
Objectives of this Lecture
- Describe the structure of DNA
- Enumerate the principles of genetic replication
- Elucidate the methods of bacteria gene transfer
- Highlight the application of Molecular genetics in the diagnosis of infections
Introduction to Genetics
- Genetics: the science that defines and analyzes heredity
- Heredity: the constancy and change in physiologic functions that form the properties of organisms
- Genotype: the hereditary potential of the organism
- Phenotype: the observable properties in an organism (e.g., resistance to antibiotic)
Central Dogma of Molecular Biology
- Transcription: the synthesis of an RNA copy (complement) of a segment of DNA
- Translation: the synthesis of a polypeptide (Proteins or Enzymes) from RNA
DNA Expression of Genetic Information
- Genetic information (DNA) is stored as a sequence of nucleotides
- Nucleotides consist of 2 types of bases: Purine (Adenine and Guanine) and Pyrimidine (Thymine, Cytosine)
- DNA molecule structure is double-stranded and complementary
- Ratio of each pair of bases (A+T) / (G+C) is constant for each species but varies widely from one species to another
RNA Expression of Genetic Information
- RNA is structurally similar to DNA, but with 2 major differences: it has Ribose instead of Deoxyribose and base Uracil instead of Thymine
- There are 3 types of RNA: Messenger (mRNA), Transfer (tRNA), and Ribosomal (rRNA)
- DNA acts as a template for mRNA synthesis
Gene Expression
- Gene: the basic physical and functional unit of heredity
- Genes are segments of DNA that carry codons specifying a particular polypeptide
- Bacterial chromosome contains a double-stranded molecule of DNA arranged in a circular form
Mechanism of Gene Expression
- Transcription: RNA polymerase forms a single polyribonucleotide strand mRNA, using DNA as a template
- Translation: mRNA and tRNA come together on the surface of a ribosome, known as ribosomal RNA (rRNA)
- Peptidyl transferase (23S RNA or ribozyme) catalyzes the formation of the peptide bond
Gene Transfer
- Bacteria are haploid organisms that use a series of primitive mechanisms for gene transfer
- There are 3 mechanisms of gene transfer: Transformation, Transduction, and Conjugation
Plasmids
- Plasmids are extra chromosomal genetic elements, circular DNA molecules present in the cytoplasm of bacteria
- Plasmids are capable of autonomous replication (independent replicons)
- Plasmids may confer properties such as drug resistance and toxigenicity to the cell
DNA Mutation
- Mutation is a random, undirected, heritable variation caused by an alteration in the nucleotide sequence at some point on the DNA of the cell
- Mutation can be spontaneous or induced by mutagens
- DNA mutations can take different formats: Addition, Deletion, Substitution, Frame-shift, Missense, Non-sense, Transversion, Suppressor
Molecular Diagnosis of Infectious Diseases
- Molecular diagnostic tests can be divided into two primary groups: tests designed to detect mutations in the human genome, and tests designed to detect infectious agents
- Microorganisms can be rapidly identified using molecular methods, to diagnose causative agents in infections
- Three component-steps are involved in molecular diagnosis: Specimen processing and nucleic acid extraction, Target amplification, and Detection/characterization of amplified product
Target Amplification
- Two primary methods are used: Nucleic acid hybridization techniques with specific molecular probes, and DNA amplification by the polymerase chain reaction (PCR)
Advantages of Molecular Diagnosis
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Ability to detect latent (non-replicating) viruses and to localize their genomes to nuclear or cytoplasmic regions within cells
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Ability to characterize antimicrobial resistance
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Prospects for molecular medicine in microbiology are vast and will have profound effects in laboratory and clinical practice### Signal Amplification
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Signal amplification techniques involve the interaction between a probe and target, which is augmented through successive rounds of nucleotide and/or antibody hybridization.
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This approach maximizes the detectable readout.
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A widespread application of signal amplification is in the detection of human papillomavirus in Pap smears.
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Signal amplification has largely given way to target-amplification of nucleic acid in current practice.
Quantifying Amplification Products
- PCR can exponentially amplify low-abundance DNA/RNA, but its diagnostic utility relies on paired methods for detecting the resultant products.
- Amplicons must be detected by simple, direct, and reproducible means to be leveraged effectively by clinical laboratories.
- Early diagnostic PCR involved electrophoresing the product through an agarose gel and staining with a DNA-binding fluorescent dye (e.g., ethidium bromide) to visualize amplicons under UV light.
- PCR can be combined with molecular probes for combined amplification/detection, using labeled oligonucleotides that bind amplicons and facilitate an empirical readout.
- Many platforms exploit matrix hybridization in both solid and liquid formats, including methods for signal generation such as antibody conjugation and chromogenesis, amplicon identification via mass spectrometry, and modulation of electronic microcircuits.
- Fluorescent probes are a widespread method for PCR detection in clinical microbiology.
Nucleic Acid Amplification
- Other than PCR, other NAATs have been developed that utilize strategies such as isothermal methods that do not require temperature cycling.
- Examples include loop-mediated isothermal amplification, nicking endonuclease amplification reaction, transcription-mediated amplification, and the helicase chain reaction.
- The COVID-19 pandemic has led to several CRISPR/cas*-based assays gaining IVD-status for the first time through emergency use authorization, in combination with loop-mediated or recombinase-aided amplification.
Sequencing in Microbial Diagnostics
- While NAATs remain the most common molecular tools within clinical microbiology laboratories, a pathogen’s genetic sequence can also provide valuable data.
- Diagnostic sequencing is assuming a growing role in the care of infections, especially given advances in sequencing technology.
- Next-generation sequencing (NGS) technologies include both short-read second-generation methods (e.g., Illumina, Ion Torrent), as well as long-read third-generation techniques.
Gene Expression and Transfer
- Gene expression involves the mechanism of gene information expression and transfer in bacteria.
- Bacterial transfer of genetic information occurs through mechanisms such as mutation and transferable drug resistance.
Molecular Diagnostics
- Molecular diagnosis of infectious diseases involves the use of techniques such as PCR, NAATs, and sequencing to detect and identify pathogens.
Central Dogma and DNA Expression
- The central dogma of molecular biology describes a two-step process: transcription – translation.
- DNA expression of information involves transcription, where two DNA strands are complementary and not parallel, and the ratio of each pair of bases (A+T) / (G+C) is constant for each species with no variation among species.
Virulence and Toxins
- Virulence is a quantitative measure of pathogenicity, and classes of microbial adhesins include glycosaminoglycan.
- Exotoxins include bacterial products released during exponential growth and are toxic to target cells.
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Description
This lecture covers the structure of DNA, principles of genetic replication, methods of bacteria gene transfer, and application of molecular genetics in infectious disease diagnosis.
