MODERN SYSTEMS OF BACTERIAL TAXONOMY DNA ANALYSIS NUCLEIC ACID HYBRIDIZATION

Traditional vs Modern Binomial taxonomy DNA sequences

Modern systems The routine laboratory identification are the microscopic observation, culturation biochemical test and serology test Latest and the most modern era of identification are molecular biological test Most of the methods relates to the content of genetic materials

Serology Studies of serum and immune responses thru serum m/o is antigenic, normally response as antibodies The reagent used in test kit is antiserum Present of m/o usually causing the clotting effects towards antiserum (+ve result) Oppositely, the absent of m/o will be diluted effects (-ve result)

Types of serology test Slide agglutination test ELISA (Enzyme-linked Immunosorbent Agglutination) Western blotting (same approach but not categorize as serological test)

Phage typing To determine which phages a bacterium susceptible to Eg. Bacteriophage (the bacterial virus) only attacking the particular strain or types of bacteria Receptor sites for bacteriophages are highly specific. Clear sites(plaques)are left when phages have killed bacterial cells. On the basis of which phages attacked a bacteria culture ,can determine which strain of that bacterial species present.

Fatty acid analysis To compare the fatty acid profiles among m/o Also known as FAME (fatty acid methyl ester) Widely used in clinical and public health laboratories

Flow cytometry To identify bacteria in sample without culturing the bacteria Cell and m/o go through a small portion Pass thru a laser and show scattering graph determining size, shape, density and surface Also consist of fluorescent detection

DNA base composition Can be group by comparing the relative oercentage of bases present in their DNA cell. Show the relatedness via % of (Guanine G and cytosine C) G complement with C, A complement with T Pair s of GC resulting pairs of AT; (GC + AT = 100%) Studies shown G-C content varies in 23-75% in bacteria

Example: Staph aureus and Clostridium tetani have similar DNA composition while Pseudomonas aeruginosa has a different DNA composition. But similar percentage of G-C content does not prove that the organisms are closely related.Because the sequence of bases may be quite different. Example: Human & B.subtilis nearly identical G-C content.

DNA Fingerprinting To determine the source of hospital-acquired infections Spread via finger tips Relate to hospital personnel or patients from the same ward Short-time period required, instead of routine process Should have the control sequences Similar sequences shows >% of relationship

Polymerase Chain Reaction (PCR) To make multiple copies of a piece of DNA enzymatically Used to Clone DNA for recombination Amplify DNA to detectable levels Sequence DNA Diagnose genetic disease Detect pathogens ANIMATION PCR: Overview ANIMATION PCR: Components

Polymerase Chain Reaction m/o cannot be cultured through conventional methods This method can be used to increase the amount of microbial DNA to levels that can be tested by gel electrophoresis Eg. From the amber of ancient period If there are primer of particular m/o in the sample, there will be amplified DNA indicates that m/o, obviously shows in the gel

Nucleic acid hybridization Southern Blotting DNA chips Ribotyping and Ribosomal RNA sequencing Flourescent In Situ Hybridization (FISH)

Introduction Each complimentary DNA containing complementary bases When heat, the hydrogen bonds separated When cool, it reunite almost back to normal Basic purpose is to identify unknown m/o Can hybridize any single-stranded nucleic acid chain; DNA-DNA, RNA – RNA, DNA-RNA

DNA BASE COMPOSITION

Nucleic acid hybridization

Nucleic acid hybridization

Southern Blotting To identify unknown m/o Using DNA probe for rapid identification Method involves breaking enzyme, then selecting a specific fragment as a probe , then the probe able to hybridize with the DNA of all particular m/o strains, but not with the DNA of closely related group m/o

DNA probe used to identify bacteria

Southern Blotting Arrangement of papers in Blot tray.

Southern blot

DNA chips Possible to quickly detect a pathogen in a host by identifying a gene that is unique to that pathogen The chips composed of DNA probe Unknown DNA label by fluorescent dye and placed in a chips Hybridization between DNA probe and DNA detect by fluorescent

DNA chip technology

Fluorescent In Situ Hybridization Fluorescent dye-labeled RNA or DNA probe are used to specifically m/o in place or in situ Cell will be treated then the probe will enter the cell easily and react to the target ribosome in the cell (in situ) To determine the identity, abundance and relative activity of m/o in an environment Also to detect bacteria that have not yet been cultured (tiny quantity)

FISH

Ribotyping and Ribosomal RNA Sequencing To determine the phylogenic relationships among organisms 3 advantages: All cells contain ribosomes RNA genes undergo <changes over time – each sequences looks ‘same’ to each other Cell do not have to be cultured in laboratory

Ribosomal sequencing

Task of the week Compare between Western Blotting and Southern Blotting. Differentiate in table form.

The end SCORE A!!!