Molecular Techniques in Microbiology

These include 9 techniques

(1) Standard polymerase chain reaction (PCR):

@ Kary Mullis invented the PCR in 1983 (USA) Given Nobel Prize in Chemistry in 1993.

@ He was able to amplify DNA by repeated cycles driven by DNA polymerase enzyme.

@ PCR technique used an automated process for DNA amplification & separation of the DNA strands.

Kary Mullis

PCR Applications:

1. DNA extraction: From cell genome DNA by selective amplification of a specific region of DNA. This is done by hybridization تهجين)) probe and DNA clones (نسيالت) techniques to create copies of DNA fragments.

2. DNA sequencing: To determine unknown PCR-amplified sequences by the insertion of a DNA sequence into a genetic material of another organism.

3. DNA-based phylogeny: To determine the evolutionary relationships among organisms.

Extracted DNA

4. Identification of uncultural or slow-growing organisms: Such as mycobacteria, anaerobic bacteria, or viruses.

5. Early diagnosis of infectious diseases:

Such as leukemia and lymphomas; detecting malignant cells at a high sensitivity.

PCR

6. Quantification viral load in a patient (HIV)

7. Diagnosis of hereditary diseases

8. Identification of genetic fingerprints. This is used in forensic sciences and paternity testing

Paternity fingerprints (1) Father. (2) Child. (3) Mother .

(2) Real-time PCR:

@ Real time means immediate. Also called quantitative PCR (qPCR)

@ It is used to amplify and quantify DNA.

@ qPCR enables at the same time both detection and quantification.

@ qPCR technique uses hybridization probe technique.

DNA probe hybridization

Applications of qPCR: 1.DNA amplification, DNA quantification, genotyping.

2. Quantification of microbial load in foods and vegetables.

3. Rapid detection of cancer, newly emerging diseases, and genetic abnormalities.

4. Microbial assessment of water quality.

5. Production of plant seedlings that are free of pathogens.

PCR cycler

(3) Dna Sequencing & Genome Data Technique:

Used to analyze the bacterial DNA sequence and mapping all its genome data.

(4) Rna Interference Technique:

Used in biological research and drug discovery

(5) 16s Rrna Sequencing Technique:

Used for bacterial identifications

Automated Genetic Analyzer for complete genome sequencing .

(6) Gel Electrophoresis Technique:

Used to separate DNA, RNA, or protein molecules by blotting through an electric field by virtue of their size, shape or electric charge.

Gel electrophoresis machine

(7) Dna Microarray Technique:

@ Used as an alternative to the electrophoresis blotting techniques.

@ This technique can detect the presence of pathogens in a sample.

@ It can determine the genetic differences between two microbial strains.

Microarrays Machine

(8) Direct Gene Detection Technique:

Used to detect genes related to drug resistance mechanisms, such as mecA gene in Staphylococcus aureus

Gene detection

(9) Pulsed-field gel electrophoresis Technique:

@ Used for separation of large DNA molecules by applying to a gel matrix electric field that periodically changes direction.

@ It is similar to standard gel electrophoresis except that instead of running the current in one direction, it is run in 3 directions.

@ Used for genotyping, genetic fingerprinting, and tracing sources of infections.

Genotyping technique