6. Técnicas de Biología Molecular para identificación y caracterización de genes RAM

Name: 6. Técnicas de Biología Molecular para identificación y caracterización de genes RAM
Uploaded: 2021-09-14T21:59:50.000Z
Duration: 30 min 56 s

Molecular Techniques for Identifying and Characterizing Resistance Genes

Introduction to Molecular Violence Techniques

The module discusses molecular techniques for identifying and characterizing genera, focusing on the genetic information that defines organisms' form, function, and behavior.

Genes serve as units of hereditary information storage and transmission, which can be passed vertically (from parent to offspring) or horizontally (between microorganisms).

Mechanisms of Antibiotic Resistance in Bacteria

Bacteria develop resistance mechanisms to survive in antibiotic-rich environments; this information is encoded in resistance genes known as RAM genes.

Resistance may arise from mutations in existing genes or acquisition of new genes from the environment. Understanding these genes aids in identifying bacterial genotypes and their epidemiology.

Detection Techniques for RAM Genes

Traditional detection methods include molecular biology techniques like PCR; however, sequencing provides more comprehensive insights into resistance gene characterization.

Advances in sequencing technology have led to first-generation (Sanger) sequencing and second/third-generation whole-genome sequencing, which will be discussed further.

Fundamentals of DNA Extraction

The extraction process involves cell lysis using detergents or heat, followed by the removal of proteins and lipids with organic solvents to isolate DNA for analysis.

Two primary methods exist: traditional methods using organic solvents and commercial kits that streamline the process while ensuring high-quality DNA extraction.

Steps Involved in Commercial DNA Extraction Kits

The extraction process includes three main steps:

Cell Lysis: Breaking down cellular structures to release DNA.

Purification: Separating DNA from proteins/lipids through washing solutions and centrifugation.

Elution: Collecting purified DNA using an elution buffer followed by centrifugation.

Assessing Quality of Extracted DNA

Evaluating extracted DNA's purity, concentration, and integrity is crucial before proceeding with further analyses; common techniques include spectrophotometry for quantification.

Absorbance at 260 nm indicates the amount of DNA present; higher absorbance correlates with greater concentrations due to the chemical nature of DNA's absorption spectrum at this wavelength.

Purity Assessment Using Spectrophotometry

To assess purity, different indices are used; a ratio between absorbances helps determine contamination levels.

A ratio between 1.8 to 2.1 suggests optimal quality free from protein contamination.

Ratios below these values indicate potential contamination issues such as proteins or phenols affecting sample integrity.

Integrity Check via Electrophoresis

Understanding DNA Visualization and PCR Techniques

DNA Visualization Techniques

The presence of a single band in agarose gel indicates intact DNA, while a smear suggests degradation.

For PCR products, a single band at a specific molecular weight is expected; multiple bands may indicate non-specific amplification.

Fundamentals of PCR

Polymerase Chain Reaction (PCR) is the most widely used technique for detecting DNA sequences by generating millions of copies through repetitive cycles.

Each cycle consists of three key steps:

Denaturation: Double-stranded DNA separates at high temperatures to form single strands.

Alignment: Primers bind to specific regions on the denatured DNA, defining the target fragment for copying.

Extension: A polymerase enzyme synthesizes new DNA strands based on the template provided by the primers.

Application of Sequencing Techniques

The Sanger sequencing method is highlighted as a traditional approach for sequencing fragments between 150 to 800 base pairs. It generates various lengths of DNA copies using special nucleotides that terminate further elongation.

Fluorescent techniques and capillary electrophoresis have modernized Sanger sequencing, allowing simultaneous detection of all four nucleotides due to their unique fluorescence properties. This enables separation based on size during electrophoresis.

Practical Uses and Limitations

Sanger sequencing is commonly used for characterizing bacterial plasmids and PCR products, especially when prior knowledge of gene sequences exists, such as with antimicrobial resistance genes like SHV1.

After extracting pure and intact bacterial DNA, specific PCR amplifications are performed followed by Sanger sequencing using fluorescent nucleotides detected by optical systems. The results are presented as an electropherogram showing peaks corresponding to nucleotide bases.

Conclusion on Molecular Detection Techniques

To detect resistance genes in bacteria, it’s essential first to extract high-quality DNA verified through molecular techniques like spectrophotometry and electrophoresis.