M03.05.05 Molecular Techniques in Medicine

Molecular techniques have revolutionized modern medicine, providing powerful tools for diagnosis, treatment, and research. These techniques allow for the analysis of DNA, RNA, proteins, and other biomolecules, offering insights into genetic disorders, infectious diseases, cancer, and personalized medicine.

Polymerase Chain Reaction (PCR)

Definition: PCR is a technique used to amplify specific DNA sequences.

Steps:

1. Denaturation: Heating the DNA sample to separate strands.
2. Annealing: Cooling to allow primers to bind to the target sequence.
3. Extension: DNA polymerase extends the primers to form a new DNA strand.

Applications:

• Diagnostics: Detecting genetic mutations, and pathogens.
• Research: Cloning, sequencing.
• Forensics: Identifying individuals from DNA samples.

Table: PCR Components

Key Points to Memorize:

• Denaturation temperature: ~95°C
• Annealing temperature: 50-65°C (depends on primer Tm)
• Extension temperature: ~72°C

Gel Electrophoresis

Definition: A technique to separate DNA, RNA, or proteins based on size and charge.

Process:

1. Preparation: The sample is loaded into a gel matrix.
2. Electrophoresis: Electric current is applied, causing molecules to migrate through the gel.
3. Visualization: The stained gel is observed under UV light or other detection systems.

Applications:

• DNA Fragment Analysis: Size estimation of PCR products.
• Protein Analysis: Identifying and purifying proteins.
• RNA Studies: Evaluating RNA integrity and size.

Table: Gel Electrophoresis Types

Key Points to Memorize:

• Agarose gel concentration: 0.7-2% for DNA
• Loading dye: Helps visualize and track sample migration
• Ethidium bromide: Common stain for visualizing nucleic acids

Real-Time PCR (qPCR)

Definition: Quantitative PCR allows for the quantification of DNA or RNA in real-time.

Advantages:

• Sensitivity: Detects low levels of nucleic acids.
• Quantification: Measures the amount of target sequence in real-time.
• Speed: Faster than traditional PCR.

Applications:

• Gene Expression Analysis: Quantifying mRNA levels.
• Pathogen Detection: Quantifying viral or bacterial load.
• Genotyping: Detecting genetic variations.

Table: qPCR Components

Key Points to Memorize:

• Ct value: Cycle threshold where fluorescence exceeds background
• SYBR Green: A common fluorescent dye
• TaqMan Probes: Specific probes for detecting DNA sequences

Next-Generation Sequencing (NGS)

Definition: NGS encompasses high-throughput sequencing technologies that allow for rapid sequencing of entire genomes or targeted regions.

Technologies:

• Illumina Sequencing: Short-read sequencing with high accuracy.
• PacBio Sequencing: Long-read sequencing for complex genomes.
• Nanopore Sequencing: Real-time sequencing with long reads.

Applications:

• Genomics: Whole genome sequencing, exome sequencing.
• Transcriptomics: RNA sequencing for gene expression profiling.
• Oncology: Detecting cancer mutations, personalized treatment.

Table: NGS Workflow

Key Points to Memorize:

• Read Length: Determines the resolution of sequencing
• Coverage: Depth of sequencing for accuracy
• Bioinformatics: Essential for data analysis and interpretation

CRISPR-Cas9

Definition: A genome editing tool that allows for precise modifications to DNA.

Mechanism:

1. Guide RNA (gRNA): Directs the Cas9 enzyme to the target DNA sequence.
2. Cas9 Enzyme: Cuts the DNA at the specified location.
3. DNA Repair: The cell’s repair machinery fixes the cut, allowing for gene editing.

Applications:

• Gene Knockout: Disabling specific genes to study their function.
• Gene Therapy: Correcting genetic mutations.
• Functional Genomics: Studying gene function and regulation.

Table: CRISPR-Cas9 Components

Key Points to Memorize:

• PAM Sequence: Protospacer adjacent motif required for Cas9 binding
• Off-target Effects: Potential unintended edits in the genome
• Applications: Wide-ranging from research to therapeutic interventions

Conclusion

Molecular techniques are indispensable in modern medicine, offering unparalleled insights and tools for diagnosis, treatment, and research. Mastery of these techniques equips medical professionals with the knowledge and skills to advance clinical practice and scientific discovery.

Key Points to Memorize:

• PCR: Steps (Denaturation, Annealing, Extension) and applications.
• Gel Electrophoresis: Types (Agarose, Polyacrylamide) and uses.
• qPCR: Quantification, sensitivity, key components.
• NGS: Technologies, workflow, applications.
• CRISPR-Cas9: Mechanism, components, clinical relevance.