Genetics Week 8 Quiz

Molecular Techniques for DNA and Protein Analysis

• Gel Electrophoresis: Method for separating DNA fragments by size after isolation from an organism.
• Southern Blotting: Used to isolate specific genes of interest post-separation of DNA fragments.
• Molecular Cloning: Technique to clone and produce large quantities of a targeted gene.
• Reverse Transcription (RT) & CR: Processes for understanding transcriptional profiles of genes, measuring gene expression levels.
• DNA Sequencing: Determines the precise sequence of nucleotides in DNA, checking for mutations.
• Recombinant Protein Expression: Produces protein products from cloned genes.
• SDS-PAGE: Technique for separating proteins based on size after tissue collection.
• Western Blotting: Isolates and detects specific proteins post-SDS-PAGE separation.

SDS-PAGE and Protein Visualization

• Role of SDS: Sodium Dodecyl Sulfate denatures proteins by unfolding them and imparts a uniform negative charge, allowing size-based separation during electrophoresis.
• Staining Agents: Coomassie Blue is commonly used to visualize protein bands on SDS-PAGE gels.
• Membrane Types: Nitrocellulose and Polyvinylidene Difluoride (PVDF) membranes are commonly used in Western blotting.
• Protein Detection: Uses antibodies; primary antibodies bind to target proteins, while secondary antibodies facilitate detection via conjugation with enzymes or fluorophores.
• Antibody Functions:
  • Primary antibodies specifically bind to target proteins.
  • Secondary antibodies bind to primary antibodies to enable visualization or detection of target proteins.
• Western Blot Clinical Application: Used for confirming HIV diagnosis by detecting anti-HIV antibodies in serum.

Sanger Sequencing

• Modified Nucleotides: Dideoxynucleotide triphosphates (ddNTPs) lack a 3’ hydroxyl group, preventing chain elongation when incorporated into the DNA strand, thus terminating synthesis.

Genomics Overview

• Definition: Genomics is the comprehensive study of an organism’s complete DNA (genome), including structure, function, evolution, and mapping.

Human Genome Project Sequencing Methods

• Hierarchical Shotgun Sequencing: Large DNA fragments are cloned, mapped, and sequenced in smaller pieces to create a physical map.
• Whole Genome Shotgun Sequencing: Entire genomes are fragmented and sequenced directly, assembling sequences using computational methods.

Genetic Variations

• Single Nucleotide Polymorphisms (SNPs): Variations at a single nucleotide position in DNA, representing common genetic variations among individuals.
• Haplotype: A specific combination of alleles or SNPs on a single chromosome that are inherited together.

DNA Profiling Techniques

• Short Tandem Repeat (STR) Analysis: Examines repeating base sequences in DNA, varying among individuals, useful for identification.
• Restriction Fragment Length Polymorphism (RFLP): Cuts DNA into fragments using restriction enzymes and separates them by gel electrophoresis, revealing individual variations.

Southern Blot vs. Western Blot

• Hybridization Probes in Southern Blot: Used to detect specific DNA sequences by binding to complementary strands.
• Western Blot Techniques: Utilizes antibodies for the detection of specific proteins.

This quiz covers the molecular techniques used in genetic analysis, focusing on methods such as gel electrophoresis, southern blotting, and molecular cloning. It encourages students to explore and apply these techniques in various scenarios related to DNA manipulation. Test your understanding of genetics and the methodologies for analyzing DNA fragments!