Molecular Genetics Overview

Molecular Genetic Techniques for Understanding Species Biology

• Molecular techniques are used to study species biology.
• Techniques for cutting and visualizing DNA molecules are described.
• DNA fragment copying using PCR is explained.
• Methods for determining the base sequence of a DNA fragment are detailed.
• Molecular techniques for analyzing gene function, including targeted mutagenesis, are discussed.
• Practical applications of molecular genetics are described.

Recombinant DNA Technology

• Recombinant DNA technology is a set of techniques used to locate, isolate alter, and study DNA segments.
• Combining DNA from two sources is common.
• This frequently involves genetic engineering, combining genes from different bacteria or inserting a human gene into a viral chromosome.

Restriction Enzymes

• Restriction enzymes are naturally produced by bacteria to defend against viruses.
• Bacteria protect their own DNA from restriction enzymes by modifying their recognition sequences, often by adding methyl groups.
• Hundreds of restriction enzymes, recognizing diverse DNA sequences, have been identified from bacteria.

Engineered Nucleases

• Engineered nucleases overcome the limitation of restriction enzymes (short recognition sequences).
• These consist of a restriction enzyme portion for nonspecific cleavage combined with a protein recognizing the specific DNA sequence.
• Examples include Zinc-Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs).

Genome Editing with CRISPR-Cas

• CRISPR-Cas systems are naturally occurring mechanisms in bacteria and archaea used for defense against invading DNA elements.
• CRISPR sequences consist of short, palindromic repeats that incorporate fragments of foreign DNA.
• CRISPR-Cas systems can be used for site-directed mutagenesis.

Separating and Viewing DNA Fragments

• Gel electrophoresis separates DNA fragments based on size and electrical charge.
• Larger fragments migrate slower.
• Dyes are used in the gel to visualize the separated DNA fragments.

Locating DNA Fragments with Probes

• Probes are DNA or RNA sequences complementary to a specific DNA or RNA sequence of interest.
• Southern blotting uses probes to identify specific DNA fragments within a mixture.
• Northern blotting identifies specific RNA fragments.
• Western blotting locates specific proteins.

PCR (Polymerase Chain Reaction)

• PCR is a technique for amplifying specific DNA fragments outside of cells (in vitro).
• It involves three steps: denaturation, annealing, and extension.
• DNA polymerase enzymes, notably Taq polymerase (isolated from Thermus aquaticus), are essential as they are stable at high temperatures.
• Automated thermal cyclers allow for rapid temperature changes essential to the PCR process.
• Contamination is a significant concern with this method.

DNA Sequencing

• DNA sequencing methods determine the order of bases within a DNA fragment (e.g., Dideoxy sequencing and Next-Generation Sequencing).
• Dideoxy sequencing uses dideoxyribonucleoside triphosphates (ddNTPs).
• Next-generation sequencing technologies typically examine multiple DNA fragments at once for rapid sequencing.

DNA Fingerprinting

• DNA fingerprinting identifies individuals based on variations in short tandem repeats (STRs).
• STRs are short DNA sequences repeated in tandem at multiple locations within the human genome.
• Variations in the number of STRs across individuals are used as genetic markers to distinguish individuals.

Analyzing Gene Function

• Techniques for analyzing gene function include forward and reverse genetics, creating random mutations, targeted mutagenesis, generating transgenic animals, generating knockout mice, and RNA interference (RNAi).
• Forward genetics involves studying a phenotype and then finding the causal gene in the organism.
• Reverse genetics begins with the gene sequence and discovers the organism's phenotype.
• Transgenic animals involve introducing foreign DNA into an organism's genome; knockout mice inactivate a specific gene.
• RNA interference (RNAi) silences gene expression by using complementary RNA molecules to target specific mRNA sequences.

Biotechnology Applications

• Molecular genetic techniques have practical applications in medicine, agriculture, pharmaceuticals, and more.
• Examples include developing pharmaceuticals, specialized bacteria for specific processes, and genetically modified organisms (e.g., crops and animals).