unit 2 genetics

Unit 2 - Genetics Part 3

Goals

The primary objectives of this unit are as follows:

  • Describe plasmids. A plasmid is a small, circular piece of DNA that is separate from the chromosomal DNA and can replicate independently. They are primarily found in bacteria, and also in archaea and eukaryotic organisms.

  • Explain how and why plasmids may be found inside bacteria. Plasmids often carry genes that confer advantageous traits, such as antibiotic resistance, to the bacteria. They can be transferred between bacterial cells, thereby enhancing survival under selective pressures like antibiotic treatment.

  • Explain how and why plasmids may spread antibiotic resistance. Plasmids can carry genes that code for proteins which inactivate antibiotics. When bacteria acquire these plasmids, they gain resistance, and through horizontal gene transfer, these plasmids can spread to other bacteria.

  • Describe transposons. Transposons, or "jumping genes," are segments of DNA that can move or transpose themselves to different positions within the genome. They can create mutations by inserting themselves into genes or regulatory regions.

  • Explain how transposons may create mutations. By inserting into coding or regulatory sequences, transposons can disrupt normal gene function or alter gene expression, potentially leading to phenotypic changes or disease.

  • Understand the fundamentals of Genetic Engineering. Genetic engineering involves laboratory techniques to modify the genetic makeup of organisms. This includes various methods for manipulating DNA to achieve desired traits or functionalities.

    • What are the steps? Key steps include isolating the target gene, preparing it for insertion, and ultimately inserting it into a target organism's genome.

    • What are restriction enzymes? How are they used in genetic engineering? Restriction enzymes, or restriction endonucleases, cut DNA at specific sequences, allowing scientists to isolate genes of interest or modify plasmids.

    • What is electrophoresis? How is it used in genetic engineering? Electrophoresis is a method for separating DNA or RNA fragments based on size, using an electric field to move the molecules through a gel matrix.

    • What is PCR? How is it used in genetic engineering? Polymerase Chain Reaction (PCR) is a technique used to amplify small segments of DNA, which is crucial for cloning and various genetic analyses.

    • What are the steps to PCR? PCR typically involves three main steps: denaturation, annealing of primers, and extension of the new DNA strand.

Overview of Gene Flow/Recombination Events

  • Definition of Horizontal Gene Transfer (HGT): Horizontal gene transfer is the transfer of genetic material between organisms of the same generation as opposed to vertical transfer from parent to offspring. This process includes mechanisms such as transformation, conjugation, and transduction.

    • A) Conjugation: Involves the direct transfer of DNA from one bacterium to another through a pilus.

    • B) Transduction: Involves the transfer of genetic material from one bacterium to another via bacteriophages (viruses that infect bacteria).

    • C) Transformation: The process by which bacteria take up free DNA from their environment.

Plasmids

  • Different Types of Plasmids: There are various kinds of plasmids, each serving unique functions:

    • Resistance Transfer Factor (RTF): Plasmids that carry antibiotic resistance genes.

    • Fertility Factor: Plasmids that enable conjugation between bacteria.

    • Advantageous Genes: Genes that provide benefits under certain conditions. If a plasmid's genes are no longer advantageous, the plasmid may be ejected by the bacterial cell.

    • Bacteriocins: Proteins coded by plasmids that can kill other target bacteria. Plasmids do not promote antibiotic production, but their presence can lead to the selection of strains that possess these plasmids when antibiotics are used.

Transposons

  • Definition and Function of Transposons: Transposons are DNA sequences that can change their position within the genome, influencing gene expression and potentially causing mutations.

    • Typical Response to Stress: Transposons often transpose in response to environmental stressors, thereby increasing genetic diversity.

    • Occurrence in Organisms: They are found in prokaryotes, eukaryotes, and viruses.

    • Magnitude of Human DNA: Approximately 40-50% of human DNA consists of retrotransposons.

    • Mechanism of Action: Transposons can synthesize RNA, which undergoes reverse transcription to form DNA that integrates into new genomic locations.

Genetic Engineering

  • Laboratory Techniques: Genetic engineering encompasses laboratory procedures used to modify the genetic constitution of organisms. The following are key aspects:

    • Steps in Genetic Engineering: The genetic engineering process can involve eukaryotic or prokaryotic DNA handling:

    • 1) Isolate Target Location (Plasmid): Identify and select a suitable plasmid for genetic modification.

    • 2) Isolate Gene of Interest (GoI): Identify and extract the gene targeted for insertion. This can be amplified using PCR techniques to increase the quantity available for manipulation.

    • 3) Prepare GoI for Insertion: The isolated gene needs to be processed for successful integration into the plasmid.

    • 4) Insert GoI into Target: The gene of interest is inserted into the plasmid or host genome.

Preparation for Insertion

  • Use of Restriction Endonucleases: Restriction enzymes (also known as restriction endonucleases) are crucial in genetic engineering for cutting DNA at specific sequences, a process referred to as:

    • Enzyme Digest: This involves the breakdown of DNA at predetermined sites to facilitate the insertion of new genes. For example, a restriction enzyme might cut at the sequence AG^AATTCGC, where the caret (^) denotes the cut site.

Electrophoresis

  • Process and Application: Gel electrophoresis utilizes an electric field and a gel such as agarose to separate DNA fragments based on size. The steps involved are:

    • 1) Create Gel: Prepare a gel of specified concentration to serve as the matrix for the DNA separation.

    • 2) Load Gene Ladder: Introduce a known size standard (gene ladder) to the gel for comparison.

    • 3) Load Samples: Place the DNA samples into the gel wells.

    • 4-5) Run Gel: Apply electricity to move the DNA and observe the separation of bands. The sizes of the bands can be assessed based on how far they travel; larger bands take longer to move through the gel.

Example Gel Analysis

  • Evaluation of Bands: When examining a gel, various enzymes such as EcoRI, BglII, and MboI are analyzed based on the size of the produced bands. The enzyme responsible for generating the largest band can be identified by its migration on the gel, similarly for the smallest band.

Polymerase Chain Reaction (PCR)

  • Steps in PCR: This technique for amplifying DNA involves three main phases:

    • A) Denaturation: The double-stranded DNA is heated to separate it into two strands.

    • B) Annealing: Short single-stranded primers bind to the separated DNA strands to define the target region for amplification.

    • C) Extension/Elongation: Taq DNA polymerase synthesizes new DNA strands complementary to the target sequence, resulting in an exponential increase in the amount of DNA.

Goals for Genetic Engineering

  • Possible Outcomes: Various objectives exist for genetic engineering, including:

    • Harvesting Genes: Extracting genes for insertion into plants or animals to enable desired traits.

    • Eliminating Undesirable Traits: Removing or modifying genes that produce harmful or non-beneficial characteristics.

    • Creating Beneficial Trait Combinations: Combining multiple traits to achieve improved organisms.

    • Protein Production: Utilizing genetic constructs to produce desired proteins such as enzymes, hormones, or vaccines.

    • Ethical Considerations: There are ongoing controversies and bioethical paradigms surrounding the implications of genetic engineering, necessitating thoughtful consideration of its use.

Exam Practice - 1

  • Analysis of Images: Evaluate whether provided images depict DNA replication, transcription, or translation by observing details such as:

    • Absence of tRNA: Indicates a lack of translation as tRNA is responsible for bringing amino acids for protein synthesis.

    • (Ribo)nucleotides: Identification of U (uracil) suggests mRNA synthesis, indicating transcription.

    • Single Strand Product: A single-stranded product implies transcription rather than replication, which results in double-stranded DNA.