4. Describe 2 of the following methods of biotechnology in detail Include all of the steps that must be taken and a reason why a scientist may use the method. 18 Marks.A] Gene Cloning PCR Gel Electrophoresis dd Nucleotide Sequencing (you do not need to describe Gel electrophoresis in this description, just say when on occurs and what it tells you)

Let's explore two biotechnology methods: Gene Cloning and PCR.<br /><br />**A) Gene Cloning**<br /><br />Gene cloning aims to create multiple identical copies of a specific DNA fragment (a gene). Scientists use this method to study genes in isolation, produce proteins for therapeutic use (like insulin), or modify organisms for specific traits.<br /><br />**Steps:**<br /><br />1. **Isolation of the Target Gene:** The DNA fragment containing the gene of interest is isolated using restriction enzymes. These enzymes act like molecular scissors, cutting DNA at specific sequences.<br />2. **Vector Preparation:** A vector, typically a plasmid (a small, circular DNA molecule found in bacteria), is chosen. The same restriction enzyme used in step 1 is used to cut the plasmid, creating a space for the target gene.<br />3. **Ligation:** The target gene and the cut plasmid are mixed together with DNA ligase, an enzyme that acts like molecular glue. Ligase joins the target gene and the plasmid, creating a recombinant DNA molecule.<br />4. **Transformation:** The recombinant plasmid is introduced into a host cell, usually bacteria. This process is called transformation. Several methods exist, including heat shock or electroporation, which make the bacterial cell membrane temporarily permeable to the plasmid.<br />5. **Selection and Screening:** Not all bacteria will take up the recombinant plasmid. The bacteria are grown on a selective medium (e.g., containing an antibiotic) that allows only the bacteria containing the plasmid to grow. Further screening methods, such as blue-white screening, can identify bacteria containing the plasmid with the inserted gene.<br />6. **Multiplication:** The selected bacteria are cultured, allowing them to multiply and replicate the recombinant plasmid containing the target gene. This creates many copies of the gene.<br /><br />**Reasons for using Gene Cloning:**<br /><br />* **Producing large quantities of a specific gene:** This is crucial for studying gene function and structure.<br />* **Producing proteins for therapeutic use:** For example, human insulin can be produced in bacteria through gene cloning.<br />* **Developing genetically modified organisms:** Genes can be cloned and introduced into organisms to confer desirable traits, such as pest resistance in crops.<br /><br /><br />**B) Polymerase Chain Reaction (PCR)**<br /><br />PCR is a technique used to amplify a specific DNA sequence, creating millions or even billions of copies from a very small starting sample. This is useful when the amount of DNA available is limited, such as in forensic science or ancient DNA studies.<br /><br />**Steps:**<br /><br />1. **Denaturation:** The DNA sample is heated to 94-96°C to separate the double-stranded DNA into two single strands.<br />2. **Annealing:** The temperature is lowered to 50-65°C (depending on the primers used). Short, single-stranded DNA sequences called primers bind to complementary sequences flanking the target DNA region. These primers act as starting points for DNA synthesis.<br />3. **Extension:** The temperature is raised to 72°C, the optimal temperature for the DNA polymerase enzyme. DNA polymerase extends the primers, using the original DNA as a template, synthesizing new complementary strands.<br />4. **Repeat:** Steps 1-3 are repeated for multiple cycles (typically 25-35). Each cycle doubles the amount of target DNA.<br /><br />**Gel Electrophoresis in PCR:**<br /><br />After PCR, gel electrophoresis is often used to visualize the amplified DNA. The DNA fragments are separated based on size, and the presence of a band of the expected size confirms successful amplification.<br /><br />**Reasons for using PCR:**<br /><br />* **Amplifying small amounts of DNA:** This is crucial in forensic science, where DNA evidence may be limited.<br />* **Diagnosing infectious diseases:** PCR can detect the presence of pathogens, even at low levels.<br />* **Studying ancient DNA:** PCR can amplify DNA from degraded samples, allowing researchers to study the genomes of extinct organisms.<br />* **Genetic testing:** PCR can be used to identify genetic mutations associated with diseases.<br /><br /><br />These descriptions provide a detailed overview of gene cloning and PCR, including the steps involved and the reasons why scientists utilize these powerful biotechnology techniques.<br />