Question

In this chapter, we focused on a number of interesting applications of genetic engineering, genomics, and biotechnology. At the same time, we found many opportunities to consider the methods and reasoning by which much of this information was acquired. From the explanations given in the chapter, what answers would you propose to the following fundamental questions: (a) What experimental evidence confirms that we have introduced a useful gene into a transgenic organism and that it performs as we anticipate? (b) How can we use DNA analysis to determine that a human fetus has sickle- cell anemia? (c) How can DNA microarray analysis be used to identify specific genes that are being expressed in a specific tissue? (d) How are GWAS carried out, and what information do they provide? (e) What are some of the technical reasons why gene therapy is difficult to carry out effectively?

Short answer

Answer: Some key considerations and challenges in carrying out effective gene therapy include the delivery of the gene, targeting specific cells, gene integration, gene regulation, and evaluating long-term effects. These challenges involve developing methods for safe and efficient gene delivery, ensuring the therapeutic gene reaches only intended cells, integrating the gene stably into the patient's genome, managing gene expression levels for optimal therapeutic effect, and monitoring potential long-term effects such as immune reactions or secondary diseases.

Step-by-step solution

(a) Experimental Evidence for Useful Gene in Transgenic Organism)

To confirm the successful introduction of a useful gene in a transgenic organism, the following evidence is usually observed: 1. Phenotypic change: Visible changes in the organism's physical appearance, behavior, or characteristics as a result of the introduced gene. 2. Gene expression: Monitor the expression of the gene by extracting RNA from the organism and measuring mRNA levels specific to the gene of interest using techniques like qPCR (quantitative Polymerase Chain Reaction). 3. Protein production: Confirm the presence of the protein produced by the introduced gene using techniques like Western Blot or Immunohistochemistry.

(b) Diagnosing Sickle-Cell Anemia in the Human Fetus)

To determine if a human fetus has sickle-cell anemia using DNA analysis, the following steps are followed: 1. Collect a sample: Obtain a sample of the fetus's cells during pregnancy by either chorionic villus sampling (CVS) or amniocentesis. 2. Extract DNA: Isolate the DNA from the collected fetal cells using standard DNA extraction techniques. 3. Perform PCR: Amplify the region of the fetal DNA that contains the gene responsible for beta-globin production (HBB) using Polymerase Chain Reaction (PCR). 4. Sequence Analysis: Analyze the amplified DNA sequence to identify any mutations present in the HBB gene, specifically the single-point mutation in the HBB gene that causes sickle-cell anemia.

(c) Identifying Genes Using DNA Microarray Analysis)

DNA microarray analysis can be used for identifying specific genes being expressed in a specific tissue with the following steps: 1. Extract RNA: Isolate the total RNA from the tissue sample. 2. Convert to cDNA: Reverse transcribe the extracted RNA into complementary DNA (cDNA) using a reverse transcriptase enzyme. 3. Label cDNA: Fluorescently label the cDNA using target-specific fluorescent dyes. 4. Hybridization: Hybridize the labeled cDNA to a DNA microarray chip containing thousands of DNA sequences complementary to the targeted genes. 5. Detection: Use a microarray scanner to detect and measure the fluorescence intensities, which correspond to the expression levels of the specific genes in the tissue sample.

(d) GWAS and Provided Information)

Genome-wide association studies (GWAS) are carried out to identify the genetic variations associated with specific traits or diseases. GWAS provide information on the following aspects: 1. Genetic variants associated with diseases or traits: The study identifies specific genetic markers (SNPs-single nucleotide polymorphisms) that are associated with a particular disease or trait. 2. Allele frequency: GWAS provide information on the frequency of the disease or trait-associated alleles in the studied population. 3. Biological pathways and gene functions: Based on the identified genetic variants, further functional studies can be carried out to understand the biological pathways and gene functions involved in the disease or trait.

(e) Technical Reasons for Difficult Gene Therapy)

There are several technical reasons why gene therapy is difficult to carry out effectively: 1. Delivery of the gene: Developing safe and efficient methods to deliver the therapeutic gene into the target cells while avoiding triggering an immune response. 2. Targeting specific cells: Ensuring the therapeutic gene reaches only the intended cells and not the healthy or non-targeted cells. 3. Gene integration: Ensuring the therapeutic gene integrates stably and correctly into the patient's genome without causing unintended disruptions or mutations. 4. Gene regulation: Managing the expression levels of the introduced gene to achieve optimal therapeutic effect, while avoiding overexpression or underexpression. 5. Long-term effects: Monitoring and assessing potential long-term effects such as immune reactions, emergence of secondary diseases, or development of resistance to the therapeutic gene.