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 does a positive ASO test for sickle-cell anemia determine that an individual is homozygous recessive for the mutation that causes sickle-cell anemia? (c) From microarray analysis how do we know what genes are being expressed in a specific tissue? (d) How can we correlate the genome with RNA expression data in a tissue or a single cell? (e) From GWAS how do we know which genes are associated with a particular genetic disorder?

Short answer

Answer: To confirm the successful integration and functioning of a useful gene in a transgenic organism, the following experimental evidence is vital: 1. Successful integration of the foreign gene into the host organism's genome. 2. Proper expression of the added gene in the host organism, leading to the production of the desired protein/enzyme. 3. Observable phenotypic changes in the host organism that had been predicted by the presence of the added gene. 4. No adverse effects on the host organism's overall health or functioning due to the addition or expression of the new gene.

Step-by-step solution

(a) Experimental evidence for a useful gene in a transgenic organism

To confirm that we have introduced a useful gene into a transgenic organism and that it performs as anticipated, we can observe the following experimental evidence: 1. Successful integration of the foreign gene into the host organism's genome. 2. Proper expression of the added gene in the host organism, leading to the production of the desired protein/enzyme. 3. Observable phenotypic changes in the host organism that had been predicted by the presence of the added gene. 4. No adverse effects on the host organism's overall health or functioning due to the addition or expression of the new gene.

(b) Positive ASO test for sickle-cell anemia

A positive ASO (allele-specific oligonucleotide) test for sickle-cell anemia determines that an individual is homozygous recessive for the mutation causing the disease by using probes that are complementary to the mutated sequence. If both alleles of the individual have the mutation, then the ASO probes will bind to both alleles, indicating that the individual is homozygous recessive for the sickle-cell anemia causing mutation.

(c) Microarray analysis for gene expression

Microarray analysis allows us to identify what genes are being expressed in a specific tissue by comparing the mRNA extracted from the tissue to a DNA microarray containing known gene sequences. In this process, the extracted mRNA is reverse transcribed into complementary DNA (cDNA), which is then labeled with a fluorescent molecule. The labeled cDNA is then hybridized to the microarray, and the intensity of fluorescence at each spot on the array is measured. The intensity of fluorescence indicates the level of expression of each gene present on the microarray in the tissue.

(d) Correlating genome with RNA expression data

Correlating the genome with RNA expression data in a tissue or a single cell can be done using techniques such as RNA-sequencing (RNA-seq) and Single-Cell RNA-sequencing (scRNA-seq). These methods allow researchers to obtain a comprehensive view of the transcriptome, identifying the genes being expressed and their levels of expression. By comparing the RNA expression data with the individual's genome, researchers can uncover biologically relevant information such as gene regulatory networks, alternative splicing events, and identify novel genes.

(e) GWAS and genetic disorders

Genome-Wide Association Studies (GWAS) help to identify genes associated with particular genetic disorders by examining the presence of specific genetic markers, known as single nucleotide polymorphisms (SNPs), in large populations of affected and unaffected individuals. Statistical analysis of the frequency of these SNPs in the population allows researchers to identify associations between certain SNPs and the genetic disorder in question. Once these associations have been established, researchers can then focus on the genes located near these SNPs to further investigate their involvement in the genetic disorder.

Key concepts

Transgenic Organisms

Transgenic organisms are living entities that have been genetically modified to carry a gene from a different species. The introduction of a foreign gene allows transgenic organisms to exhibit new traits or perform new functions that are not typically found in their species. To confirm the success of this genetic alteration, scientists look for direct evidence:

• Integration of the gene within the organism's genome is confirmed via molecular techniques such as PCR and DNA sequencing.
• The expression of the foreign gene is verified by detecting its RNA or the protein it encodes for through assays like Western blot or enzyme activity tests.
• Predicted phenotypic changes are observed to ensure the gene's functionality, for example, increased resistance to pests in crops or the production of therapeutic proteins in animals.
• The organism's health and development are monitored to ensure there are no detrimental effects from the genetic modification.

Each step serves as a critical checkpoint, ensuring the transgenic organism not only contains the new gene but is also benefiting from its presence, thus achieving the desired outcome of genetic engineering.

Allele-Specific Oligonucleotide Test

The allele-specific oligonucleotide (ASO) test is a diagnostic tool used to detect specific mutations in DNA that are indicative of certain genetic disorders, such as sickle-cell anemia. This test uses short DNA sequences, or oligonucleotides, designed to match exactly with a particular mutated DNA sequence. When a patient is homozygous recessive for a condition like sickle-cell anemia, both alleles contain this mutation:

• The ASO probes are labeled with a detectable tag and added to the patient's DNA.
• If the mutation is present, the probes will bind perfectly, producing a detectable signal.
• The intensity of this signal is measured, indicating if one or both alleles have the mutation.

If a strong signal is detected from both alleles, it confirms the individual is homozygous recessive. The ASO test's specificity allows for rapid and accurate detection of genetic mutations at the molecular level.

Microarray Analysis

Microarray analysis is a powerful tool used to measure gene expression across a genome. This technique involves:

• Isolating mRNA from a tissue sample.
• Converting it to cDNA.
• Labeling the cDNA with fluorescent tags.
• Hybridizing the tagged cDNA onto a microarray chip dotted with known DNA sequences.
• Scanning the chip to detect fluorescence, which indicates which genes are active.

By analyzing fluorescence patterns, scientists can deduce the levels of gene expression within a specific tissue. This provides insight into the biological processes occurring and can lead to discoveries related to disease, development, and responses to environmental changes.

RNA-Sequencing (RNA-seq)

RNA-sequencing, commonly referred to as RNA-seq, is a next-generation sequencing method allowing researchers to examine the complete transcriptomic landscape of a cell or tissue. This technique reveals which genes are being transcribed and to what extent.

• RNA-seq starts by extracting RNA from a biological sample.
• The RNA is then converted to cDNA and fragmented.
• Sequencing adaptors are ligated to the fragments, and they are sequenced using high-throughput technology.

The obtained sequences are mapped to a reference genome, which helps correlate RNA expression data with genomic information. The result of RNA-seq is a comprehensive dataset depicting gene expression levels, uncovering gene regulation patterns, and possibly identifying previously unknown genes or splice variants.

Genome-Wide Association Studies (GWAS)

Genome-wide association studies (GWAS) are research methods used to find genetic variations associated with specific diseases by scanning the genomes of many individuals. This approach involves:

• Looking for single nucleotide polymorphisms (SNPs) across the genomes of both affected and unaffected individuals.
• Using statistical methods to identify which SNPs are more frequent in those with the disease compared to those without.
• Investigating the genes near significant SNPs to understand their potential role in the disease.

This large-scale analysis can reveal genetic predispositions to diseases and is crucial for developing personalized medicine approaches. GWAS findings help drive further research into the biology of diseases and the development of new therapeutic strategies.