When the S. cerevisiae genome was sequenced and surveyed for possible genes, only about 40% of those genes had been previously identified in forward genetic screens. This left about 60% of predicted genes with no known function, leading some to dub the genes fun (function unknown) genes. As an approach to understanding the function of a certain fun gene, you wish to create a loss-of-function allele. How will you accomplish this?

Verified step by step guidance

1. Identify the target gene sequence in the S. cerevisiae genome that you wish to create a loss-of-function allele for.
2. Design a DNA construct that will disrupt the target gene. This could involve inserting a selectable marker gene, such as an antibiotic resistance gene, into the coding sequence of the target gene.
3. Use homologous recombination to introduce the DNA construct into the yeast cells. This process will replace the wild-type allele with the disrupted allele.
4. Select for yeast cells that have successfully incorporated the disrupted allele by growing them on media containing the antibiotic corresponding to the resistance gene used in the construct.
5. Verify the loss-of-function mutation by performing a functional assay or sequencing to confirm that the target gene is no longer functional.

Key Concepts

Loss-of-Function Alleles

Loss-of-function alleles are mutations that result in the complete or partial inactivation of a gene. These alleles can be generated through various methods, such as gene editing techniques like CRISPR-Cas9, which introduces targeted mutations. By studying organisms with these alleles, researchers can infer the gene's function based on the phenotypic changes observed.

Gene Editing Techniques

Gene editing techniques, such as CRISPR-Cas9, allow scientists to make precise alterations to an organism's DNA. This technology utilizes a guide RNA to direct the Cas9 enzyme to a specific location in the genome, where it creates a double-strand break. The cell's repair mechanisms then attempt to fix this break, often leading to insertions or deletions that can disrupt gene function, thus creating loss-of-function alleles.

Functional Genomics

Functional genomics is the field of study that aims to understand the relationship between genes and their functions within an organism. It employs various techniques, including gene knockout and overexpression studies, to elucidate the roles of genes, especially those with unknown functions. By analyzing the effects of loss-of-function alleles, researchers can gain insights into the biological pathways and processes in which these genes are involved.