1 Deletion Mapping

Deletion mapping is a specialized genetic mapping technique used to determine the location of a
specific gene on a chromosome. It is particularly useful when the location of alleles, variants of a
recessive gene, are known to be located within a specific region, but their specific location is
unknown. The process involves two strains of organisms: a donor strain and a recipient deletion
mutant strain. The donor strain carries a point mutation, a small change in the gene sequence,
which affects the function of the gene under investigation. The recipient deletion mutant strain
has a portion of its genetic material intentionally deleted, including the region where the gene of
interest resides. When these two strains are crossed, if the point mutation in the donor strain
coincides with the deletion in the recipient strain—meaning they affect the same base pair of the
gene—then the wild-type function of the gene cannot be restored. However, if the point mutation
in the donor strain is located outside of the deleted region in the recipient strain, the donor strain
can restore the wild-type function of the gene through recombination. Recombination is the
process by which chromosomes break and rejoin, leading to the formation of new combinations
of genes. In this case, the wild-type sequence from the donor strain that corresponds to the
deleted region in the recipient strain can invade and repair the deleted region. This repair enables
the restoration of gene function, indicating that the point mutation lies outside of the deleted
region.

2 Deletion Mapping Methodology

Deletion mapping involves several steps to pinpoint the location of a gene within a chromosome.
Let's break down each step using Drosophila eye gene as an example:
2.1 Designing a Deletion Series

This step involves designing a series of deletions that span the chromosome region containing
the gene of interest. Each deletion should cover a different portion of the chromosome, ensuring
that the entire region is covered. For example, if we're interested in a gene involved in eye
development in Drosophila, we would design a series of deletions that cover the chromosome
region known to contain genes related to eye development.
2.2 Generating Deletion Mutants

Once the deletion series is designed, deletion mutants need to be generated. This is typically
done using techniques such as radiation or chemical mutagenesis to induce random deletions in
the chromosome. In the case of Drosophila, flies with induced deletions are screened for the
desired deletions in the target region.
2.3 Phenotypic Analysis

The next step involves analyzing the phenotypes of the deletion mutants. This includes observing
any changes in physical characteristics or traits associated with the gene of interest. In our
example, researchers would observe the eye development phenotype in each deletion mutant.
Mutants with abnormal eye development could indicate the deletion of a gene related to eye
development.
2.4 Correlating Phenotypes with Deletions

Once phenotypic analysis is complete, researchers correlate the observed phenotypes with
specific deletions. Mutants exhibiting similar phenotypes are likely to have deletions affecting
the same gene or genes within the region. For instance, if multiple deletion mutants exhibit
defects in eye development, it suggests that the deleted region contains genes critical for eye
development.
2.5 Fine Mapping and Identification

After narrowing down the region containing the gene of interest, further fine mapping techniques
are employed to precisely identify the location of the gene within the deletion. Techniques such
as complementation tests, which involve crossing deletion mutants with known mutations in the
gene of interest, can help pinpoint the gene's location.
Once the gene is identified, its sequence can be analyzed to understand its function and how it
contributes to the observed phenotype.

3 Deletion Mapping and in-situ Hybridization

In-situ hybridization is a complementation analysis which is a powerful genetic tool used to
determine whether two different mutations affecting the same phenotype occur in the same or
different genes. When applied to a deletion and a mutant strain in Drosophila melanogaster (fruit
flies), it can also help locate the position of a mutation relative to a deletion on a chromosome.
3.1 Crossing Deletion and Mutant Strains

In complementation analysis, researchers cross two different strains: one carrying a deletion
(deletion stock) and the other carrying a specific mutation (mutant stock). The resulting offspring
will carry one chromosome with deletion and the other with mutation. These flies are referred to
as "mutation/deletion flies."
3.2 Phenotypic Observation

Researchers observe the phenotype of mutation/deletion flies. The phenotype could be any
observable characteristic, such as eye color, wing shape, or behavior, depending on the mutation
under study. If the mutation/deletion files exhibit the mutant phenotype, it indicates that the
mutation and the deletion are not complementing each other. In other words, the deletion does
not complement the mutation. This suggests that the mutation falls within the deleted region.
Conversely, if the mutation/deletion flies exhibit a wild-type phenotype, it suggests that the
mutation and the deletion complement each other. This means that the deletion restores the
normal function of the gene affected by the mutation, indicating that the mutation must be
located outside the deleted region.
3.3 Interpretation

If the mutation fails to complement the deletion and the flies show the mutant phenotype, it
suggests that the mutation must be located within the region that has been deleted. This indicates
that the gene affected by the mutation is likely located within the deleted region, and the fly lacks
a wild-type copy of the gene. If the mutation complements the deletion and the flies show the
wild-type phenotype, it suggests that the mutation must be located outside the deleted region.
This indicates that the gene affected by the mutation is located elsewhere on the chromosome,
and the fly still possesses at least one functional copy of the gene.
By using this complementation analysis, researchers can precisely determine whether a mutation
lies within, or outside a given deletion on a chromosome, providing valuable insights into the
genetic organization and mapping of the genome.

Figure 3-1 In-situ Hybridization

The image illustrates the two-step process of denaturing and hybridizing DNA molecules.
Denaturation: Disruption of the hydrogen bonds between complementary strands of double-stranded DNA,
separating them into single strands. This is usually achieved by applying heat.
Hybridization: The process of single-stranded DNA molecules with complementary base sequences coming
together and forming double-stranded DNA.
The bottom of the image shows a probed DNA sequence, which could be a segment of interest being investigated for
the presence of a complementary sequence in the denatured DNA.
4 Mapping Deletions Within a Gene

4.1 Large deletions

These are significant removals of a segment of the gene sequence. The image depicts them using
double-headed arrows with numbers on either side indicating the deleted region. For example,
the leftmost deletion removes a segment between position 1272 and 1241. This removes 1272 -
1241 + 1 = 32 nucleotides from the original sequence.
4.2 Smaller deletions

These involve removing a shorter segment of the gene sequence. They are shown using single-
headed downward arrows with numbers on either side signifying the deleted region. For
instance, the smaller deletion on the right removes a segment between position 1605 and 1509,
eliminating 1605 - 1509 + 1 = 96 nucleotides.
4.3 Point Mutation

The text "Point mutations within that narrow region" sits below a horizontal line labeled "SITE"
and a vertical line labeled "090." This suggests a possible point mutation within a specific,
narrow region of the gene. However, the exact nature of the point mutation isn't provided in the
image.
4.4 Problem

https://www.youtube.com/watch?v=LnhG7_oDwRY