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Table of Contents
Why Do Genetics
More Basic Concepts
Mutagenesis in vitro
Effects of Mutations
Two Factor Crosses
Other Mapping Methods
Problem Set 1
Problem Set 2
VIII C. DELETION MAPPING
The above types of mapping use the frequencies of appearance of different phenotypes to determine the linear order of mutations. The following method of mapping tests the possibility of the restoration of the wild-type genotype. As such it asks the simpler question "whether or not", rather than "how often".
Consider a cross between a donor and recipient where one strain contains a deletion of part of the region of interest and the other strain contains a point mutation in the region of interest. Operationally, this would involve mixing a culture of the recipient with either DNA, transducing pahge, or a donor strain, and plating on medium where only recombinants would yield colonies. If the point mutation and some portion of the deletion coincide (affect the same base pair), then there is no way to restore a wild-type genotype by a recombinational event. If the point mutation and the deletion mutation do not coincide, then there will be some frequency of recombination events between the two, generating a wild-type sequence. There are a number of reasons why this is the best way to do fine structure mapping:
The first and most difficult step in doing deletion mapping is the generation of the deletion mutations themselves. There are a variety of ways of doing this:
Unless the deletions used have been physically characterized, the actual use of deletions in genetic mapping involves a circular argument and protocol. One takes a number of point or insertion mutations in the region of interest as well as a number of putative deletions and crosses them in all possible combinations. The presence of a deletion mutation is verified geneticallyif it fails to recombine with one or more point mutations that can be shown to recombine with each other (this might serve as the genetic definition of a deletion). Thus one is using the point mutations to establish the presence and identity of various deletion mutations and using the deletion mutations to order the point mutations. If one is presented with a table of recombination data, one can typically identify that mutations are deletion mutations by the very fact that they fail to recombine with more than one point mutation.
The figure below gives some sample Mapping data and illustrates two ways that deletions are commonly drawn. Such data is approached by first determining which mutations are deletions (mutations which fail to recombine with two or more mutations which themselves can recombine) and then using these to assort the other mutations into regions called deletion intervalsas defined by those deletions.
It should be recognized that one is actually scoring linkage to a deletion when one is doing deletion analysis. Zero recombinants indicates that the point mutation is 100% linked to the deletion. The reliability of saying a mutation falls under a deletion is a function of how many events are scored and therefore how tight the linkage must be. Finally, while deletions are a physical loss of a specific region of DNA, classical deletion mapping says very little about the physical size of the regions involved. A deletion map turns out to be a series of clusters of point mutations, with each cluster defined by the end point of one or more deletions. Thus, the presence of such a "deletion interval" is a function both of one's ability to generate a deletion end point as well as one's ability to find point mutations in that region to allow recognition of different deletion end points.
A sort of "inverse deletion" mapping can be performed with physically characterized regions of DNA. These can be introduced into various mutant backgrounds to ask if the cloned region is capable of recombining with the mutation to give a wild-type genotype. If a positive result is seen, the mutation is "covered" by the cloned region.
While sequencing can identify mutations (as in, bases changes relative to the wild type), it cannot tell whether a given change is responsible for a mutant phenotype. For example, if the sequenced region has two mutations, it is unclear which is "causative" of the phenotype. In contrast, in genetic mapping the only mutations that are "seen" are those that cause a mutant phenotype. As a consequence, a preliminary round of mappingprior to sequencing not only identifies the region to be sequenced but also verifies that the mutations is causative of the phenotype of interest.
[See sample problems 21-22A]
The methods noted in sections VIII D & E are useful for identifying the approximate location of a gene or mutation on either a genetic or physical map of the entire genome.
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