- Why are maps required for the sequencing of genomes? If a map of a genome was unavailable, what would be the major difficulties in obtaining a genome sequence?
A genome map provides a guide for the sequencing experiments by showing the positions of genes and other distinctive features. If a map is unavailable then it is likely that errors will be made in assembling the genome sequence, especially in regions that contain repetitive DNA.
- How has PCR made the analysis of RFLPs much faster and easier? What was required to map RFLPs prior to the utilization of PCR?
The primers for the PCR are designed so that they anneal either side of the polymorphic site, and the RFLP is typed by treating the amplified fragment with the restriction enzyme and then running a sample in an agarose gel. Before the invention of PCR, RFLPs were typed by Southern hybridization, which is time-consuming.
- How does the linkage between genes provide a critical component to genetic mapping? Discuss how genetic markers can be linked to provide maps of individual chromosomes.
If a pair of genes display linkage then they must be on the same chromo- some. If crossing-over is a random event then the recombination frequency between a pair of linked genes is a measure of their distance apart on the chromosome. The recombination frequencies for different pairs of genes can be used to construct a map of their relative positions on the chromosome.
- Why is a double homozygote used for test crosses in linkage analysis experiments? Why is it preferable that the homozygote alleles be recessive for the traits being tested?
The double homozygote will produce gametes that are all the same genetically and if they are recessive then this parent will not contribute to the phenotype of the offspring.
- Genetic mapping techniques require at least two alleles for a given marker, while physical mapping techniques do not rely on the presence of alleles to map genomes. Discuss how the technique of fluorescent in situ hybridization can be used to map genome locations even if there is no genetic variation present at a given position.
FISH uses a fluorescently labeled DNA fragment as a probe to bind to an intact chromosome. The binding position can be determined and this information used to create a physical map of the chromosome.
- How does a scientist prepare a clone library of DNA from just a single chromosome?
Individual chromosomes can be separated by flowcytometry. Dividing cells are carefully broken open so that a mixture of intact chromosomes is obtained. The chromosomes are then stained with a fluorescent dye. The amount of dye that a chromosome binds depends on its size, so larger chromosomes bind more dye and fluoresce more brightly than smaller ones. The chromosome preparation is diluted and passed through a fine aperture, producing a stream of droplets, each one containing a single chromosome. The droplets pass through a detector that measures the amount of fluorescence, and hence identifies which droplets contain the particular chromosome being sought. An electric charge is applied to these drops, and no others, enabling the droplets containing the desired chromosome to be deflected and separated from the rest.
- What are the ideal features of a DNA marker that will be used to construct a genetic map? To what extent can RFLPs, SSLPs, or SNPs be considered “ideal” DNA markers?
The text indicates that the ideal features include high frequency in the genome being studied, ease of typing, and the presence of multiple alleles. This implies that SSLPs should be the “ideal” markers, but in reality SNPs are more popular. A discussion of this apparent paradox demands consideration of the relative importance of each of the three criteria, and in particular a realization that the critical feature of an “ideal” marker is high density.
- What features would be desirable for an organism that is to be used for extensive studies of heredity?
Many teachers will remember tackling this question during their own student days, and the answer has not changed: short generation time, large number of offspring, easily scored phenotypes, and such like. It is instructive to consider to what extent genomics has added new criteria to this list: is a complete genome sequence a useful feature of an organism to be used in studies of heredity?
- Which is more useful—a genetic or a physical map?
This is a very open-ended question that is designed to prompt discussion of a number of topics that are covered in later chapters. The discussion might begin by asking what purpose the map is intended to fulfill. A map designed to aid a sequencing project might not be the same as one designed to enable individual genes to be cloned. If it is concluded that for sequencing purposes a physical map is more useful, and in fact a genetic map has little or no direct value (which is a reasonable inference to make from a reading of Chapter 4), then the discussion could turn to how easy or otherwise it would be to locate the genes in a genome sequence, and to assign functions to those genes, without any prior knowledge of where the genes are. These issues are discussed in Chapter 5.