18. Molecular Genetic Tools

Methods for Analyzing DNA

Open Question
The National Institutes of Health created the Genetic Testing Registry (GTR) to increase transparency by publicly sharing information about the utility of their tests, research for the general public, patients, health-care workers, genetic counselors, insurance companies, and others. The Registry is intended to provide better information to patients, but companies involved in genetic testing are not required to participate. Should company participation be mandatory? Why or why not? Explain your answers.
Open Question
Should the FDA regulate direct-to-consumer genetic tests, or should these tests be available as a 'buyer beware' product?
Open Question
Would you have your genome sequenced, if the price was affordable? Why or why not? If you answered yes, would you make your genome sequence publicly available? How might such information be misused?
Open Question
You have isolated another cDNA clone of the CRABS CLAW gene from a cDNA library constructed using the vector shown in Problem 18. The cDNA was directionally cloned using the EcoRI and XhoI sites. You sequence the recombinant plasmid using primers complementary to the T7 and T3 promoter sites flanking the MCS (the positions of these sequences are shown in the figure in Problem 18). The first 30 to 60 bases of sequence are usually discarded since they tend to contain errors.Can you identify the start of the coding region in the end of the gene? What does the sequence preceding the start codon represent?
Open Question
You have isolated another cDNA clone of the CRABS CLAW gene from a cDNA library constructed using the vector shown in Problem 18. The cDNA was directionally cloned using the EcoRI and XhoI sites. You sequence the recombinant plasmid using primers complementary to the T7 and T3 promoter sites flanking the MCS (the positions of these sequences are shown in the figure in Problem 18). The first 30 to 60 bases of sequence are usually discarded since they tend to contain errors.Can you identify which sequence portions are derived from the vector (specifically the MCS) and which are derived from the cDNA clone?
Open Question
You have isolated another cDNA clone of the CRABS CLAW gene from a cDNA library constructed using the vector shown in Problem 18. The cDNA was directionally cloned using the EcoRI and XhoI sites. You sequence the recombinant plasmid using primers complementary to the T7 and T3 promoter sites flanking the MCS (the positions of these sequences are shown in the figure in Problem 18). The first 30 to 60 bases of sequence are usually discarded since they tend to contain errors.Will the long stretch of T residues in the T3 sequence exist in the genomic sequence of the gene?
Open Question
Following the tragic shooting of 20 children at a school in Newtown, Connecticut, in 2012, Connecticut's state medical examiner requested a full genetic analysis of the killer's genome. What do you think investigators might be looking for? What might they expect to find? Might this analysis lead to oversimplified analysis of the cause of the tragedy?
Open Question
Private companies are offering personal DNA sequencing along with interpretation. What services do they offer? Do you think that these services should be regulated, and if so, in what way? Investigate one such company, 23andMe, at http://www.23andMe.com, before answering these questions.
Open Question
Genomic DNA from the nematode worm Caenorhabditis elegans is organized by nucleosomes in the manner typical of eukaryotic genomes, with 145 bp encircling each nucleosome and approximately 55 bp in linker DNA. When C. elegans chromatin is carefully isolated, stripped of nonhistone proteins, and placed in an appropriate buffer, the chromatin decondenses to the 10-nm fiber structure. Suppose researchers mix a sample of 10-nm–fiber chromatin with a large amount of the enzyme DNase I that randomly cleaves DNA in regions not protected by bound protein. Next, they remove the nucleosomes, separate the DNA fragments by gel electrophoresis, and stain all the DNA fragments in the gel.How do the expected results support the 10-nm–fiber model of chromatin?
Open Question
Genomic DNA from the nematode worm Caenorhabditis elegans is organized by nucleosomes in the manner typical of eukaryotic genomes, with 145 bp encircling each nucleosome and approximately 55 bp in linker DNA. When C. elegans chromatin is carefully isolated, stripped of nonhistone proteins, and placed in an appropriate buffer, the chromatin decondenses to the 10-nm fiber structure. Suppose researchers mix a sample of 10-nm–fiber chromatin with a large amount of the enzyme DNase I that randomly cleaves DNA in regions not protected by bound protein. Next, they remove the nucleosomes, separate the DNA fragments by gel electrophoresis, and stain all the DNA fragments in the gel.Explain the origin of DNA fragments seen in the gel.
Open Question
A family consisting of a mother (I-1), a father (I-2), and three children (II-1, II-2, and II-3) are genotyped by PCR for a region of an autosome containing repeats of a 10-bp sequence. The mother carries 16 repeats on one chromosome and 21 on the homologous chromosome. The father carries repeat numbers of 18 and 26.Following the layout of Figure 7.26c, which aligns members of a pedigree with their DNA fragments in a gel, draw a DNA gel containing the PCR fragments generated by amplification of DNA from the parents (I-1 and I-2). Label the size of each fragment.
Open Question
You have identified an enhancer trap line (see Figure 14.17) generated by P element transposition in Drosophila in which the marker gene from the enhancer trap is specifically expressed in the wing imaginal disc.How can you identify the gene adjacent to the insertion site of the enhancer trap?
Open Question
You have identified an enhancer trap line (see Figure 14.17) generated by P element transposition in Drosophila in which the marker gene from the enhancer trap is specifically expressed in the wing imaginal disc.

How would you show that the expression pattern of the enhancer trap line reflects the endogenous gene expression pattern of the adjacent gene?
Open Question
A family consisting of a mother (I-1), a father (I-2), and three children (II-1, II-2, and II-3) are genotyped by PCR for a region of an autosome containing repeats of a 10-bp sequence. The mother carries 16 repeats on one chromosome and 21 on the homologous chromosome. The father carries repeat numbers of 18 and 26.

Identify all the possible genotypes of children of this couple by specifying PCR fragment lengths in each genotype.
Open Question
A family consisting of a mother (I-1), a father (I-2), and three children (II-1, II-2, and II-3) are genotyped by PCR for a region of an autosome containing repeats of a 10-bp sequence. The mother carries 16 repeats on one chromosome and 21 on the homologous chromosome. The father carries repeat numbers of 18 and 26.

What genetic term best describes the pattern of inheritance of this DNA marker? Explain your choice.