18. Molecular Genetic Tools

Methods for Analyzing DNA

Open Question
A hereditary disease is inherited as an autosomal recessive trait1. The wild-type allele of the disease gene produces a mature mRNA that is 1250 nucleotides (nt) long. Molecular analysis shows that the mature mRNA consists of four exons that measure 400 nt (exon 1), 320 nt (exon 2), 230 nt (exon 3), and 300 nt (exon 4). A mother and father with two healthy children and two children with the disease have northern blot analysis performed in a medical genetics laboratory. The results of the northern blot for each family member are shown here.Identify the genotype of each family member, using the sizes of mRNAs to indicate each allele. (For example, a person who is homozygous wild type is indicated as '1250/1250.')
Open Question
A 2-kb fragment of E. coli DNA contains the complete sequence of a gene for which transcription is terminated by the rho protein. The fragment contains the complete promoter sequence as well as the terminator region of the gene. The cloned fragment is examined by band shift assay (see Research Technique 8.1). Each lane of a single electrophoresis gel contains the 2-kb cloned fragment under the following conditions:Lane 1: 2-kb fragment aloneLane 2: 2-kb fragment plus the core enzymeLane 3: 2-kb fragment plus the RNA polymerase holoenzymeLane 4: 2-kb fragment plus rho proteinDiagram the relative positions expected for the DNA fragments in this gel electrophoresis analysis.
Open Question
A 2-kb fragment of E. coli DNA contains the complete sequence of a gene for which transcription is terminated by the rho protein. The fragment contains the complete promoter sequence as well as the terminator region of the gene. The cloned fragment is examined by band shift assay (see Research Technique 8.1). Each lane of a single electrophoresis gel contains the 2-kb cloned fragment under the following conditions:Lane 1: 2-kb fragment aloneLane 2: 2-kb fragment plus the core enzymeLane 3: 2-kb fragment plus the RNA polymerase holoenzymeLane 4: 2-kb fragment plus rho proteinExplain the relative positions of bands in lanes 1 and 3.
Open Question
Variable number tandem repeats (VNTRs) are repeating DNA sequences of about 15–100 bp in length, found both within and between genes. Why are they commonly used in forensics?
Open Question
Although the capture and trading of great apes has been banned in 112 countries since 1973, it is estimated that about 1000 chimpanzees are removed annually from Africa and smuggled into Europe, the United States, and Japan. This illegal trade is often disguised by simulating births in captivity. Until recently, genetic identity tests to uncover these illegal activities were not used because of the lack of highly polymorphic markers (markers that vary from one individual to the next) and the difficulties of obtaining chimpanzee blood samples. A study was reported in which DNA samples were extracted from freshly plucked chimpanzee hair roots and used as templates for PCR. The primers used in these studies flank highly polymorphic sites in human DNA that result from variable numbers of tandem nucleotide repeats. Several offspring and their putative parents were tested to determine whether the offspring were 'legitimate' or the product of illegal trading. The data are shown in the following Southern blot.

Examine the data carefully and choose the best conclusion.

a. None of the offspring are legitimate.
b. Offspring B and C are not the products of these parents and were probably purchased on the illegal market. The data are consistent with offspring A being legitimate.
c. Offspring A and B are products of the parents shown, but C is not and was therefore probably purchased on the illegal market.
d. There are not enough data to draw any conclusions. Additional polymorphic sites should be examined.
e. No conclusion can be drawn because 'human' primers were used. <>
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.