Figure 10-1 shows that specific DNA fragments can be synthesized in vitro prior to cloning. What are two ways to synthesize DNA inserts for recombinant DNA in vitro?

In Figure 10-4, why is cDNA made only from mRNA and not also from tRNAs and ribosomal RNAs?

Redraw Figure 10-6 with the goal of adding one EcoRI end and one XhoI end. Below is the Xhol recognition sequence.

Redraw Figure 10-7 so that the cDNA can insert into an XhoI site of a vector rather than into an EcoRI site as shown

In Figure 10-10, determine approximately how many BAC clones are needed to provide 1 × coverage of

In Figure 10-14, why does DNA migrate to the anode (+ pole)?

In Figure 10-17a, why are DNA fragments of different length and all ending in an A residue synthesized?

As you will see in Chapter 15, most of the genomes of higher eukaryotes (plants and animals) are filled with DNA sequences that are present in hundreds, even thousands, of copies throughout the chromosomes. In the chromosome-walking procedure shown in Figure 10-19, how would the experimenter know whether the fragment he or she is using to “walk” to the next BAC or phage is repetitive? Can repetitive DNA be used in a chromosome walk?

Redraw Figure 10-23 to include the positions of the single and double crossovers.

In Figure 10-25, why do only plant cells that have T-DNA inserts in their chromosomes grow on the agar plates? Do all of the cells of a transgenic plant grown from one clump of cells contain T-DNA? Justify your answer.

In Figure 10-27, what is the difference between extrachromosomal DNA and integrated arrays of DNA? Are the latter ectopic? What is distinctive about the syncytial region that makes it a good place to inject DNA?

From this chapter, make a list of all the examples of (a) the hybridization of single-stranded DNAs and (b) proteins that bind to DNA and then act on it.

Compare and contrast the use of the word recombinant as used in the phrases (a) “recombinant DNA” and (b) “re-combinant frequency.”

Why is ligase needed to make recombinant DNA? What would be the immediate consequence in the cloning process if someone forgot to add it?

In the PCR process, if we assume that each cycle takes 5 minutes, how manyfold amplification would be accomplished in 1 hour?

The position of the gene for the protein actin in the hap-loid fungus Neurospora is known from the complete genome sequence. If you had a slow-growing mutant that you suspected of being an actin mutant and you wanted to verify that it was one, would you (a) clone the mutant by using convenient restriction sites flanking the actin gene and then sequence it or (b) amplify the mutant gene by using PCR and then sequence it?

You obtain the DNA sequence of a mutant of a 2-kb gene in which you are interested and it shows base differences at three positions, all in different codons. One is a silent change, but the other two are missense changes (they encode new amino acids). How would you demonstrate that these changes are real mutations and not sequencing errors? (Assume that sequencing is about 99.9 percent accurate.)

In a T-DNA transformation of a plant with a transgene from a fungus (not found in plants), the presumptive transgenic plant does not express the expected phenotype of the transgene. How would you demonstrate that the transgene is in fact present? How would you demonstrate that the transgene was expressed?

How would you produce a mouse that is homozygous for a rat growth-hormone transgene?

Why was cDNA and not genomic DNA used in the commercial cloning of the human insulin gene?

After Drosophila DNA has been treated with a restriction enzyme, the fragments are inserted into plasmids and selected as clones in E. coli. With the use of this “shotgun” technique, every DNA sequence of Drosophila in a library can be recovered.

In any particular transformed eukaryotic cell (say, of Saccharomyces cerevisiae), how could you tell if the transforming DNA (carried on a circular bacterial vector)

In an electrophoretic gel across which is applied a powerful electrical alternating pulsed field, the DNA of the haploid fungus Neurospora crassa (n = 7) moves slowly but eventually forms seven bands, which represent DNA fractions that are of different sizes and hence have moved at different speeds. These bands are presumed to be the seven chromosomes. How would you show which band corresponds to which chromosome?

The protein encoded by the cystic fibrosis gene is 1480 amino acids long, yet the gene spans 250 kb. How is this difference possible?

In yeast, you have sequenced a piece of wild-type DNA and it clearly contains a gene, but you do not know what gene it is. Therefore, to investigate further, you would like to find out its mutant phenotype. How would you use the cloned wild-type gene to do so? Show your experimental steps clearly.

Why is it necessary to use a special DNA polymerase (Taq polymerase) in PCR?

For each of the following experimental goals, is PCR or gene cloning preferable and why?

In Northern blotting, electrophoresis is used to resolve which biological molecules? What type of probe is used to identify the target molecule(s)?

One feature that virtually all plasmid vectors have in common is the polylinker (also called a multiple cloning site). Explain what a polylinker is and why it is such an important feature.

A second feature that virtually all plasmid vectors have in common is the selectable marker. Explain what this is and why it is such an important feature.

Prototrophy is often the phenotype selected to detect transformants. Prototrophic cells are used for donor DNA extraction; then this DNA is cloned and the clones are added to an auxotrophic recipient culture. Successful transformants are identified by plating the recipient culture on minimal medium and looking for colonies. What experimental design would you use to make sure that a colony that you hope is a transformant is not, in fact,

A cloned fragment of DNA was sequenced by using the dideoxy chain-termination method. A part of the autoradiogram of the sequencing gel is represented here.

The cDNA clone for the human gene encoding tyrosinase was radioactively labeled and used in a Southern analysis of EcoRI-digested genomic DNA of wild-type mice. Three mouse fragments were found to be radio active (were bound by the probe). When albino mice were used in this Southern analysis, no genomic fragments bound to the probe. Explain these results in relation to the nature of the wild-type and mutant mouse alleles.

Transgenic tobacco plants were obtained in which the vector Ti plasmid was designed to insert the gene of interest plus an adjacent kanamycin-resistance gene. The inheritance of chromosomal insertion was followed by testing progeny for kanamycin resistance. Two plants typified the results obtained generally. When plant 1 was backcrossed with wild-type tobacco, 50 percent of the progeny were kanamycin resistant and 50 percent were sensitive. When plant 2 was back-crossed with the wild type, 75 percent of the progeny were kanamycin resistant and 25 percent were sensitive. What must have been the difference between the two transgenic plants? What would you predict about the situation regarding the gene of interest?

A cystic-fibrosis mutation in a certain pedigree is due to a single nucleotide-pair change. This change destroys an EcoRI restriction site normally found in this position. How would you use this information in counseling members of this family about their likelihood of being carriers? State the precise experiments needed. Assume that you find that a woman in this family is a carrier, and it transpires that she is married to an unrelated man who also is a heterozygote for cystic fibrosis, but, in his case, it is a different mutation in the same gene. How would you counsel this couple about the risks of a child’s having cystic fibrosis?

Bacterial glucuronidase converts a colorless substance called X-Gluc into a bright blue indigo pigment. The gene for glucuronidase also works in plants if given a plant promoter region. How would you use this gene as a reporter gene to find the tissues in which a plant gene that you have just cloned is normally active? (Assume that X-Gluc is easily taken up by the plant tissues.)

The plant Arabidopsis thaliana was transformed by using the Ti plasmid into which a kanamycin-resistance gene had been inserted in the T-DNA region. Two kanamycin-resistant colonies (A and B) were selected, and plants were regenerated from them. The plants were allowed to self-pollinate, and the results were as follows: