ANSWERS TO REVIEW QUESTIONS – CHAPTER 14
1.
What are the roles of the vector and insert in the generation of a recombinant DNA clone?
(pp. 291–294)
Vectors are plasmid, bacteriophage, cosmid or chromosomal DNA molecules into which donor DNA
can be ligated. The vector must have one or more unique restriction sites into which the donor DNA
can be inserted, an origin of replication and a selectable marker. The insert joins the sticky cut ends of
the DNA to form a hydrogen-bonded duplex. The DNA ligase enzyme then catalyses the formation of
phosphodiester bonds to join the two fragments together.
2. Describe the various types of vector used in constructing DNA molecules. (pp. 291–294)
Plasmid vectors are small double-stranded DNA moleculars that exist in bacterial cells independent of
the bacterial chromosome and have one or more antibiotic resistance genes for selection in the host.
Bacteriophage vectors can be double- or single-stranded viruses that infect bacteria and contain
sequences that are not essential for infection and which can be replaced by the DNA to be cloned.
Cosmids are hybrids of plasmids and the bacteriophage  (lambda) with both an antibiotic resistance
gene and sequences to allow assembly of the DNA into intact virus particles. Artificial chromosome
vectors (BACs and YACs) are segments of bacterial and yeast chromosomes with sites for the insertion
of very large genomic fragments.
3.
Describe the preparation of a genomic clone library. Why are libraries of genomic DNA and
cDNA useful? (pp. 294–297)
Refer to Figures 14.7–14.9.
How can a specific gene, such as the -globin gene, be isolated from a cDNA library? (pp.
294–297)
Refer to Figure 14.8.
4.
5.
Several human genes produce protein products that have potential therapeutic uses. The
cloned DNA can be inserted into an expression vector and the recombinant protein
expressed in E. coli. However, the RNA products of human genes contain introns that cannot
be excised in prokaryotes. How can this problem be overcome to produce the protein in E.
coli? (p. 294)
Using a cDNA clone inserted into the expression vector will overcome the problem of introns in human
genes that are not removed in E. coli, as the mRNA used to make the cDNA clone will already have
had the introns removed during the processing of the pre-mRNA molecule.
6.
How is DNA sequence amplification achieved with the polymerase chain reaction? (pp.298–
299)
Refer to Figure 14.10.
7.
What are the most useful sequence polymorphisms in forensic analysis? Why are they so
useful? (pp. 304–306)
Microsatellites or variable number tandem repeats are the most useful polymorphisms for forensic
analysis. This is because there are very many places where these sequences occur in the genome and
there are often many different sizes of each fragment in the human population. This means that there
should be many differences between individuals and so a pattern can serve as a molecular fingerprint. It
is also easy to get enough DNA for analysis of these polymorphisms from very small samples of blood
or a few hair follicles.
8.
Explain how polymorphic DNA markers and chromosome walking can be used to isolate
eukaryotic genes. (pp. 304–305)
Polymorphic DNA markers can be used as recombination markers along human chromosomes, and by
linkage analysis the position of genes can be determined in relation to the markers. To isolate a gene
with an unknown function, DNA markers are found closely linked to the gene and then overlapping
genomic clones are identified to ‘walk’ along the chromosome until the piece of DNA containing the
gene can be isolated by screening with new markers. This is still usually a large piece of DNA and so
transcripts expressed from this region are identified and then analysed to see whether they contain
mutations that would account for the different effects seen in healthy or diseased individuals.
9.
What techniques are available for genetic transformation of animal or plant cells? (pp. 288–
294)
Transformation of animal and plant cells requires the DNA to be inserted into the cells and then
integrated into the chromosomes. In plants there is a natural vector that does this, Agrobacterium
tumefaciens, but this does not work for all plants, and so a common method is to get the DNA into a
cell by mechanical means (micro-injection, electroporation or particle bombardment) and then rely on
random integration into the host cell chromosomes. This is similar for animal cells, but microinjection
is the commonest method in this case. The biggest problem is the regeneration of a whole organism
from the few cells that are transformed, as well as problems associated with the random integration of
the new genes, sometimes in more than one place in the genome.
10. Explain how large amounts of a human hormone can be produced using recombinant DNA.
(pp. 302–308)
Genes encoding important human proteins can be cloned into expression vectors and used to produce
proteins with amino acid sequences that are identical to the natural proteins (e.g. blood clotting factor
used to treat haemophilia).