Chapter 20: DNA Technology and Genomics
Interactive Questions – Answers
20.1 The third sequence, because it has the same sequence running in opposite directions. The
enzyme would probably cut between G and A, producing AATT and TTAA sticky ends.
20.2 a. bacterial plasmid
b. lacZ gene
c. restriction site
d. ampR (ampicillin resistance) gene
e. gene of interest
f. complementary sticky ends
g. human DNA fragments
h. recombinant plasmid
i. recombinant bacteria
j. plate with ampicillin and X-gal
1. Plasmid and human DNA are isolated.
2. Both DNAs are cut with same restriction enzyme. Single cut in plasmid disrupts lacZ
gene; multiple fragments of human DNA formed.
3. Fragments are mixed and some foreign fragments base-pair wit plasmid. DNA ligase
seals ends
4. Recombinant plasmids transform acteria with mutation in their lacZ gene.
5. Plate bacteria on agar containing ampicillin and X-gal.
Cells containing recombinant plasmid are identified by their ability to grow in presence of
the antibiotic and by their white color. Blue colonies contain plasmids hat resealed and thus
have a functioning lacZ gene. Identify clones carrying gene of interest with nucleic acid
probe or by protein product.
20.3 A genomic library contains copies of DNA segments from the entire genome. Thus, all
genes should be represented, along with the regulatory sequences and introns. A cDNA library
allows you to sequence only the exons of a gene, and also indicates which genes are expressed
either in different cell types or at different stages of development in the same cell type.
20.4 a. By amplifying the gene prior to cloning, the later task of identifying clones carrying the
desired gene is simplified.
b. There is a limit to the number of accurate copies that can be made due to the
accumulation of relatively rare copying errors. Large quantities of a gene are better
prepared by DNA cloning in cells, an the gene may be expressed in the cells.
20.5 Southern blotting procedure:
a. restriction enzyme treatment of samples
b. gel electrophoresis
c. blotting
d. hybridization with labeled probe
e. autoradiography
Crime samples contain blood from the victim and, presumably, the perpetrator. After
identifying the bands from the victim, the remaining fragments match those of Suspect
2’s fragment profile.
20.6 The public consortium followed a hierarchy of three stages: (1) genetic (linkage) mapping
that established about 200 markers/chromosome; (2) physical mapping that clones and ordered
smaller and smaller overlapping fragments (using YAC or BAC vectors for cloning the large
fragments); and (3) DNA sequencing of each small fragment, followed by assembly of the
overall sequence. The Celera whole-genome shotgun approach omitted the first two stages.
Each chromosome was cut into small fragments, which were cloned in plasmid or phage vectors.
The sequence of each fragment was determined, and powerful computers assembled the
overlapping fragments t determine the overall sequence.
20.7 a. Comparing noncoding sequences in the human genome with those in the much smaller
yeast genome has revealed highly conserved sequences that have turned out to be
important regulatory control sequences in both. The functions of some human disease
genes have been identified through the study of similar yeast genes.
b. Once the sequence and organization of the smaller genome is determined, it can be used
as a framework for organizing the sequences from the larger genome.
20.8 a. Before, members of the extended family had to be tested to determine the variant of the
RFLP marker to which the disease-causing allele was linked. Then RFLP analysis was
done with the patient’s blood to determine which marker, and thus which allele, he or she
had inherited. Now the cloned gene can be used as a probe, and hybridization analysis
can detect the abnormal allele.
b. The major difficulty is to assure that proper control mechanisms are present so that the
gene is expressed at the proper time, in the proper place, and to the proper degree.
Insertion of the therapeutic gene must not harm other cell functions. Ethical
considerations include whether genetic engineering should be done on germ cells, thus
influencing the genetic makeup of future generations.
Structure Your Knowledge – Suggested Answers
1. a. Bacterial enzymes that cut DNA at restriction sites, creating “sticky ends” that can
base-pair with other fragments. Use: make recombinant DNA, form restriction fragments
used for many other techniques
b. Mixture of molecules applied to gel in electric field; molecules separate, moving at
different rates due to charge and size Use: separate restriction fragments into pattern of
distinct bands, fragments can be removed from gel and retain activity or can be identified
with probes
c. mRNA isolated from cell is treated with reverse transcriptase to produce a
complementary DNA strand, and then a double-stranded DNA gene, minus introns and
control regions. Use: creates genes that are easier to clone in bacteria, produces library of
genes that are active in cell
d. Radioactively or fluorescently labeled single-stranded DNA or mRNA used to basepair with complementary sequence of DNA or RNA. Use: locate gene in clone of
bacteria, identify bands on gels, diagnose infectious diseases
e. DNA fragments separated by gel electrophoresis, transferred by blotting onto paper,
labeled probe added, rinsed, autoradiography. Use: analyze DNA for homologous
sequences, DNA fingerprinting
f. Single-stranded DNA fragments are incubated with four nucleotides, DNA polymerase,
and four labeled dideoxy nucleotides that interrupt synthesis, samples separated by size
sequence of nucleotides read from sequence of fluorescent tags. Use: determine
nucleotide sequence
g. Polymerase chain reaction: DNA is mixed with heat-resistant DNA polymerase,
nucleotides, and primers having complementary sequences for targeted DNA section and
repeatedly heated to separate, cooled to pair with primers and replicate. Use: rapidly
produce multiple copies of a gene of section of DNA in vitro
h. Restriction fragments analyzed by Southern blotting to compare different band patterns
caused by DNA differences in restriction sites. Use: DNA fingerprints for forensic use,
map chromosomes using RFLP markers, diagnose genetic diseases
i. Fluorescently labeled cDNA is made from a cell’s mRNA and applied to a DNA
microarray of single-stranded DNA from many different genes attached to a glass grid.
The intensity and location of fluorescence indicates gene expression in the cell. Use: test
thousands of genes simultaneously to compare gene expression in different tissues or at
different developmental stages or conditions.
2. Agricultural applications: (1) production of vaccines and hormones, which will improve
the health or productivity of livestock, (2) improvement of the genomes of agricultural
plants and animals, (3) “pharm” animals or plants to produce human proteins, (4)
development of plant varieties that have genes for resistance to diseases, herbicides, and
insects
Medical applications: (1) development of vaccines, (2) diagnosis of genetic diseases, (3)
treatment of genetic disorders, (4) production of insulin, human growth factor, TPA, and
other useful products
Answers to Test your Knowledge – Multiple Choice Answers
1. c: produce a cut (usually staggered) at specific restriction sites on DNA
2. b
3. e: does all of the above
4. e: GAATTC
CTTAAG
5. c: the sequencing and systematic study of whole genomes
6. c: destruction of natural oil deposits
7. c: Ti plasmid
8. a: is a virus that is nonpathogenic
9. a: The signals that control gene expression are different and prokaryotic promoter regions
must be added to the vector
10. b: a cell produces mRNA for only a small portion of its genes
11. c: They signal the attachment of RNA polymerase
12. b: reverse transcriptase
13. d: DNA polymerase
14. a: restriction enzyme
15. c: can grow on plates with tetracycline but not with ampicillin
16. d: DNA fingerprinting
17. b: a portion of the amino acid sequence of that protein
18. d: CAG
19. c: STRs (simple tandern repeats)
20. b: More than one polypeptide can be produced from a gene by alternative splicing
21. a: a transgenic animal that produces large quantities of a pharmaceutical product
22. a: separation of fragments by gel electrophoresis
23. e: all of the above
24. d: a, b, and c
25. d