Chapter 13
Nucleic Acid Biotechnology Techniques
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SUMMARY
Section 13.1
In order to study nucleic acids, we must have a way to separate and identify
them.
The most common separation technique is gel electrophoresis. DNA pieces
Section 13.2
Restriction endonucleases are enzymes produced by bacteria that hydrolyze the
phosphodiester backbone of DNA at specific sequences.
Section 13.3
Cloning refers to creating a genetically identical population.
DNA can be combined by using restriction enzymes that create sticky ends in the
DNA. This recombinant DNA will have a target DNA sequence that the
Section 13.4
Genetic engineering is the process of inserting genes of interest into specific
organisms for either a medical or purely scientific benefit.
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Section 13.5
A DNA library is a collection of clones of an entire genome.
The genome is digested with restriction enzymes and the pieces are cloned into
Section 13.6
Polymerase Chain Reaction (PCR) is a sophisticated, automated technique for
amplifying DNA from very small amounts of sample.
The DNA to be amplified is mixed with specific primers, dNTPs, and a heat
Section 13.7
A DNA fingerprint is created by digesting DNA with restriction enzymes,
separating the pieces on a gel, and then visualizing some of the pieces by using
Section 13.8
DNA can be sequenced by using several techniques. One of the most common is
called the chain termination method.
Nucleic Acid Biotechnology Techniques 3
Section 13.9
As more DNA sequences become available, it becomes possible to compare
those sequences. Of particular interest is any pattern that may emerge from
genes that encode proteins with similar functions.
Important medical applications are emerging, and new methods are making it
possible to analyze large quantities of data. Complete protein:protein interaction
LECTURE NOTES
This chapter covers a number of common laboratory techniques for isolating and
manipulating nucleic acids. The material here serves as an excellent supplement to an
associated lab course or component. One to two lectures may be devoted to this
LECTURE OUTLINE
I. Purification and detection of nucleic acids
A. Separation techniques – gel electrophoresis
B. Detection methods
II. Restriction endonucleases
A. Palindromic cut sites
B. “Sticky” versus blunt ends
III. Cloning
A. Recombinant/Chimeric DNA
B. Use of sticky ends
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C. Bacteriophage and plasmid vectors
1. Transformation
IV. Genetic engineering
A. Natural recombinant DNA
B. Bacteria as “protein factories”
C. Protein expression vectors
VIII. DNA sequencing
IX. Genomics & proteomics
ANSWERS TO EXERCISES
13.1 Purification and Detection of Nucleic Acids
1. Safety, no need for special licensing, and convenience of disposal.
2. DNA is labeled with 32P and run on a gel. The gel is placed next to X-ray paper,
13.2 Restriction Endonucleases
4. The use of restriction endonucleases with different specificities gives overlapping
sequences that can be combined to give an overall sequence.
5. Restriction endonucleases do not hydrolyze a methylated restriction site.
Nucleic Acid Biotechnology Techniques 5
8. An endonuclease is an enzyme that cuts nucleic acid chains in the middle, as
9. They are all palindromes (ignoring punctuation and spacing in the latter two
cases), analogous to palindromic sequences of bases in DNA. Just as the five
10. GGATCC, GAATTC, AAGCTT (remember that these are listed 5′ to 3′, so you
must read the complementary strand 5′ to 3′ to see that the sequence is the
same).
11. HaeIII cuts at a sequence of four bases, cuts in the middle of the sequence, and
12. Sticky ends are short regions of single-stranded DNA extending from the ends of
double-stranded DNA molecules. These are produced by some restriction
13. An advantage of using HaeIII is that it yields blunt ends. Thus, one could
combine DNA cut with this enzyme with any other DNA that also had blunt ends.
13.3 Cloning
14. A portion of exogenous DNA is introduced into a suitable vector, frequently a
bacterial plasmid, and many copies of the DNA are produced when the bacteria
grow. Viruses are also commonly used as vectors.
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17. The key feature of a plasmid capable of blue/white screening is the gene for the
-subunit of the enzyme -galactosidase. These plasmids are used with a strain
of E. coli that are deficient in the -subunit of this enzyme. -Galactosidase can
18. Restriction enzymes to cut DNA, DNA ligase to rejoin DNA, a suitable vector to
carry the foreign DNA, a cell line to accept the vector, and a way of selecting for
the correct transformants.
19. Since most recombinant DNA occurs with bacterial and viral vectors, a big
concern is that a mutated virus or bacteria will be released that can infect other
13.4 Genetic Engineering
20. To increase disease resistance, resistance to pests, shelf life, level of nitrogen
22. The corn being grown in the field has been genetically engineered. The gene that
was introduced came from the bacterium Bacillus thuringensis.
23. LDH 3 has the subunit composition H2M2. Each of the subunits is coded for by a
separate gene, so in order to clone LDH 3, one would have to clone the gene for
24. An expression vector, such as pET 5 plasmid, has the components of any normal
cloning vector (e.g., origin of replication, selectable marker, multiple cloning site),
Nucleic Acid Biotechnology Techniques 7
25. A fusion protein is a combination of a protein coded for by an expression vector
and the target gene. A common one is a histidine tag and enterokinase, which
26. The bovine growth hormone is a protein that is denatured and digested in the
intestinal tract. Also, all cow’s milk contains some of the hormone.
27. The DNA sequence to be inserted in the bacterial plasmid to direct the
28. Isolate the DNA that codes for the growth factor by means of suitable probes.
Introduce the DNA into a bacterial genome. Allow the bacteria to grow and to
produce human growth hormone.
in bacteria, there will be no risk of prion contamination.
13.5 DNA Libraries
30. A DNA library is a collection of cells that carry cloned pieces of the entire DNA
genome of an organism. A cDNA library is made by taking the mRNA from an
organism, converting it to cDNA, and cloning that for the library. In this way, the
active DNA sequence is stored.
31. If a DNA library is to represent the total genome of an organism, it must contain
13.6 The Polymerase Chain Reaction
33. The polymerase chain reaction depends on repeated cycles of separation of
DNA strands followed by annealing of primers. The first step requires a
significantly higher temperature than the second, giving rise to the requirement
for strict temperature control.
34. Part of the procedure of the polymerase chain reaction requires the use of high
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36. The contaminating DNA as well as the desired DNA is amplified at each stage of
the polymerase chain reaction, giving rise to an impure product.
37.
(a) The primers have very different G–C contents.
(b) The forward primer will have significant secondary structure with itself (hairpin
13.7 DNA Fingerprinting
40. The polymerase chain reaction can increase the amount of a desired DNA
sample by a considerable factor, making possible definite identification of DNA
13.8 Sequencing DNA
42. 5′GATGCCTACG3′
43. Two factors are involved here. First, large polymers must be cleaved into smaller,
manageable fragments for sequencing. Enzymes (endoproteases) that cleave
proteins, while showing some specificity, are far from absolutely specific, and
44. DNA often has introns in the gene, so knowing the DNA sequence may give the
wrong answer for the final protein sequence. Also, proteins are modified
reflected in the DNA.
45. Open-ended answer.
Nucleic Acid Biotechnology Techniques 9
46. Benefits: A person at risk for future heart disease could be more careful with diet
and exercise. Such a person might also take a drug beforehand that would help
13.9 Genomics and Proteomics
47. The genome is the total DNA of a cell, containing all the genes of that organism.
The proteome is the total complement of proteins.
48. A proteomic analysis has been done on the fruit fly Drosophila melanogaster.
49. Using robotic technology, a slide or “chip” is loaded with thousands of specific
single-stranded DNA sequences. RNA is collected from samples to be tested and
50. Yeast could be grown under the two conditions and the mRNA collected. The
mRNA could then be converted to cDNA and each population could be labeled
51. Cancerous cells have altered metabolism at the genetic level. The gene
expression patterns seen in patients with known types of cancer act like a
52. DNA microarrays have thousands of bound single-stranded DNA spots. They are
used to test for the presence of the corresponding mRNA in a biological sample
via cDNA produced from the mRNA. Protein arrays, on the other hand, have