8

Question 1

biotech

Answer 1

—the use of microorganisms to make practical

products

Question 2

mutagens

Answer 2

Mutagens are physical and chemical agents that produce
mutations (changes in nucleotide sequence of a genome). Scientists
deliberately use mutagens to create changes in microbes’
genomes so that the microbes’ phenotypes are changed. They
then select for and culture cells with characteristics considered
beneficial for a given biotechnological application.

Question 3

cDNA

Answer 3

Reverse transcriptase creates a flow of genetic information in the opposite direction from the
flow in conventional transcription: it uses an RNA template to
transcribe a molecule of DNA, which is called complementary
DNA (cDNA) because it is complementary to an RNA template.

Question 4

Because hundreds to millions of copies of mRNA exist for every
active gene,

Answer 4

it is frequently easier to produce a desired gene by
first isolating the mRNA molecules that code for a particular
polypeptide and then use reverse transcription to synthesize a
cDNA gene from the mRNA template.

Question 5

probes

Answer 5

Probes are nucleic acid molecules
with a specific nucleotide sequence that have been
labeled with radioactive or fluorescent chemicals so that
their locations can be detected. The use of probes to locate
specific sequences of nucleotides is based on the fact that
any given nucleotide sequence will preferentially bond to
its complementary sequence.

Question 6

antiesnse

Answer 6

Antisense nucleic acid molecules have nucleotide
sequences that bind to and interfere with genes and
mRNA molecules.

Question 7

naming restriction enzyems

Answer 7

Researchers name restriction enzymes with three letters (denoting
the genus and specific epithet of the source bacterium)
and Roman numerals (to indicate the order in which enzymes
from the same bacterium were discovered). In some cases, a
fourth letter denotes the strain of the bacterium.

Question 8

restrictione nzyems

Answer 8

Such
enzymes cut DNA molecules and are restricted in their action—
they cut DNA only at locations called restriction sites. Restriction
sites are specific nucleotide sequences, which are usually
palindromes3
—they have the same sequence when read forward
or backward.

Question 9

In nature, bacterial cells use restriction enzymes

to

Answer 9

protect themselves from phages by cutting phage DNA into
nonfunctional pieces. The bacterial cells protect their own DNA
by methylation4
of some of their nucleotides, hiding the DNA
from the restriction enzymes

Question 10

sticky ends

Answer 10

makes staggered cuts of the two strands of DNA, producing
fragments that terminate in mortise-like sticky ends.
Each sticky end is composed of up to four nucleotides that
form hydrogen bonds with its complementary sticky end
(Figure 8.2a). Scientists can use these bits of single-stranded
DNA to combine pieces of DNA from different organisms
into a single recombinant DNA molecule

Question 11

blunt ends

Answer 11

when rest enzymes cut
both strands of DNA at the same point. It is more difficult to make recombinant
DNA from blunt-ended fragments because they are not
sticky, but they have a potential advantage—blunt ends are
nonspecific. This enables any two blunt-ended fragments,
even those produced by different restriction enzymes, to be
combined easily

Question 12

vectors

Answer 12

To deliver a gene
into a cell, researchers use vectors, which are nucleic acid molecules,
such as viral genomes, transposons, and plasmids

Question 13

Genetic vectors share several useful properties:

Answer 13

Small enough to manipulate in a lab

Survive inside cells

Contain recognizable genetic marker

Ensure genetic expression of gene

Question 14

Generally, __ and __ are able to carry __

genes than can __.

Answer 14

Generally, viruses and transposons are able to carry larger

genes than can plasmids.

Question 15

process to produce vector

Answer 15

After a given restriction
enzyme cuts both the DNA molecule containing the gene
of interest (in this example, the human growth hormone gene)
and the vector DNA (here a plasmid containing a gene for antibiotic
resistance as a marker) into fragments with sticky ends 1 ,
ligase anneals the fragments to produce a recombinant plasmid
2 . After the recombinant plasmid has been inserted into a bacterial
cell 3 , the bacteria are grown on a medium containing
the antibotic 4 ; only those cells that contain the recombinant
plasmid (and thus the human growth hormone gene as well) can
grow on the medium

Question 16

gene library

Answer 16

a collection
of bacterial or phage clones—identical descendants—each
of which contains a portion of the genetic material of interest. In
effect, each clone is like one book in a library in that it contains
one fragment (typically a single gene) of an organism’s entire
genome.

Question 17

generate gene library

Answer 17

isolate genome of organism. using restriction enzymes
to generate fragments of the DNA of interest and then using
ligase to synthesize recombinant vectors. They insert the vectors
into bacterial cells, which are then grown on culture media.

Question 18

pcr

Answer 18

The polymerase chain reaction (PCR) is a technique by which
scientists produce a large number of identical molecules of DNA
in vitro. Using PCR, researchers start with a single molecule of
DNA and generate billions of exact replicas within hours.

Question 19

pcr steps

Answer 19

denaturation, primig, extneison

Question 20

denaturation

Answer 20

Exposure to heat (about 94°C) separates the
two strands of the target DNA by breaking the hydrogen
bonds between base pairs but otherwise leaves the two
strands unaltered.

Question 21

priming

Answer 21

A mixture containing an excess of DNA primers
(synthesized such that they are complementary to nucleotide
sequences near the ends of the target DNA), DNA
polymerase, and an abundance of the four deoxyribonucleotide
triphosphates (A, T, G, and C) is added to the target
DNA. This mixture is then cooled to about 65°C, enabling
double-stranded DNA to re-form. Because there is an excess
of primers, single strands are more likely to bind to a primer
than to one another

Question 22

extension

Answer 22

Raising the temperature to about 72°C increases
the rate at which DNA polymerase replicates each strand to
produce more DNA

Question 23

thermocycler

Answer 23

The process can be automated using a thermocycler, a device
that automatically performs PCR by continuously cycling all
the necessary reagents—DNA, DNA polymerase, primers, and
triphosphate deoxynucleotides—through the three temperature
regimes.

Question 24

electrophoresis

Answer 24

a technique that involves
separating molecules based on their electrical charge, size,
and shape.

Question 25

Electroporation

Answer 25

Electroporation involves
using an electrical current to puncture microscopic holes
through a cell’s membrane so that DNA can enter the cell
from the environment

Question 26

protoplast

Answer 26

cells

whose cell walls have been enzymatically removed.

Question 27

protoplast fusion

Answer 27

When protoplasts encounter
one another, their cytoplasmic membranes may fuse to
form a single cell that contains the genomes of both “parent”
cells. The DNA from the two fused cells recombines
to form a recombinant molecule.

Question 28

injection

Answer 28

• gene gun to fire tiny
  tungsten or gold beads coated with DNA into a target cell
  (Figure 8.9c). The cell eventually eliminates the inert metal
  beads.
• In microinjection, a geneticist inserts DNA into a
  target cell with a glass micropipette having a tip diameter
  smaller than that of the cell or nucleus

Question 29

genetic mapping

Answer 29

One application of these tools and techniques is genetic mapping,
which involves locating genes on a nucleic acid molecule.
Genetic maps provide scientists with useful facts, including
information concerning an organism’s metabolism and growth
characteristics, as well as its potential relatedness to other microbes

Question 30

restriction fragmentation

Answer 30

used for mapping the relative locations of genes
in plasmids and viruses, researchers compare DNA fragments resulting
from cleavages by several restriction enzymes to determine
each fragment’s location relative to the others. If the researchers
know the locations of specific genes on specific fragments, then elucidation
of the correct arrangement of the fragments will reveal the
relative locations of the genes on the entire DNA molecule.

Question 31

genomics

Answer 31

the

sequencing and analysis of the nucleotide bases of genomes.

Question 32

vaccines contain

Answer 32

Vaccines contain antigens—foreign substances such as weakened
bacteria, viruses, and toxins that stimulate the body’s immune
system to respond to and subsequently remember these
foreign materials. In effect, a vaccine primes the immune system
to respond quickly and effectively when confronted with
pathogens and their toxins.

Question 33

subunit vaccines

Answer 33

Scientists now use recombinant DNA technology to produce
safer vaccines. Once they have inserted the gene that
codes for a pathogen’s antigens into a vector, they can inject the
recombinant vector or the proteins it produces into a patient.
Thus, the patient’s immune system is exposed to a subunit of
the pathogen—one of the pathogen’s antigens—but not to the
pathogen itself. Such subunit vaccines are especially useful in
safely protecting against pathogens that either cannot be cultured
or cause incurable fatal diseases.

Question 34

genetic screening

Answer 34

Laboratory technicians
use DNA microarrays to screen patients, prospective parents,
and fetuses for such mutant genes. This procedure, called
genetic screening, can also identify viral DNA sequences in a
patient’s blood or other tissues.

Question 35

gene therapy

Answer 35

missing or defective genes are replaced with normal
copies. Scientists remove a few genetically defective cells—for
example, cells that produce a defective protein—from a patient,
insert normal genes, and replace the cells into the patient, curing
the disease.

Question 36

dna fingerprinting

Answer 36

identifying individuals

or organisms by their unique DNA sequences.

Question 37

dna fingerprinting process

Answer 37

procuring a sample of DNA,
making multiple copies of it via PCR, cutting the copies with restriction
enzymes, and separating the fragments by gel electrophoresis
to produce a unique pattern.

Question 38

uses of genomics / genome mapping

Answer 38

Drug development

Vaccine development

Relate DNA sequence data to protein function

Question 39

xenotransplants

Answer 39

animal cells, tissues, or organs introduced

into the human body.

Question 40

Clinical microbiologists use (3) in diagnostic applications.

Answer 40

Clinical microbiologists use PCR, fluorescent genetic probes,
and DNA microarrays in diagnostic applications.

Question 41

gel electrophoresis

Answer 41

to isolate fragments of DNA molecules that can
then be inserted into vectors, multiplied by PCR, or preserved
in a gene library.

Question 42

gel

Answer 42

The gel is typically composed
of a purified sugar component of agar, called agarose, which acts
as a molecular sieve that retards the movement of DNA fragments
down the chamber and separates the fragments by size.

Question 43

south3rn blot

Answer 43

a method, called the
Southern blot, to transfer DNA from agarose gels to nitrocellulose
membranes, which are less delicate. (Scientists could
also use probes to localize specific sequences in electrophoresis
gels, but because gels are flimsy and easily broken and deform
as they dry, it is difficult to probe gels.)

Question 44

southern blot process

Answer 44

gel electrophoresis, then
The DNA is denatured into single
strands with NaOH. Once the DNA fragments have been
separated by size, the liquid in the electrophoresis gel is blotted
out 2 . DNA is transferred and bonded with heat to a nitrocellulose
membrane 3 . Radioactive probes complementary to DNA sequences of interest are added 4 . The probes expose photographic
film, revealing the DNA of interest 5

Question 45

array

Answer 45

An
array consists of molecules of single-stranded DNA, either
genetic DNA or cDNA, immobilized on glass slides, silicon
chips, or nylon membranes.

Question 46

dna microarray

Answer 46

Robots, similar to those that
construct computer chips, deposit PCR-derived copies of
hundreds of thousands of different DNA sequences in precise
locations on the array. In any case, single strands of fluorescently
labeled DNA in a sample washed over an array adhere only to
locations on the array where there are complementary DNA
sequences

Question 47

Scientists use DNA microarrays in a number of ways, including
the following:

Answer 47

Monitoring gene expression

Diagnosis of infection

Identification of organisms in an environmental sample