Chapter 20: DNA tools and biotechnology

Question 1

Define nucleic acid hybridization

Answer 1

Base pairing of one strand of nucleic acid to another complementary strand

Question 2

Describe the process of “next-generation sequencing”

Answer 2

DNA fragments are amplified/copied to yield identical fragments

a template strand for each fragment is immobilized
complimentary strand is synthesized

Question 3

How are plasmids utilized in DNA cloning?

Answer 3

scientists isolate plasmids from bacterial cells and alter them, resulting in a recombinant DNA molecule
the plasmid is returned tot he bacteria, producing a recombinant bacterium

Question 4

Define cloning vector

Answer 4

a DNA molecule that can carry foreign DNA into a host cell and be replicated there

Question 5

How are restriction enzymes used to make a recombinant DNA plasmid?

Answer 5

an enzyme will cut the sugar-phosphate backbone at each site
A DNA fragment from another source is added, base pairing of sticky ends produces various combinations
DNA ligase seals the strands

Question 6

The three steps of PCR are

Answer 6

1. heat to denature
2. cool to anneal/h bond primers to sequences at end of target sequence
3. DNA polymerase extends primers, then replication occurs

Question 7

What is an expression vector? What is it typically used for?

Answer 7

a cloning vector with a highly active bacterial promoter upstream of a restriction site where the eukaryotic gene can be inserted into the correct reading frame

Question 8

What is electroporation?

Answer 8

a method for introducing recombinant DNA into eukaryotic cells
a brief electrical pulse applied to a solution containing cells creates temporary holes in plasma membranes through which DNA can enter

Question 9

What are some potential difficulties in using plasmid vec-
tors and bacterial host cells to produce large quantities of
proteins from cloned eukaryotic genes?

Answer 9

Some eukaryotic genes are too large to be incorporated into bacterial plas-
mids. Bacterial cells lack the means to process RNA transcripts into mRNA, and
even if the need for RNA processing is avoided by using cDNA, bacteria lack
enzymes to catalyze the post-translational processing that many eukaryotic
proteins require to function properly. (This is often the case for human proteins,
which are a focus of biotechnology.)

Question 10

Define a nucleic acid probe

Answer 10

A short, single stranded nucleic acid complementary to the mRNA of interest

Question 11

Define in situ hybridization

Answer 11

A technique that allows MRNA to be seen in place

Question 12

Define reverse transcriptase polymerase chain reaction, or RT-PCR?

Answer 12

can be used to compare gene expression between samples

turns sample sets of mRNAs into double-stranded DNAs with corresponding sequences
first, the enzyme reverse transcriptase is used to synthesize a complementary DNA copy
the result is a double stranded complementary DNA (cDNA)
PCR and electropheris is performed

Question 13

RNA-seq is used to … and performed by…

Answer 13

analyze the expression of many genes
1.mRNAs are isolated
2.mRNAs are cut into small fragments
3. mRNAs are reverse transcribed into cDNAs
4. cDNAs sequenced
5. Short sequences mapped by computer

Question 14

Define in vitro mutagenesis

Answer 14

A technique to edit the genetic material of cells in a predictablew ay

specific mutations are introduced into a cloned gene. the mutated gene is returned to the cell in a way that disables/”knocks out” the normal cellular copies of the same gene

Question 15

Define genome wide association studies

Answer 15

large scale analyses where researchers look for genetic markers

Question 16

A SNP is

Answer 16

a single base-pair site where variation is found in at least 1% of the population

Question 17

SNPs can be detected by a variety of techniques, including

Answer 17

RNA-seq and PCR

Question 18

If most SNPs are in noncoding regions, how are they associated with a disease?

Answer 18

Suggests that the gene whose mutation causes the disease is located v clsoe to the SNP on the chromosome (genetic linkage)

Question 19

Describe the role of complementary base pairing during
RT-PCR, RNA sequencing, and DNA microarray analysis.

Answer 19

.Reverse transcriptase uses mRNA as a template to synthesize the first strand of cDNA, also involved when DNA polymerase synthesizes the second
strand of the cDNA.
RT-PCR, the primers must base-pair with
their target sequences, Also the DNA polymerase
relies on complementary base pairing to the template strand to add new nucleotides during synthesis of the fragments. In DNA microarray analysis, the labeled
cDNA probe binds only to the specific target sequence due to complementary
nucleic acid hybridization . In RNA-seq, when
sequencing the cDNAs, base complementarity plays a role in the sequencing
process.

Question 20

Describe the experiment performed by John Gurdon

Answer 20

a less differentiated nucleus from an embryo was placed into an enucleated cell. Most developed into tadpoles. A tadpole nucleus was palced into an enucleated cell. Most stopped developing.

Question 21

Why are mammalian clones often slightly different?

Answer 21

environmental influences

Question 22

Define induced pluripotent stem cells

Answer 22

Deprogrammed cells that have returned to their undifferentiated state via a retrovirus

Question 23

iPS cells have two major potential uses…

Answer 23

cells from diseased patients have been reprogrammed to become iPS
regenerative medicine

Question 24

What is the advantage of using stem cells for gene therapy
or gene editing?

Answer 24

Stem cells continue to reproduce themselves, ensuring that the corrective gene
product will continue to be made.

Question 25

As a physician, you have a patient with symptoms
that suggest a hepatitis A infection, but you have not been
able to detect viral proteins in the blood. Knowing that hepa-
titis A is an RNA virus, what lab tests could you perform to
support your diagnosis? Explain the results that would sup-
port your hypothesis.

Answer 25

Because hepatitis A
is an RNA virus, you could isolate RNA from the blood and try to detect copies of
hepatitis A RNA by RT-PCR. You would first reverse-transcribe the blood mRNA
into cDNA and then use PCR to amplify the cDNA, using primers specific to
hepatitis A sequences. If you then ran the products on an electrophoretic gel, the
presence of a band of the appropriate size would support your hypothesis. Alter-
natively, you could use RNA-seq to sequence all the RNAs in your patient’s blood
and see whether any of the sequences match up with that of hepatitis A. (Since
you are only seeking one sequence, though, RT-PCR is probably a better choice.)

Question 26

In DNA technology, the term vector can refer to
(A) the enzyme that cuts DNA into restriction fragments.
(B) the sticky end of a DNA fragment.
(C) a SNP marker.
(D) a plasmid used to transfer DNA into a living cell

Answer 26

a plasmid used to transfer DNA into a living cell

Question 27

Which of the following tools of DNA technology is incorrectly
paired with its use?
(A) electrophoresis—separation of DNA fragments
(B) DNA ligase—cutting DNA, creating sticky ends of restric-
tion fragments
(C) DNA polymerase—polymerase chain reaction to amplify
sections of DNA
(D) reverse transcriptase—production of cDNA from mRNA

Answer 27

DNA ligase—cutting DNA, creating sticky ends of restric-
tion fragments

Question 28

Plants are more readily manipulated by genetic engineering
than are animals because
(A) plant genes do not contain introns.
(B) more vectors are available for transferring recombinant
DNA into plant cells.
(C) a somatic plant cell can often give rise to a complete plant.
(D) plant cells have larger nuclei

Answer 28

a somatic plant cell can often give rise to a complete plant

Question 29

A paleontologist has recovered a bit of tissue from the 400-year-
old preserved skin of an extinct dodo (a bird). To compare
a specific region of the DNA from a sample with DNA from
living birds, which of the following would be most useful for
increasing the amount of dodo DNA available for testing?
(A) SNP analysis
(B) polymerase chain reaction (PCR)
(C) electroporation
(D) gel electrophoresis

Answer 29

polymerase chain reaction (PCR)

Question 30

Which of the following is true of cDNA produced using human
brain tissue as the starting material?
(A) The procedure to make it requires amplification by the poly-
merase chain reaction.
(B) It is produced from pre-mRNA using reverse transcriptase.
(C) It can be labeled and used as a probe to detect genes ex-
pressed in the brain.
(D) It includes the introns of the pre-mRNA

Answer 30

It can be labeled and used as a probe to detect genes ex-
pressed in the brain.

Question 31

Expression of a cloned eukaryotic gene in a bacterial cell
involves many challenges. The use of mRNA and reverse
transcriptase is part of a strategy to solve the problem of
(A) post-transcriptional processing.
(B) post-translational processing.
(C) nucleic acid hybridization.
(D) restriction fragment ligation.

Answer 31

post-transcriptional processing.