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Chapter 10

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In the laboratory, DNA molecules can be cut at specific sequences using:
restriction nucleases.
On average, which restriction nuclease would cut a random DNA sequence into a greater number of pieces?
A restriction nuclease that recognizes a four-nucleotide-pair sequence
How does a bacterium protect its DNA from cleavage by its own restriction nucleases?
The bacterium chemically modifies its own DNA sequences, thereby preventing recognition by the nucleases.
Gel electrophoresis separates DNA fragments from each other by virtue of which feature?
Length
When a mixture of DNA fragments of different sizes is loaded onto an agarose gel and a voltage is applied, the DNA will migrate in the gel. Which molecules will migrate the fastest?
The shortest
When DNA fragments are being separated by gel electrophoresis, toward which electrode do the DNA fragments move?
The positive Electrode
Incorporating radioactive isotopes into DNA prior to gel electrophoresis is one method to:
visualize the separated DNA bands in the gel.
Nucleic acid hybridization, which can be used to detect any given DNA or RNA sequence in a mixture of nucleic acid fragments, relies on the fact that:
a single strand of DNA or RNA will form a double helix with another nucleic acid strand of the complementary nucleotide sequence.
In Southern blotting, DNA fragments are first separated from each other by:
gel electrophoresis
The restriction enzyme HindIII cuts DNA at the sequence AAGCTT. In the human genome, approximately how often will this target sequence be encountered?
About 750,000 times
A circular plasmid DNA contains target sites for both HaeIII and EcoRI. The plasmid was digested with each enzyme separately and both enzymes together. The resulting products were separated by gel electrophoresis as shown in the figure. For comparison, the uncleaved plasmid was electrophoresed in the first lane of the gel. The relative sizes of the DNA fragments are indicated above each band. How many HaeIII sites are present in the plasmid?
1
A circular plasmid DNA contains target sites for both HaeIII and EcoRI. The plasmid was digested with each enzyme separately and both enzymes together. The resulting products were separated by gel electrophoresis as shown in the figure. For comparison, the uncleaved plasmid was electrophoresed in the first lane of the gel. The relative sizes of the DNA fragments are indicated above each band. Which is NOT true of the EcoRI sites in the plasmid?
The EcoRI sites are the same distance from the HaeIII site.
Nucleic acid hybridization is an extremely sensitive method for detecting specific nucleotide sequences: even a single mismatch between a DNA probe and its target nucleotide sequence will alter the stability of the double-stranded molecules that result. This sensitivity can be exploited when using a DNA probe to detect genetic variants that heighten the risk of disease. For example, a gene encoding a receptor protein that binds to the neurotransmitter dopamine has been linked to an increased risk of alcohol abuse. Individuals that carry a variant form of this gene, in which a single A has been replaced by a C, are more prone to developing alcoholism.
To determine whether an individual carries the "normal" version of this gene or the risky variant, investigators design a DNA probe that is a perfect complement for the risky variant. This probe will also hybridize with the normal gene, but the match is not perfect.
Investigators analyze the temperature-dependent stability of the probe hybridized to single-stranded genomic DNA. In this experiment, the extent of hybridization is monitored with the use of a dye that fluoresces strongly when it intercalates between the bases of double-stranded DNA. Which of the curves shown has a higher melting point? Which represents the risky variant and which the normal gene?
Curve 2 has a higher melting point, which means that it represents the risky variant
A linear fragment of DNA, 3000 nucleotides (3 kb) in length, is incubated with the restriction nucleases EcoRI and HpaII. The resulting fragments are separated by gel electrophoresis and the DNA bands are visualized by staining with a molecule that fluoresces intensely when it is bound to DNA.
Based on this gel, how many recognition sequences for each enzyme are present in the original fragment?
The data are insufficient to answer the question.
A linear fragment of DNA, 3000 nucleotides (3 kb) in length, is incubated with the restriction nuclease HpaII. The resulting fragments are separated by gel electrophoresis and the DNA bands are visualized by staining with a molecule that fluoresces intensely when it is bound to DNA. Based on this gel, how many recognition sequences for HpaII are present in the original fragment?
one
What does the term DNA cloning refer to?
The act of making many identical copies of a DNA molecule
Which allows scientists to join together two DNA fragments?
DNA ligase
Which of the following statements is NOT typically true of a plasmid?
It inserts the gene of interest into the bacterial chromosome.
What does a DNA library consist of?
A collection of cloned DNA fragments
Which DNA library lacks introns and regulatory sequences?
A cDNA library
When making a DNA library from a starting material of mRNA, which enzyme is required to copy the mRNA molecules into DNA molecules?
Reverse transcriptase
Which of the following libraries would be expected to be essentially the same?
Genomic libraries made from mouse liver and kidney cells
What is the starting material for making a cDNA library?
mRNA
If you want to clone a gene so that you may deduce the amino acid sequence of the protein from the DNA, what type of library would be most useful to start from?
A cDNA library
Which of these common recombinant DNA procedures does NOT require the use of an enzyme?
Nucleic acid hybridization
The coding region of any gene can be cloned from a cDNA library made from any tissue.
false
Once a genomic library has been prepared, how is the bacterial clone that contains the DNA fragment of interest generally identified?
Hybridization with a labeled probe
Which of the following is NOT true?
PCR can be used to sequence a genome.
True:
PCR amplifies a DNA sequence.

PCR can be used to clone a gene.

PCR can be used to detect the presence of a virus in a blood sample.
In order to design primers for the polymerase chain reaction, what must be known about the DNA of interest?
The sequence at its ends
In the polymerase chain reaction, what is used to separate the two strands of a double-stranded DNA molecule?
high heat
Using PCR, after 30 rounds of replication beginning with a single template molecule of double-stranded DNA, how many copies of the target sequence would you expect?
About 1 billion
What serves as the original template material for the polymerase chain reaction?
DNA or RNA
In order to detect an infectious microorganism by PCR, what is required as a primer?
Short sequences complementary to a part of the microbe's genome
Investigators are attempting to clone a gene that is bracketed by the following sequence:
ATGAAATCTACGTTTCAC......CCCCCAGTACCCCCCTTA
Which of the following pairs of primers could be used to direct the amplification of this gene? (All sequences are written 5' to 3'.)
ATGAAATCTACGTTTCAC and TAAGGGGGGTACTGGGGG
An investigator is setting up a procedure for doing paternity testing in his laboratory. To see whether his reagents and protocol are working properly, he obtains DNA samples from his wife and child and, using three sets of PCR primers, he amplifies three different STRs—regions in the DNA where individuals differ in the number of repeats present in each sequence. When such STRs are separated by electrophoresis, they generate a "DNA fingerprint" that can be used to help identify an individual.
For comparison, the investigator collects a DNA sample from himself and from four other people who work in the lab. He amplifies the same three STRs and then runs all of the samples on a single gel. The first two lanes contain the DNA fingerprint of the mother (M) and child (C). Based on these results, which sample belonged to the child's father?
1
Which technique or reagent would be least likely to reveal whether a patient has been infected by a virus?
Green fluorescent protein
One of the drawbacks of PCR is that it can only be used to generate genomic clones, not cDNA clones.
false
Sanger sequencing can be used to
determine the nucleotide sequence of any purified DNA fragment.
All sequencing methods require DNA amplification.
false
One relatively new, third-generation sequencing method allows:
sequencing of a single molecule of DNA by detecting the shapes of its nucleotides.
By comparing a nucleotide sequence to sequences available in public databases, one CANNOT:
determine the gene's precise role in the physiology or development of the organism.
Which of the following technologies allows us to study the expression patterns of thousands of genes at the same time?
DNA microarrays
Which of the following techniques allows a researcher to identify the location of particular RNAs in a cell?
In situ hybridization
A reporter gene:
(A) can reveal when a gene is expressed.

(B) can reveal where a gene is expressed.

(C) is a gene whose activity can be easily monitored.

(D) is a gene whose product can be visually monitored
Which of the following is considered a reporter protein?
Green fluorescent protein
Which of the following refers to the appearance or behavior of an individual?
Phenotype
The "classical genetic approach" works best with organisms that reproduce rapidly and can be analyzed individually in the laboratory.
true
Which of the following terms describes a mouse in which a particular gene has been genetically engineered?
Knockout mouse
Transgenic animals are used to model human diseases in which mutant genes play a major part because:
transgenic approaches in humans are unethical
Plants are resistant to manipulation by recombinant DNA techniques.
false
What extra sequences are found in expression vectors that are not found in typical cloning vectors?
Promoter sequences that direct the production of large quantities of protein
RNAi is a form of reverse genetics.
true
Creating a targeted "gene knockout" is an example of a classical genetic approach to determining the function of a gene.
false
RNAi is particularly easy in this organism, because the animals can be fed E. coli that has been genetically engineered to produce the double-stranded RNAs that trigger RNAi.
C. elegans
Investigators are studying a gene whose expression is controlled by a set of three regulatory DNA sequences that lie next to one another upstream of the gene's coding sequences. To begin to dissect how these sequences influence the gene's expression, the investigators construct a series of reporter genes that include different combinations of these regulatory elements. These combinations, along with the effect they have on the expression of the gene, are shown in the diagram. Based on these data, what can be concluded about these regulatory DNA sequences?
Segment A is necessary for expression of the gene.
Investigators are studying gene X, whose expression is controlled by a set of three regulatory DNA sequences that lie upstream of the gene's coding sequences. In an intact organism, gene X is expressed in cell types B, E, and F.
To begin to dissect how these regulatory sequences influence the gene's expression in different cell types, the investigators construct a series of recombinant DNA molecules in which different combinations of these regulatory elements control the expression of a reporter gene, Y. These recombinant molecules are then tested for expression in cells. The effects they have on the expression of the reporter gene Y are shown in the diagram.
Based on these data, what can be concluded about these regulatory DNA sequences?
Segment 1 turns off gene X in cell D
RNA sequencing, or RNA-Seq, can be used to track the expression of genes in different tissues. The technique counts the number of times a particular RNA sequence occurs in a given sample. These "reads" reveal the relative abundance of different mRNAs in a particular cell type.
For many mRNAs, the RNA-Seq reads show an uneven pattern. Shown here is an RNA-Seq analysis of a single mRNA from liver compared with the mRNA sequence from brain. The height of the red lines indicates the number of sequencing reads and the structure of the mRNA is shown below, with the coding sequences indicated in light blue and the untranslated regions in purple. What could explain this pattern?
The mRNA is alternatively spliced in the liver compared with the brain
Genetic engineering in plants is made simpler by the fact that:
transgenic plants can be grown from almost any type of plant cells transfected with DNA in culture.
Shotgun sequencing is the approach of choice for sequencing large genomes.
false
In clone-by-clone sequencing, individual clones are first placed in order based on:
the restriction sites they contain
Automated Sanger sequencing differs from the original method in that it:
uses a mixture of chain-terminating nucleotides, each with its own label.