Chapter 20: Recombinant DNA Technology, Mastering Genetics Flashcards
What is the function of restriction endonucleases in bacteria?
They provide a defense mechanism against infection by viruses.
Restriction endonucleases recognize and degrade viral DNA, thus preventing viral infections.
TRUE or FALSE?
Restriction endonucleases cut DNA at specific recognition sequences and then bond two strands covalently with the same “sticky ends.”
False
Restriction endonucleases cut DNA at specific sequences, but DNA ligase must be used to bond two strands covalently with the same “sticky ends.”
BamHI cuts the sequence 5′ G|GATCC 3′. Which of the following sequences would not be recognized by this enzyme?
BamHI cuts the sequence 5′ G|GATCC 3′. Which of the following sequences would not be recognized by this enzyme?
This sequence does not contain the BamHI recognition site.
Which of the following DNA sequences is one strand of a restriction enzyme recognition sequence?
5’ AAACCC 3’
5’ GGGTTT 3’
5’ GGGGGG 3’
5’ GGATCC 3’
5’ GGATCC 3’
The 5’ → 3’ sequence of the complementary strand would be the same as the 5’ → 3’ sequence of this strand; i.e., this sequence is symmetrical about the midpoint.
X-Gal is included in the growth medium on which cells transformed with bacterial plasmids are grown. The reason X-Gal is included is to _______.
identify bacteria that contain a recombinant plasmid
Colonies produced from cells containing a recombinant plasmid are white, whereas colonies from cells containing a nonrecombinant plasmid are blue.
TRUE or FALSE?
Within a six-base DNA recognition sequence, an enzyme that cuts between the 3rd and 4th bases from the 5’ end will generate blunt ends.
True
Restriction endonucleases are especially useful if they generate “sticky” ends. What makes an end sticky?
single-stranded complementary tails
Assume that a given plasmid vector to be used in a cloning experiment contains 4000 base pairs of DNA. Assume also that the restriction endonuclease Cuj cuts this plasmid at the following sites (starting from an arbitrary zero point): 1000, 1500, and 3000.
Given complete digestion of the plasmid with the endonuclease so that only linear fragments are produced, what sizes of DNA are expected?
500 bp, 1500 bp, and 2000 bp
TRUE or FALSE?
To isolate a bacterium with a plasmid that carries a desired DNA fragment cloned within the ampicillin resistance gene, we should grow bacteria in a medium that contains ampicillin.
False
TRUE or FALSE?
In general, the main goal of cloning is to include as many different genes as possible in a single cloning vector.
False
TRUE or FALSE?
A common term for a plasmid or other DNA element that serves as a cloning vehicle is vector.
True
Following are 4 processes common to most cloning experiments:
A) transforming bacteria
B) plating bacteria on selective medium
C) cutting DNA with restriction endonucleases
D) ligating DNA fragments
Place components of this list in the order in which they would most likely occur during a cloning experiment.
- Cutting DNA with restriction endonucleases.
- Ligating DNA fragments.
- Transforming bacteria.
- Plating bacteria on selective medium.
Some vectors such as pUC18 and others of the pUC series contain a large number of restriction enzyme sites clustered in one region. Which term is given to this advantageous arrangement of restriction sites?
multiple cloning site
What term is used to refer to the process in which DNA can be introduced into host bacterial cells?
transformation
Assume that one conducted a typical cloning experiment using a typical plasmid, transformed an appropriate host bacterial strain, and plated the bacteria on an appropriate X-gal medium. Blue and white colonies appeared. Which of the two types of colonies, blue or white, would most likely contain the recombinant plasmid?
white colonies
TRUE or FALSE?
Phage λ can carry larger DNA fragments than plasmids.
True
Phage vectors can carry DNA fragments of about 20 kb, whereas plasmids can only carry DNA of less than 15 kb.
Which of the following elements is not found in a plasmid?
Lambda arms
Lambda arms are regions that flank the inserted foreign DNA in phage λ vectors.
A DNA fragment is introduced into the lacZ gene of a plasmid, which also contains a tetracycline resistance gene. What is the appearance of bacteria transformed with this plasmid if they are spread on plates containing tetracycline and Xgal?
White colonies that are resistant to tetracycline
The presence of blue colonies means that the plasmid taken up by these bacteria is recombinant, since the lacZ gene was disrupted.
Why are filters overlaid with X‑ray film when screening a cDNA library?
To visualize probe hybridization events
The X‑ray film allows probe hybridization events on the filter to be visualized if a radioactive probe is used.
TRUE or FALSE?
Chromosome walking is used mainly to isolate a gene when the sequence of the gene is known.
False
Chromosome walking is used mainly when the gene of interest has not yet been cloned but its approximate location on a chromosome is known.
One of the primary reasons for generating a large number of clones in a eukaryotic genomic library is that ________.
each vector can take up only a relatively small fraction of the eukaryotic DNA
Which type of DNA library represents the genes expressed by a given cell at a certain time?
cDNA
cDNA libraries represent expressed genes from specific cell types under specific conditions. Noncoding sequences are not included.
Nucleic acid blotting is widely used in recombinant DNA technology. In a Southern blot, one generally ________.
hybridizes filter-bound DNA with a DNA probe
Northern blots are used to study what type of molecule?
RNA
RNA fragments are separated using gel electrophoresis and blotted onto a nylon membrane. Labeled nucleic acid fragments are used to probe the filter and identify hybridizing sequences.
What is a probe in molecular biology?
A DNA or an RNA molecule used in hybridization reactions
A probe is a labeled single strand of DNA or RNA used to locate its complementary sequence. They are commonly used in southern and northern blots, as well as in library screening.
Which of the following molecules is not required for a PCR reaction?
Ligase
Ligase is not required for a PCR reaction. The enzyme used during PCR is a thermostable DNA polymerase.
TRUE or FALSE?
The thermostability of Taq polymerase is required during the annealing phase of PCR.
False
The annealing phase takes place at the lowest temperature of PCR. Taq polymerase is derived from bacteria that live in hot springs, so the enzyme is thermostable, meaning that its enzymatic properties can withstand the high temperatures needed for denaturation.
What is the purpose of raising the temperature to 90–95°C at the beginning of each cycle of PCR?
To separate the double‑stranded DNA
The temperature is raised to denature the double‑stranded DNA molecule into single strands.
The role of the primers in PCR is _______.
to define the target region and provide a 3’ end that can be extended by taq polymerase
Primers bind to end of the target DNA strands, then taq polymerase synthesizes a new strand using the target DNA as a template.
If there are five molecules of DNA containing the target region at the beginning of a PCR reaction, how many copies of the target will be present after three rounds of amplification?
40
The number of target sequences is doubled with each replication cycle.
Immediately after the primers have annealed to the target sequence, _______.
the temperature is raised so that taq polymerase can extend the primers
The temperature is raised to 70–75∘C, the temperature over which taq polymerase is optimally active.
TRUE or FALSE?
During a PCR, heat is provided to inactivate the polymerase enzyme.
False
In a typical PCR, primers are used to cleave specific regions of the DNA template.
False
The products of restriction digestion can be visualized by gel electrophoresis, which separates fragments based on their size.
True
Restriction digestion produces fragments of DNA, and the sizes of these fragments can be determined by gel electrophoresis using standard DNA fragments of known size.
A 1.5‑kb fragment of DNA is cloned into a plasmid vector that is 5.5 kb long at the EcoRI site, and the plasmid vector is then used to transform bacteria. If the plasmid DNA is then extracted from a single bacterial colony and digested with EcoRI, what digestion products will be produced if the plasmid contains the fragment?
One 1.5‑kb fragment and one 5.5‑kb fragment
EcoRI digestion will produce two fragments corresponding in size to the 1.5‑kb fragment cloned into the plasmid plus the 5.5‑kb plasmid itself.
Digestion of a 1.1‑kb fragment of DNA with BamHI produces two fragments of 700 bp and 400 bp. Digestion of the same 1.1‑kb fragment with XhoI produces two fragments of 300 bp and 800 bp. Digestion with both enzymes produces three fragments of 100 bp, 300 bp, and 700 bp. Which of the following statements is true about the DNA fragment?
The XhoI site is located within the BamHI 400‑bp fragment.
Since double digestion produces a 100‑bp fragment and a 300‑bp fragment, the XhoI site must be located within the BamHI 400‑bp fragment.
Which of the following statements about ddNTPs is true?
They have a hydrogen at the 3′ carbon of the sugar.
ddNTPs terminate synthesis because there is no 3′‑hydroxyl group onto which DNA polymerase can add nucleotides.
DNA fragments that are 600 bp long will migrate more quickly through a sequencing gel than fragments that are 150 bp long.
False
Small DNA fragments have less hindrance in moving through the gel, so they migrate more quickly than larger fragments.
Which of the following statements about manual Sanger sequencing is true?
The DNA sequence obtained is complementary to the template strand.
The DNA fragments produced in sequencing reactions are synthesized by DNA polymerase to be complementary to the template strand.
A ddNTP, used often in DNA sequencing, lacks a(n) ________ at the ________ and ________ carbons.
OH; 2’; 3’
The function of a ddNTP in DNA sequencing is to methylate guanine.
False
In which of the following biochemical reactions is it common to use ddNTPs (dideoxyribonucleoside triphosphates)?
DNA sequencing
Pyrosequencing is a method of DNA sequencing that uses beads as a matrix for PCR.
True
The use of restriction enzymes
In recombinant DNA technology, restriction enzymes are used to isolate DNA segments (often genes) and recombine them with other pieces of DNA. A restriction enzyme recognizes a specific DNA base sequence—called a restriction site or recognition site—and cuts eachstrand of the DNA at a particular point within that site.
Most restriction sites are symmetrical: On each strand within the site, the sequence of bases is the same when read in the 5’ → 3’direction. The restriction site for the enzyme PvuI is an example of a symmetrical site.
What are sticky ends?
A restriction enzyme cuts each strand of a DNA molecule at a particular point within the enzyme’s restriction site. If the cuts are staggered—that is, if the cut points on the two strands are not directly opposite each other—the resulting DNA fragments have single-stranded ends.These ends are called “sticky ends” because they can base-pair with complementary single-stranded ends on other DNA fragments.
How can scientists produce a DNA fragment that contains a specific gene?
To clone a specific gene, scientists start with a much longer stretch of DNA containing the gene and use a restriction enzyme to cut theDNA into many fragments. Only one of these fragments—the one that contains the whole gene—is useful for cloning. This fragment must have two sticky ends to be integrated into a cloning vector, which happens in the next step.
To produce a DNA fragment that contains the whole gene and has two sticky ends, where must the enzyme’s restriction sites belocated?
outside the gene on both sides, but not inside the gene
Restriction Enzymes, Recombinant DNA, and Gene Cloning
DNA cloning is a technique for producing many copies of a DNA segment for use in research, medicine, agriculture, or other applications.
In this tutorial, you will explore the procedures involved in cloning an imaginary gene in humans. DNA cloning is based on the following sequence of steps:
- use of restriction enzymes to cut DNA into fragments
- insertion and ligation of DNA fragments into a cloning vector
- transformation and selection of recombinant bacteria
- isolation of desired recombinant bacteria: screening the library
Using restriction enzymes to cut DNA
The ability of restriction enzymes to cut DNA at specific sites makes DNA cloning possible.
The diagram below shows a section of human DNA that contains an imaginary gene for video game proficiency (the vgp gene), shown in red. Shaded areas mark the restriction sites (also called recognition sites) of four restriction enzymes: EcoRI, HaeIII, BamHI, and HindIII. Arrows indicate where each enzyme cuts the two DNA strands.
Which enzyme(s) will produce a DNA fragment that contains the entire vgp gene (shown in red) and has “sticky ends”?
BamHI, HindIII
Cloning the vgp gene requires an enzyme that has restriction sites on both sides of that gene but not within the gene. Two enzymes, HindIII and BamHI, satisfy that requirement, while also producing DNA fragments that have sticky ends. Sticky ends make it possible for the fragments to combine with the DNA of a cloning vector, such as a plasmid.
BamHI cuts the plasmid outside the ampR gene, leaving the gene intact and functional, which is important for the next step in the cloning procedure. Once BamHI opens the plasmid, the sticky ends it produces in the plasmid can base-pair with those on BamHI human DNA fragments. If DNA ligase is then added, it will covalently join the sugar-phosphate backbones of the plasmid and the fragments, producing recombinant plasmids.
Note that human DNA contains many BamHI restriction sites in addition to the two shown in Part A. Therefore, only a small fraction of the recombinant plasmids will have the vgp gene, like the plasmid shown above. Most of the recombinant plasmids will have a fragment of human DNA that does not contain the vgp gene, and would not be of interest to the scientists.
Which enzymes would cut the human DNA on both sides of the vgp gene, but not inside the gene?
BamHI , Haelll and HindIIl
Which enzymes would cut the plasmid without disrupting the function of the amp^R gene?
BamHI, EcoRI, and HaeIII,
Which enzymes would produce sticky ends when cutting both the human DNA and the plasmid?
BamHI, EcoRI, and HindIII,
Which one restriction enzyme satisfies all three of the following requirements:
Would cut the human DNA on both sides of the vgp gene, but not inside the gene.
Would cut the plasmid without disrupting the function of the amp^R gene.
Would produce sticky ends when cutting both the human DNA and the plasmid.
BamHI
What kind of plasmid will ONLY grow in medium without ampicillin:
no plasmid!
What kind of plasmid will grow in both media?
nonerecombinant plasmid,
recombinant plasmid but no vgp gene,
recombinant plasmid with vgp gen
Only bacteria that were transformed (picked up a plasmid) can grow in a medium containing ampicillin. Each transformed bacterium reproduces, forming a clone of cells.
The complete set of clones is called a plasmid library. Some clones in the library have nonrecombinant plasmids, some have recombinant plasmids without the vgp gene, and a small fraction have recombinant plasmids with the vgp gene.
To screen a library of bacterial colonies for clones that carry a specific gene, a relatively short, single-stranded nucleic acid probe is hybridized to the DNA of that gene. Often, the probe is a DNA strand synthesized by reverse transcriptase from the mRNA encoded by the gene.
Bases in the probe hydrogen-bond to complementary bases in the gene. If the probe has been labeled with a radioactive isotope or a fluorescent tag, researchers can identify the bacterial clones that contain the DNA to which the probe has bound. Those clones can be grown in large quantities, allowing many copies of the gene to be isolated for use in research or other applications.
Modern techniques
Developed in 1970’s
Virtual revolution in biotechnology
First commercial approval was 1982 for bacterial produced human insulin
Has led to an explosion of this industry
In recombinant DNA technology, restriction enzymes are used to isolate DNA segments (often genes) and recombine them with other pieces of DNA. A restriction enzyme recognizes a specific DNA base sequence—called a restriction site or recognition site—and cuts each strand of the DNA at a particular point within that site.
Most restriction sites are symmetrical: On each strand within the site, the sequence of bases is the same when read in the 5’ → 3’ direction. The restriction site for the enzyme PvuI is an example of a symmetrical site.
A restriction enzyme cuts each strand of a DNA molecule at a particular point within the enzyme’s restriction site. If the cuts are staggered—that is, if the cut points on the two strands are not directly opposite each other—the resulting DNA fragments have single-stranded ends.
These ends are called “sticky ends” because they can base-pair with complementary single-stranded ends on other DNA fragments.
How can scientists produce a DNA fragment that contains a specific gene?
To clone a specific gene, scientists start with a much longer stretch of DNA containing the gene and use a restriction enzyme to cut the DNA into many fragments. Only one of these fragments—the one that contains the whole gene—is useful for cloning. This fragment must have two sticky ends to be integrated into a cloning vector, which happens in the next step.
To produce a DNA fragment that contains the whole gene and has two sticky ends, where must the enzyme’s restriction sites be located?
outside the gene on both sides, but not inside the gene
An enzyme will cut the human DNA on both sides of the vgp gene only if it has restriction sites to the left and right of the gene.
An enzyme will cut inside the vgp gene only if it has a restriction site within the gene.
A gene in a bacterial plasmid consists of a continuous sequence of bases that is transcribed into mRNA, which is translated into a protein. Thus, the function of the gene is to code for a specific protein.
In the case of the ampR gene, the gene codes for a protein that enables bacteria to grow in media containing the antibiotic ampicillin.
Suppose that a restriction enzyme cut the plasmid within the ampR gene and that an additional piece of DNA were inserted into the gene’s base sequence.
How would that affect the mRNA and ultimately the protein encoded by the ampR gene?
Both the mRNA and the protein would be abnormal.
Sticky ends are single-stranded stretches at the ends of a DNA molecule that are formed when restriction enzymes make staggered cuts in DNA.
Sticky ends are single-stranded stretches at the ends of a DNA molecule that are formed when restriction enzymes make staggered cuts in DNA.
Which of the following correctly describes a transformed bacterium?
a bacterium that has taken up external DNA, such as a plasmid
How do ampicillin-sensitive bacteria become resistant to ampicillin?
The third step in the cloning procedure involves incubating ampicillin-sensitive bacteria with plasmids, some of which contain the vgp gene.
How do some of these bacteria become resistant to ampicillin?
by being transformed
More than 200 restriction enzymes have been found in bacteria to date.
Many restriction endonucleases, including BamHI, recognize a palindromic sequence.
Restriction enzymes cut DNA at specific recognition sites.
Many restriction sites are symmetrical about their midpoint.
X-gal is a colorless substrate for the enzyme ____
X-gal is a colorless substrate for the enzyme beta-galactosidase
A restriction enzyme that leaves either 5’ or 3’ overhangs produce “sticky ends.”
Restriction enzymes that leave no overhang produce blunt ends.
Recombinant DNA is created by combining DNA molecules that are not found together naturally. This manipulation of DNA is achieved through the use of restriction endonucleases, which are enzymes that recognize specific palindromic DNA sequences.
Once both strands of the DNA within that sequence are cut by the restriction enzyme, the complementary tails of the DNA fragments can be annealed together by DNA ligase. More than 200 restriction enzymes have been identified in bacteria to date.
Cloning involves the insertion of DNA fragments into vectors, which are agents that transfer genetic material from one cell to another.
Vectors are used to propagate and manipulate DNA fragments in bacteriophages and bacteria.
Recombinant DNA is created by digesting DNA molecules with restriction enzymes and inserting them into carrier molecules called vectors.
Vectors can be derived from many sources, including bacterial plasmids and phages.
Phage lambda (λ) is a bacteriophage whose central gene cluster can be replaced with a large foreign DNA fragment.
The phages are used to infect bacterial cells, where they replicate and form plaques from which the cloned DNA can be recovered.
Smaller DNA fragments can be cloned into plasmids–naturally occurring extrachromosomal DNA molecules that replicate autonomously within bacterial cells. Up to 1000 copies of a plasmid may be produced within a single bacterial cell, so many copies of the cloned DNA can be generated.
Plasmid vectors generally contain a polylinker composed of unique restriction sites to facilitate the insertion of a foreign DNA fragment and genetic markers to allow for the easy detection of bacterial colonies that contain recombinant plasmids.
The efficiency of transformation of bacterial plasmids starts to decrease at around 15 kb.
A piece of foreign DNA generally disrupts and inactivates the gene into which it is inserted.
A clone library is a set of DNA segments derived from an individual.
It may represent an entire genome, a single chromosome, or a set of genes that are actively transcribed in a single cell type. A clone library can be generated and the techniques used to isolate specific clones from the library.
cDNA libraries use mRNA as a starting point and thus represent only the expressed (transcribed) sequences under a given set of conditions. Because most eukaryotic mRNAs have poly-A tails at their 3’ ends, poly-dT oligonucleotides can be used as primers for the enzyme reverse transcriptase to synthesize cDNA strands complementary to the mRNAs. After degradation of the mRNA strands in mRNA/cDNA duplexes, DNA polymerase is used to synthesize complementary DNA strands. This results in double-stranded cDNA molecules that can be cloned into a suitable vector for the generation of the library.
Most methods for screening a cloned library involve hybridization with a DNA probe to identify specific clones, which then can be isolated and grown for further analysis. If the gene of interest has not been cloned but its approximate location on a chromosome is known, however, a technique known as chromosome walking can be used for several rounds of screening to recover overlapping clones from a genomic library until the gene in question has been reached.
_______ involves the successive isolation of overlapping clones along a chromosome until the gene of interest is reached.
Chromosome walking involves the successive isolation of overlapping clones along a chromosome until the gene of interest is reached.
Northern and Southern blots are used to identify_______.
Northern and Southern blots are used to identify nucleic acid sequences.
Probes are used in several different molecular techniques, including southern and northern blots.
……
The polymerase chain reaction (PCR) is a rapid, cell-free method of DNA cloning that has extended the power of recombinant DNA research.
The technique can exponentially amplify specific DNA sequences.
PCR allows rapid direct amplification of specific target sequences of DNA that initially may be present in infinitesimally small quantities. The 3 steps in a PCR cycle are denaturation, annealing, and extension.
DNA molecules are first denatured by heating at 90-95ºC to dissociate the double-stranded molecules into single strands. During annealing, synthetic primers (oligonucleotides 15-30 nucleotides long) bind specifically to sequences flanking the segment to be amplified. These primers act as starting points for the synthesis of new DNA strands complementary to the target DNA. During extension, a heat-stable form of DNA polymerase, such as Taq polymerase, extends the primers by adding nucleotides in the 5’ to 3’ direction, making a double-stranded copy of the target DNA. Each PCR cycle theoretically doubles the number of copies of the target molecule, such that 25 cycles may result in more than a millionfold increase in the number of DNA copies.
DNA sequencing is a method used to determine the nucleotide sequence of a DNA fragment.
This is the theory behind the most common method of sequencing and how it can be performed either manually or using an automated sequencer.
The most commonly used DNA sequencing method, developed by James Sanger, involves the use of DNA polymerase to synthesize DNA strands repeatedly from a primer that binds close to the DNA to be sequenced. Fragments of different length are produced because strand synthesis terminates when a dideoxynucleotide (ddNTP) is incorporated into the growing strand. Sanger sequencing can be done manually or by using an automated sequencer.
In manual sequencing, 4 separate reactions are performed, 1 for each of the 4 ddNTP bases. These reactions are electrophoresed in separate lanes on a sequencing gel, which can then be read to reveal the base sequence of the DNA fragment.
An automated sequencer uses a single reaction mixture in which each of the 4 ddNTP terminating bases is labeled with a different fluorescent dye. The reaction is loaded in a single lane of a sequencing gel, and a laser reads the dye color terminating each fragment band and produces a graph from which the sequence is read directly.
If the plasmid contains the fragment, EcoRI will cut in two places.
…..
One of the first steps in characterizing a DNA clone is to produce a restriction map, which gives the position of restriction sites on a fragment of DNA.
Restriction mapping is a technique for determining the location of restriction sites on a fragment of DNA.
The DNA is digested with restriction enzymes, and the positions of the restriction sites are inferred from the sizes of the digested products.
DNA can be digested with restriction enzymes singly or in combination to determine the position of the restriction sites relative to one another.
The digested fragments can be resolved by gel electrophoresis, which separates fragments based on their size.
The fragment sizes (generally measured in thousands of base pairs, kb) are determined by comparison with DNA standards of known lengths.
