Chapter 17

Review the protocol typically used to construct and purify polyhistidine-tagged proteins by ranking the following steps in the order that they would occur when the procedure is carried out.

1. Clone gene of interest into expression vector 2. Introduce constructed plasmid in E. coli or other suitable host 3. Culture bacterial host so that cells produce his-tagged molecules 4. Lyse cells and apply extract to nickel- or cobalt-coated residues 5. Wash column with buffer to remove unbound proteins 6. Release his-tagged proteins with imidazole solution 7. Recover and visualize his-tagged proteins via gel electrophoresis

Review the steps of constructing a genomic library by ranking the following steps in the order that they would typically occur.

1. Genome cleaved into small fragments by restriction endonucleases 2. Fragments introduced into plasmid vectors that have been cut with the same restriction endonuclease 3. Auxotrophs transformed with recombinant plasmids 4. Transformed bacteria plated on media with selective agent (typically antibiotic) 5. Resulting colonies replica plated on medium that selects transformed auxotrophs 6. Isolate and grow resulting transformed auxotrophs

Place the steps involved in seamless cloning in the correct order according to the figure.

1. Linearize vector and amplify target DNA using primers with vector sequences 2. Mix vector, and insert DNA with T5 exonuclease to create 3' overhangs 3. 3' sticky ends hybridize to recircularize the vector 4. DNA polymerase completes the molecule and DNA ligase seals the gaps

A molecular biologist is interested in purifying a recombinant protein by His-tagging, but the protein and vector both lack histidine residues. In this case, the molecular biologist could use which of the following technique(s) to achieve purification?

All of the choices are correct.

Which of the following can be used as vectors for cloning DNA fragments?

All of the choices are correct.

Why are genomic libraries useful?

An organism's entire genome is found in a collection of vectors. Researchers can study isolated groups of genes. DNA fragments are produced that represent the entire genome.

Restriction endonucleases were discovered by _________.

Arber and Smith

The human genome project depended upon using _________ that could accommodate large DNA fragments.

BACs

Which of the following types of cloning vector can carry the largest amount of foreign DNA?

Bacterial artificial chromosome

Evaluate the statements below and choose those that correctly apply to Cas9 endonucleases.

Cas9 is ofund in bacteria and archaea, but not eukaryotes; After hydrolysis the double strands of DAN has blunt ends; The precision of Cas9 is determined by the homology of the guide RNA to a specific DNA sequence; Cas9 is an apoenzyme it requires a guide RNA to function

I am a ribonucleoprotein; I contain both a polypeptide and an RNA molecule. My RNA binds to unique sequences on a DNA molecule, targeting them for cleavage by my polypeptide. I occur naturally in microbes to protect against viral attack, and I can be engineered in the lab for precision DNA editing. What am I?

Cas9 nuclease

The PCR reaction is heated to around 68-72° Celsius to allow __________________ to synthesize new complementary DNA strands.

DNA Polymerase

The three steps that take place in each cycle during PCR occur in which order?

DNA denaturation, annealing, and synthesis

Which of the following PCR procedures includes all of the others?

DNA is amplified for one cycle

If DNA were a positively charged rather than negatively charged, what change to the gel electrophoresis procedure must be made?

DNA must be loaded at the positive pole rather than at the negative pole.

Which is the most frequently chosen prokaryotic host for use in cloning techniques?

Escherichia coli

Which of the following bacterial hosts should be used to avoid degradation of DNA that is introduced via a cloning vector?

Escherichia coli that lack endonucleases

Examine pUC19 polylinker site and the target DNA molecule then identify the best restriction sites to use to insert the provided DNA from the options given.

HindIII and BamHI

Which situation(s) might warrant the removal of a histidine tag when purifying a recombinant protein?

Histidine residues inhibit protein folding, thereby decreasing the functionality of the protein.

You and a friend are student assistants in a research laboratory that investigates the anticancer properties of proteins isolated from marine organisms. Your friend mentions that she is using a BAC vector to insert shark DNA into Escherichia coli, but after repeated attempts, has found that the bacterial cells fail to synthesize the encoded protein. What is your advice?

If the shark DNA is unmodified, it contains introns that are not recognized by bacteria, therefore protein synthesis will not occur.

Arrange the statements in their proper order to assess your knowledge of the gene cloning process.

Isolate DNA to be cloned. Use a restriction enzyme or PCR to generate fragments of DNA. Generate a recombinant molecule by inserting DNA fragments into a cloning vector. Introduce recombinant molecule into new host, which expresses the gene.

What item is not one of the three major characteristics that a vector should possess?

LacZ gene

When separating DNA fragments by gel electrophoresis, what is the purpose of including molecular weight markers?

Markers are used as a control to determine the relative size of restriction fragments.

The PCR method was developed by _________.

Mullis

Which of the following statements is true regarding the use of oligonucleotide primers in PCR?

Only a portion of the oligonucleotide primers need to hybridize to the 3' template DNA strand; the 5' "overhang" can contain a restriction site that will be copied during the second and all subsequent cycles of PCR.

Which of the following terms is most closely related to genetic complementation?

Phenotypic rescue

Recombinant DNA technology does not rely on which of the following enzymes?

RNA methylase

During the investigation of a convenience store robbery, witnesses report that the perpetrator exited through a main entry door without wearing gloves. The police are holding a prime suspect in custody while samples from the door are collected and analyzed for a DNA match. Since the door was handled by many people before the robbery, how will the forensics department distinguish the suspect's DNA from the DNA of others using PCR?

Sequence-specific primers to the suspect's DNA will be used for PCR amplification.

Which is a true statement regarding the size of PCR products?

Since the number of DNA products ending exactly between both primers increases with each cycle, when PCR is completed, the majority of products are of similar size.

Which of the following is true about restriction endonucleases?

Some make a blunt cut on the two DNA strands so that there are no single-strand regions and some make staggered cuts on the DNA so that single-strand ends are formed that can be used to insert foreign DNA cut with the same enzyme.

What items are needed in order to carry out real-time PCR (qPCR) to determine the amount of a specific DNA sequence in a sample?

Taq polymerase, Thermocycler, Fluorescence reporter, Instrument to detect/measure fluorescence

The enzyme reverse transcriptase was discovered by _________.

Temin and Baltimore

Which of the following statements is false regarding the process of seamless cloning?

The 5' ends of the oligonucleotide primers contain a recognition site for a restriction enzyme that allows sticky ends to form between the inserted DNA and the vector.

Which of the following best describes the basis for separation of DNA fragments during agarose gel electrophoresis?

The smallest fragments will migrate fastest.

Which of the following is not true of cloning vectors?

They contain at least two replication origins.

A molecular biologist treats a 600-bp length of linear DNA with HindIII restriction endonuclease. Following gel electrophoresis, the biologist observes that there is only one band on the gel and it corresponds with the migration distance of the 200-bp molecular weight marker. Assuming that no procedural errors were made, what can be concluded from these results?

Two HindIII recognition sequences were present in the original DNA

Which is most analogous to the role of GFP in recombinant DNA technology?

Using dyes to stain and detect bacteria under light microscopy from the sputum of a patient diagnosed with tuberculosis.

Sputum from a patient with a history of tuberculosis due to Mycobacterium tuberculosis bacteria is collected and sent to the microbiology lab for analysis. X-ray analysis and an acid-fast smear made from the sputum indicate that the patient has active tuberculosis. The physician has requested that the sputum be analyzed for pathogen load, but because colonies of Mycobacterium typically take between 3 to 8 weeks to appear on agar, results from traditional culture methods are often delayed. If you were the microbiologist in this case, what would you do?

Using the sputum, apply real-time PCR with primers to Mycobacterium tuberculosis DNA to estimate the pathogen load, then follow up with a traditional culture.

Which cloning vector should be used to express a 500-kb DNA fragment in Saccharomyces cerevisiae?

YAC

Very large DNA fragments, about 1,000 kb, are cloned in ________; these DNA vectors contain telomeres at each end, an origin of replication and selectable markers.

YACs

A microbiologist would like to use a noncompetent genus of streptococcal bacteria, Enterococcus faecalis, as a cloning host to express genes from Streptococcus pneumoniae, which is naturally competent. Is this possible?

Yes; electroporation or chemical transformation can be used to make Enterococcus competent, and then genes from Streptococcus can be introduced via a cloning vector.

The polymerase chain reaction, also called PCR, is a process used to ________ DNA sequences.

amplify

Plasmid vectors often contain __________ genes that can be used to screen for recombinants.

antibiotic resistance

Cas9 endonuclease

attaches to a guide RNA, targets a 20 base pair sequence of DNA, creates blunt ends after hydrolysis

Restriction endonucleases are produced by _________.

bacteria

Often in commercial plasmids, "newly" inserted DNA will disrupt a gene known as _________ resulting in an easy identification of the bacteria containing the modified plasmid.

beta-galactosidase

The polymerase chain reaction (PCR) can be used to produce __________ of copies in a few hours.

billions

Plasmids are typically _______ DNA molecules that are replicated _________ of the chromosomes in bacteria.

circular, independently

Plasmids are ________ DNA molecules that are typically found in bacteria. Plasmids can be introduced into other host cells by a lab process called _________ or a bacterial process known as _________.

circular, transformation, conjugation

After many PCR cycles, billions of _________ of the target DNA sequence have been synthesized

copies

Hybrids of plasmids and phage vectors, ________ contain the "cos site" of a bacteriophage and many attributes of plasmids, including selectable markers.

cosmids

The PCR reaction is heated to 95° Celsius to begin the next _______

cycle

The target DNA is ____________ by heating to 95° Celsius that results in single strands of nucleic acids.

denatured

Movement of charged molecules in an electrical field, which is used to separate nucleic acid fragments for recombinant DNA work, is called _________.

electrophoresis

To determine if a suspect was present at the scene, a crime scene investigator gathers a small biological sample collected at a murder scene. Which type of PCR would be the best to use for this task?

end point PCR

An enzyme that cleaves internal phosphodiester bonds of a DNA molecule is a(n) _________.

endonuclease

A(n) __________ vector contains promoters that result in high-level transcription of the gene cloned within a multicloning site.

expression

Cloning a gene involves all of the following EXCEPT _________.

expression of the vector and the gene in a cell-free environment

The advantage of homologous recombination is that __________.

following a splice with Cas9, the ends of the chromosome can be seamlessly joined with a piece of donor DNA that has been engineering to contain flanking sequences homologous to the broken chromosome ends

Which of the following was first produced commercially using recombinant DNA technology?

human insulin

In order to express eukaryotic genes in a bacterium, the __________ must first be removed.

introns

The green fluorescent protein is produced in nature by a _________ species.

jellyfish

Plasmid DNA having one EcoRI recognition sequence is treated with EcoRI restriction endonuclease. Following gel electrophoresis, how many bands should be visible on the gel?

one

Artificial chromosomes replicate __________ during each cell cycle.

only once

In order to replicate, plasmids must have a special DNA sequence called the _________ of replication, and those plasmids that can replicated in several different bacteria are called _________ vectors.

origin, shuttle

_________ is a bacterial plasmid vector.

pUC19

In order to insert "new" DNA into plasmids efficiently, commercial plasmids have ____________ sites with several restriction sites allowing easy molecular modifications and insertion.

polylinker

Which of the following components of PCR ensures amplification of the target DNA sequence only, despite the presence of many other DNA sequences?

primers

The PCR reaction is cooled to about 50° Celsius to allow the ______________ to _____________ to the target DNA sequence on the single-stranded DNA molecules.

primers and anneal

A __________ is a DNA molecule used in hybridization reactions to detect the presence of a particular gene in separated DNA fragments.

probe

Restriction endonucleases in bacteria may have evolved in order to _________.

protect the bacteria from infection by viruses

A PCR procedure that allows a determination of the amount of a particular DNA fragment that is present in a sample is called _________.

real-time PCR

Restriction endonuclease

recognize and bind to a palindromic sequence, bind to a sequence 4-8 base pairs long, creates either blunt or sticky ends after hydrolysis

Once inserted into a host bacterial cell, the presence of many plasmids may be revealed by _______________ to various antibiotics.

resistance

Cas9 endonuclease differs from a restriction enzyme in that __________.

restriction enzymes recognize a 4-8bp target sequence in the DNA and directly cleave at that site, whereas Cas9 contains a guide RNA that is complementary to a specific DNA site and a polypeptide that cleaves once hybridization is complete

Complementary DNA (cDNA) probes are produced using _________.

reverse transcriptase

Antibiotics incorporated into the culture medium can _________.

select against organisms that have not incorporated the plasmid

A(n) __________ vector is a plasmid that can be replicated in several different organisms because it has at least one origin of replication that will function in each host.

shuttle

Which of the following is not part of a yeast artificial chromosome (YAC)?

the F factor

When a eukaryotic gene is cloned into a bacterium, the advantage of a complementary DNA (cDNA) gene being used instead of fragments of genomic DNA is that _________.

the introns have been removed from the cDNA gene but not from the genomic fragment

Scientists are using GFP to monitor the expression of genes involved in the infection process of the bean pathogen Colletotrichum lindemuthianum. This is an example of inserting the GFP gene as a ________ fusion in order for it to function as a reporter gene.

transcriptional

As a newly hired molecular biologist for a company that produces genetically modified seeds, your first project is to ensure that a GM cotton plant carrying a bacterial insecticide gene is expressed in tissues of seedlings as well as mature plants. To better understand regulation of the gene, you decide to use GFP fusion to detect activity of the promoter and also use GFP fusion to determine the location of the protein in plants tissues at various stages of growth.

transcriptional; translational

Plasmid cloning vector DNA is usually introduced into bacterial hosts by _________.

transformation

Scientists are using GFP to find where the enzyme urease is located within cells of Proteus mirabilis, a causative agent of urinary tract infections. Use of the GFP gene in this manner is an example of a ________ fusion.

translational

GFP is encoded by a single gene that is easily cloned and expressed in a host organism.

true

The Cas9 nuclease is able to cleave target DNA at a single precise site by __________.

using a guide RNA that hybridizes to a unique sequence on the target DNA molecule

A DNA molecule used to carry a foreign gene into a host organism is called a _________.

vector