Question 1

One of the problems with plasmids like pUR278 is that there is no true marker for determining whether or not the plasmid contains the inserted gene since the cloning site does not lie within any specific gene. Which of the following would be an extremely useful tool for rapid selection of those bacteria that have taken up a plasmid with the desired DNA insert?

Accepted Answer

Including the gene for a fluorescent protein such as GFP in the inserted DNA would allow for rapid selection of bacteria that have taken up the plasmid with the desired DNA insert, as the bacteria expressing the fluorescent protein can be easily identified by fluorescence microscopy. The other options either do not provide a direct way to identify bacteria with the desired DNA insert or can result in selection of bacteria with plasmids that do not contain the desired insert.

Question 2

Reporter genes such as those for green fluorescent protein (GFP) are found on many commercial plasmids. These genes are useful to determine:

Accepted Answer

A

Question 3

The correct sequence for colony hybridization experiments is:
A.A replica of the bacterial colonies is obtained on an absorbent disC.
B.Autoradiography of the disc reveals probe complementary DNA.
C.Host bacteria with plasmid are plated and allowed to grow overnight.
D.The disc is treated with alkali.
E.The disc is reacted with labeled probe.

Accepted Answer

The correct sequence involves plating host bacteria with plasmid (C), obtaining a replica on an absorbent disc (A), treating the disc with alkali to denature DNA (D), reacting the disc with a labeled probe (E), and finally autoradiography to reveal probe complementary DNA (B).

Question 4

A genomic DNA library is:

Accepted Answer

A genomic DNA library is a collection of clones containing a representative sample of all the DNA sequences from a particular organism. These clones collectively represent the entire genome of the organism, hence the name "genomic DNA library." Option A is incorrect because short fragments of DNA would not be sufficient to represent the entire genome. Option B describes a different technique called oligonucleotide hybridization. Option D describes a DNA probe used to detect a specific sequence, not a library. Option E describes a plasmid, not a library.

Question 5

If the pBR322 were treated with PstI in order to insert a gene into the plasmid, which of the following would be correct regarding cells that were transformed with the resulting vector?

Accepted Answer

PstI cuts the pBR322 plasmid between the ampicillin and tetracycline resistance genes, creating two fragments with each of the resistance genes on a separate fragment. After insertion of the gene of interest, cells transform with the plasmid will only be resistant to tetracycline since the ampicillin resistance gene is on the other fragment that was not inserted. Therefore, cells that contain the desired vector would be resistant to tetracycline but sensitive to ampicillin.

Question 6

The advantages of using the pET plasmid system include ____.

Accepted Answer

The pET plasmid system has the advantage of genes being under the control of a T7 promoter (choice A) and the T7 polymerase being under the control of the lac operon and only active when induced by a specific agent such as IPTG (choice D). The other choices (B and C) are not advantages of using the pET plasmid system.

Question 7

The correct sequence of procedures in the Southern blotting (hybridization) technique is:
A.hybridization with radioactive probE.
B.agarose gel electrophoresis and visualize bands.
C.transfer (blot) to nitrocellulose filter.
D.digest DNA with restriction nucleases.
E.expose filter to X-ray film, develop and observe.

Accepted Answer

Southern blotting involves digesting DNA with restriction enzymes, separating the fragments by agarose gel electrophoresis, transferring them to a nitrocellulose filter, hybridizing with a radioactive probe, and then detecting the probe by exposing the filter to X-ray film.

Question 8

All are characteristic of plasmids EXCEPT:

Accepted Answer

Plasmids cannot perpetuate themselves without a host organism as they require host cell machinery to replicate and express their genes.

Question 9

Shuttle vectors have the property that they:

Accepted Answer

Shuttle vectors are designed to replicate in two different types of cells, usually bacteria and yeast. This allows for the manipulation of genes in one organism and the ability to express them in another organism. Shuttle vectors typically contain an origin of replication for each of the two cell types, as well as selectable markers such as antibiotic resistance genes. They may also contain promoters for the expression of the gene of interest. However, the ability to incorporate large DNA fragments or the presence of more than one antibiotic resistance gene are not necessarily characteristics of shuttle vectors.

Question 10

All are features of a plasmid useful for cloning EXCEPT:

Accepted Answer

A expressed sequence tag (EST) is a short sequence of a cDNA that's useful for identifying genes or studying their expression, but it is not directly involved in cloning. All other choices - cloning site, replicator, and selectable marker - are essential features of a plasmid used for cloning.

Question 11

All are true for cDNA libraries EXCEPT:

Accepted Answer

Oligo (dT)-cellulose chromatography, not oligo (dA), is used to isolate mRNA templates by binding to the poly-A tail of mRNA.

Question 12

To position a gene in an expression vector downstream from a promoter (i.e., perform directional cloning), it is best to:

Accepted Answer

Restricting the gene and the plasmid with different endonucleases creates sticky ends that are complementary to each other and allow for directional cloning of the gene into the plasmid. This method ensures that the gene is inserted in the correct orientation relative to the promoter.

Question 13

Looking at all of the genes that are activated during a major metabolic shift or during embryogenesis and development of organisms is called:

Accepted Answer

The term functional genomics is used to describe the study of the entire set of genes within an organism and their function. This includes the analysis of all genes that are activated during major metabolic shifts or during embryogenesis and development of organisms. Fractional expression and genetic fractionation are not commonly used terms in genomics, and mutagenesis refers to the process of deliberately inducing mutations in genes.

Question 14

To express a eukaryotic protein in E. coli, the eukaryotic cDNA must be cloned in a(n) ____ that contains ____ and ____.

Accepted Answer

To express a eukaryotic protein in E. coli, an expression vector is required that contains a promoter to initiate transcription of the cDNA and a ribosomal-binding site to start translation of the mRNA into protein. Choice B refers to hybridization, which is not applicable in this context. Choice C refers to the components of mRNA and not an expression vector. Choice D refers to the structure of eukaryotic genes and not an expression vector.

Question 15

In the Southern hybridization procedure, the gel after electrophoresis is treated with NaOH and then neutralized before blotting. What is the primary function of the alkaline treatment?

Accepted Answer

The primary function of the alkaline treatment with NaOH in Southern hybridization is to denature the double-stranded DNA into single-stranded DNA (ssDNA). This is necessary for hybridization with a labeled probe. The NaOH treatment breaks the hydrogen bonds between the base pairs, leading to a separation of the double-stranded molecule into single-stranded DNA molecules, which are then transferred onto the blotting membrane.

Question 16

All are steps in the sequence for construction of a chimeric plasmid EXCEPT:

Accepted Answer

Reannealing the ends of the vector back together would negate the purpose of inserting foreign DNA, as it would close the plasmid without incorporating the new DNA segment.

Question 17

If DNA fragments of about 4 kb are to be cloned, which vector would be most useful?

Accepted Answer

Plasmids are suitable vectors for cloning DNA fragments of about 4 kb, as they can typically accommodate inserts up to 10 kb.

Question 18

A method used to insert or transform cells with a plasmid is to:

Accepted Answer

Treating the bacteria with Ca²⁺ (a method known as calcium chloride transformation) helps to make the cell membrane more permeable, facilitating the uptake of plasmids. Briefly heating the bacteria to 42°C helps to further increase permeability and promote plasmid uptake. This method is commonly used for transformation of bacteria such as E. coli.

Question 19

Hybrid proteins or fusion proteins are produced by:

Accepted Answer

Hybrid proteins or fusion proteins are produced by translation of recombinant sequences from expression vectors carrying cDNA inserts cloned directly into the coding sequence of a vector-born protein-coding gene. This describes the process of creating a fusion protein using recombinant DNA technology. The other options do not accurately describe the process of producing fusion proteins.

Question 20

All are characteristic of YACs (yeast artificial chromosomes) EXCEPT:

Accepted Answer

All of the statements in the given options are true and characteristic of YACs.

Quiz 12: Recombinant Dna, Cloning, Chimeric Genes, and Synthetic Biology