Recombinant molecules

When discussing the chromosomal DNA of an organism, the term "genomic equivalent" refers to
the set of genes essentially identical to those of a different organism.
False! This would more accurately be described as "homologous genes" of two organisms.
the amount of DNA necessary to ensure that the DNA contains at least one copy of each gene of the organism.
Correct! This is an important number that is dependent on the size or complexity of the genome.
the amount of DNA that is packaged into each of the sex cells of a diploid organism.
False! This is the haploid set of the chromosomal sequences, only one copy of each of the chromosomal allelic pairs.
a single region of the DNA that is very similar in sequence and genetic function in a different organism.
False! This describes what would more accurately be described as "homologous genes" or "homologous sequences".

When attempting to isolate a specific DNA fragment from a complex mix of chromosomal DNA fragments, the "yield problem" is a concept that refers to
the fact that as a genome increases in size, the yield of any particular DNA fragment represents a decreasing percentage of the total DNA.
Correct! As a genome increases in size, the relative percentage of a particular DNA fragment becomes proportionately smaller, complicating purification of a specific fragment away from others.
the poor recovery of DNA from a band excised from an agarose gel.
False! There is generally a differential yield during elution of a DNA fragment from a gel, with small fragments more easily recovered than large fragments.
the observation that complex genomes do not yield well to digestion with restriction endonucleases.
False! Concentrated DNA solutions often encountered with complex genomes may retard restriction enzyme activity, but this can be corrected by dilution of the DNA or removal of contaminants.
the fact that as a genome decreases in size, the yield of DNA isolated becomes smaller, making small genomes difficult with which to work.
False! As genomes decrease in size, the relative yield of each DNA fragment increases, making smaller genomes easier with which to work.

"Molecular cloning" is a DNA fragment isolation strategy that is dependent on
the ability to easily purify a specific DNA fragment.
False! This strategy was specifically developed to overcome the yield problem and allow rapid purification of DNA fragments that could not be easily purified.
extrachromosomal propagation of DNA molecules.
Correct! Extrachromosomal propogation in a host is a key to amplifying the novel recombinant molecules.
the generation of novel recombinants including DNA fragments from more than one DNA molecule.
Correct! These recombinants overcome the yield problem by increasing the relative percentage of each DNA fragment in the recombinant molecule relative to the relative percentage of the fragment in the total genome.
the use of restriction enzymes and DNA ligases.
Correct! These enzymes allow the generation of the recombinant molecules that are the key to the strategy.

Molecular vectors are DNA molecules that are
able to replicate independently of the host chromosome.
Correct! This attribute is key to the amplification of the constructed recombinant molecules consisting of the vector with one or more extra DNA inserts.
always very small, circular molecules.
False! Many cloning vectors have been developed from the 50,000 bp linear bacteriophage lambda.
must be able to replicate well in the organism whose DNA will be cloned in the vector.
False! Vectors must replicate well in some host organism used for amplification of the recombinant molecules, but the host need not be the source of the DNA inserted into the vector.
are entirely synthetic DNA molecules developed to assist in molecular cloning strategies.
False! Vectors are assembled from pieces of naturally occurring genes with minor sequence modifications to enhance utility of performance of the molecules.

Naturally occurring DNA molecules that have served as the source of the functional genes in vectors include
no natural gene sequences are present in vectors, since they were assembled from entirely synthetic DNA sequences.
False! The majority of the sequences in vectors are derived from natural genes.
bacterial plasmids and episomes.
Correct! These elements provide the basic replication functions for many vectors.
drug resistance genes originally present in transposons.
Correct! These sequences provided the selective markers used to detect the presence of the vector in a host cell.
bacteriophage and viruses.
Correct! These sequences provided both the ability to replicate and a detection method, via formation of plaques of infected cells, for many vectors.
multiple cloning sites that consist of a small region of DNA containing several adjacent, often overlapping recognition sites for different restriction enzymes.
False! These sequences, known as multiple cloning sites (mcs) are present in many vectors, but are the one totally synthetic DNA feature of most vectors.

The replication origins, the DNA sequences that allow vectors to replicate in a cell, are generally
universal and allow the vector to replicate in a wide variety of hosts.
False! Vectors designed to replicate in significantly different hosts (shuttle vectors) must possess more than one origin of replication.
have a limited host range determined by the host range of the DNA sequence that was used in the construction of the vector.
Correct! Most widely used vectors have a rather narrow host range.
able to efficiently transfer from the first host cell to recieve the vector, rapidly spreading throughout the host culture to amplify the desired recombinant.
False! This feature is often true for vectors derived from bacteriophage and viruses, but not for vectors derived from non- infectious or self-transmissible DNA sources.
can be caused to amplify and increase their copy number in a bacterial host cell by the addition of certain antibiotics like chloramphenicol.
False! Although this is true for a limited number of vectors, such as those derived from the bacterial plasmid ColE1, this is not true for many vectors.

The insertional inactivation of a vector phenotypic marker is often used to
allow a vector containing an extra DNA insert to be easily distinguished from a vector containing no added DNA insert.
Correct! The most common use of this principle is based on the inability of a recombinant vector molecule to generate a blue bacterial colony.
allows the selective elimination of host cells that do not contain the vector.
False! This is allowed by the selective marker always present on the vector.
select recombinant molecules by complementation of a chromosomal gene function.
False! Complementation can under some circumstances be used to select specific recombinant molecules.
select a specific size class of DNA insert to be inserted into a vector.
False! Vectors derived from some viruses, such as bacteriophage lambda replacement vectors, can be used to selectively clone specific size classes of DNA fragments.

Vectors derived from the bacteriophage M13 are novel because
only these vectors produce an infectious, packaged version of the DNA.
False! Many vectors derived from viruses, such as bacteriophage lambda derivatives, share this property.
only these vectors are readily identifed in a bacterial host by their conferred antibiotic resistance.
False! Plasmid vectors are generally detected by their conferred antibiotic resistance, while M13 vectors are generally detected by the formation of plaques in a host cell lawn.
these vectors can be induced to make a single-stranded copy of one the strands of a cloned DNA fragment insert.
Correct! M13 and other closely related bacteriophage produce a single-stranded circular DNA that can be used for certain protocols, such as DNA sequence determination.
only these vectors can integrate into the chromosomal DNA and be maintained as an episome, a chromosomal integrate capable of excision and independent replication.
False! Unlike bacteriophage lambda, M13 is not capable of efficient chromosomal integration.

Recombinant molecules that are constructed in vitro can be introduced into host cells by the method of
transduction.
False! This term more accurately applies to a natural transfer of DNA from one host to another, often mediated by non- viral DNA packaged in a virus particle.
transfection.
Correct! Most often used to describe the chemical introduction of DNA derived from a vector that is infectious, such as a bacteriophage or virus.
transformation.
Correct! The most common transformation methods use a combination of chemical treatment and heat to induce a host to take up DNA.
electroporation.
Correct! This method uses electric current to carry DNA into a host cell.

A gene library for an organism is
a listing of all possible genes of that organism.
False! This list could only include all genes with identified phenotypes.
a collection of all the known derivatives of a particular gene (allelic variations) that occur naturally in an organism.
False! This would be a table of allelic variants.
a collection of a sufficient number of recombinant molecules to ensure that all genes of a particular organism are present in the collection.
False! It is not possible not guarantee that all genes will be present in the library, since not all recombinants may be stable in the host used to prepare the collection of recombinants.
 a collection of a sufficient number of recombinant molecules to ensure that all genes of a particular organism statistically calculated to be present in the collection.
Correct! Although the statistical calculation can be used to suggest a desired gene should be present in the collection, the molecule may nevertheless be absent.

A cDNA library consists of
a collection of recombinants containing DNA inserts corresponding to the mRNAs purified from an organism.
Correct! Only those genes that were being expressed at the time of RNA purification will be present in the library.
a collection of recombinants containing the mRNA molecules purified from an organism.
False! The mRNA molecules must be converted to DNA by reverse transcriptase prior to constructing recombinants.
a collection of recombinants containing DNA inserts corresponding to all of the genes that might be expressed by an organism.
False! A cDNA library contains only DNA sequences corresponding to RNA molecules that were being expressed at the time the RNA was purified, generally only a few percent of all possible genes.
a collection of recombinants containing DNA inserts corresponding to all of the RNAs purified from an organism.
False! The sythesis of cDNA by reverse transcriptase requires an RNA template preimed with an oligonucleotide primer; only tose RNA moleculaes that can be primed will be present in a cDNA library.

The RNA structural difference that allows the easy construction of cDNA libraries from eukaryotic but not most prokaryotic cells is
the inability of reverse transcriptase to elongate DNA on a primed prokaryotic mRNA template.
False! Reverse transcriptase will elongate a primed prokaryotic mRNA, but each mRNA will require a specific primer due to the general absence of a 3' poly-A sequence.
the presence of a 5' cap structure on eukaryotic but not prokaryotic mRNA's.
False! This structure does not significantly effect cDNA library construction.
the presence of alternatively spliced eukaryotic RNA molecules.
False! Alternatively processed RNA molecules are more common in eukaryotic than in prokaryotic cells, but this difference is not important to cDNA library construction.
the presence of a 3' poly-A tail on many eukaryotic mRNA's but few prokaryotic mRNA's.
Correct! This structure provides the necessary annealing point for the oligo-dT primer that can be used to prime many different eukaryotic mRNA molecules for the reverse transcriptase cDNA synthesis reaction.

Methods that might be used to screen or serve through a library for a recombinant containing a specific gene include
complementation analysis.
Correct! However, this approach that a host chromosomal mutant defective in the desired activity be transformed with a recombinant that restores the activity, not routinely useful for most genes.
phenotypic screening.
Correct! However, this approach that the production by the recombinant of a phenotypic activity that can be detected in the host organism, not routinely useful for most genes.
hybridization analysis.
Correct! A method of widespread application that requires a hybridization probe, a nucleotide sequence closely related to the gene of interest.
screening with antibodies against the desired gene product.
Correct! However, the recombinants must have been constructed in an expression vector and host system that allows protein to be produced from the cloned DNA inserts, usually as additions to a carrier protein.

The screening procedure in which a hybridization probe is used to examine bacterial transformants for the presence of a specific recombinant is referred to as a
Southern blot.
False! This method uses a nucleotide sequence probe to examine DNA fragments separated by gel electrophoresis, then transferred to and immobilized on a membrane support.
Northern blot.
False! This method uses a nucleotide sequence probe to examine RNA immobilized to a membrane support.
Western blot.
False! This method uses antibodies to examine proteins immobilized to a membrane support.
Eastern blot.
False! No such animal.
colony blot or plaque lift.
Correct! The first refers to screening bacterial colonies, while the second refers to screening viral plaques present in a lawn of host cells.

The screening procedure in which an antibody directed against a desired protein is used to examine bacterial transformants for the presence of a specific recombinant is referred to as a
Southern blot.
False! This method uses a nucleotide sequence probe to examine DNA fragments separated by gel electrophoresis, then transferred to and immobilized on a membrane support.
Northern blot.
False! This method uses a nucleotide sequence probe to examine RNA immobilized to a membrane support.
Western blot or immunoblot.
Correct! This method uses antibodies to examine proteins immobilized to a membrane support.
Eastern blot.
False! No such animal.

Characterization of the recombinant plasmid vector in a bacterial transformant requires that the plasmid be at least partially purified away from bacterial
chromosomal DNA.
Correct! The desired plasmid consists of only a few percent of the total DNA in the cell; contaminating chromosomal DNA will obscure the presence of the plasmid DNA.
RNA.
Correct! Contaminating RNA can inhibit restriction enzyme action on DNA and can obscure plasmid DNA bands during gel electrophoresis.
proteins.
Correct! Protein contaminants can inhibit restriction enzyme characterization and may include DNAse that degrades plasmid DNA during the analysis.
polysachharides
Correct! Many polysaccharides are inhibitors of restriction enzyme activity.

The miniprep or miniscreen procedure, a rapid method of purifying a small amount of plasmid DNA sufficient for restriction enzyme characterization of DNA inserts, takes advantage of which chemical features to preferentially purify plasmid DNA away from bacterial chromosomal DNA?
The much more rapid reannealing of plasmid DNA when transferred from alkaline to acidic or neutral conditions.
Correct! The covalently closed circular DNA renatures very rapidly during the procedure, while the larger chromosomal DNA retains significant amounts of single-stranded structure.
The presence of RNA in association with the bacterial chromosomal DNA.
False! Although RNA is associated with the chromosomal DNA, this is not a major factor during the preferential extraction of plasmid DNA.
The tendency of denatured DNA to form a precipitate in the presence of acetate and the detergent sodium dodecyl sulfate.
Correct! The denaturation/renaturation step in the method leaves the chromosomal DNA in substantially denatured state, and it precipitates in the presence of the SDS-acetate complex while the plasmid DNA remains soluble.
The preferential precipitation of plasmid DNA by isopropyl alcohol.
False! This step mainly precipitates DNA while leaving proteins soluble in the alcohol phase.

In spite of the apparent presence of sufficient numbers of recombinants to statistically ensure that a particular gene should be present in a library, exhaustive screening sometimes fails to identify the desired recombinant. This can be explained because
the expression protein or RNA products from certain cloned DNA fragments can be lethal to the host cell.
Correct! The expression of a cloned lysozyme gene, for example, would kill a bacterial host cell.
some DNA fragments are incapable of replication in the host cell.
Correct! Bacterial DNA replication enzymes, for example, cannot always efficiently replicate eukaryotic DNA sequences.
some DNA fragments are instable in the host cell and rapidly recombine or degrade to different forms.
Correct! The repetetive sequence elements common in eukaryotic DNA are prone to recombination when propagated in bacterial hosts.
the non-random sequence of DNA may prevent the appearance of some fragments in a library.
Correct! The absence of restriction enzyme cleavage sites used in library construction might prevent the generation of a clonable DNA fragment that contains the desired gene.

The variety of cloning vectors that has been developed allows the molecular cloning and amplification of DNA fragments in
only bacterial hosts.
False! Cloning systems also exist for a wide variety of microbial eukaryotic and higher eukaryotic hosts.
only microbial hosts.
False! Cloning systems also exist for a wide variety of eukaryotic hosts.
only animal cells.
False! Most routine DNA cloning continues to be performed in microbial systems.
only plant cells.
False! The first man-made cloning systems were developed in the bacterium Escherichia coli, although many scientists have described the Ti plasmid present in strains of Agrobacterium tumefaciens as the first natural cloning system for inserting foreign DNA into plants.
a wide variety of both prokaryotic and eukaryotic cells.
Correct! DNA can be cloned and manipulated in many different cell types, from bacteria, yeast, and other microbes through a wide range of eukaryotic cell types, including both plant and animal species.

In comparison to natural recombination processes, the primary advantages of the methodology called "recombinant DNA" include
the ability to efficiently recombine DNA from different organisms.
Correct! It has been said that all recombinants created in a tube via cloning techniques can also be created in vivo, but the efficiency of the natural process is so poor that identification and isolation of the desired recombinant would be too prohibitive for practical use.
the ability to rapidly select and applify a desired DNA fragment.
False! Although this technology is powerful, the construction of a library, screening for a desired DNA sequence, and isolation of the desired recombinant can be both time-consuming and labor-intensive.
the ability to isolate a desired DNA fragment away from other, undesired DNA fragments.
Correct! The technology overcomes the problem of isolating a single DNA fragment away from other DNA fragments of a genome.
a degree of precision that allows recombination to be directed at the level of individual nucleotide bases.
Correct! Natural recombination process appear to be dependent of regions of homology, or sequence similarity, while recombinant DNA technology directs recombination at the level of restriction enzyme nucleotide sequence cleavage sites.