Restriction enzymes and characterizing DNA.

A genetic map is
a listing of all possible genes of an organism.
False!  Only those genes identified by the existence of mutations can be positioned on a genetic map.
a relative positioning of genes relative to each other as defined by the genetic nearness of mutations to one another.
Correct!  A genetic map consists of a relative positioning of genes as defined by linkage analysis of mutations.
an absolute correlation of genes with their locations on chromosomal DNA.
False!  Distances on a genetic map are actually relative and do not generally have absolute correlation with DNA distance.
a listing of only the genes that have been identified by mutations.
False!  A genetic map involves positioning those genes that have been identified by mutation analysis.

A restriction map is
a positioning of genes on the chromosomal DNA molecule relative to restriction sites in the DNA.
False!  This positioning involves correlation of the genetic map with the restriction map.
a map that shows the locations of genes that are not subject to mutation and whose analysis is subsequently restricted.
False!  The locations of genes that are not subject to mutation are very difficult to identify.
the positioning relative to each other of restriction sites in a DNA molecule.
Correct!  A restriction map provides the locations of restriction sites for use as reference points along a DNA molecule.
a listing of all known restriction enzymes and the sequences those enzymes recognize and cleave.
False!  However, tables of restriction enzyme recognition sites are widely available in enzyme supply catalogs.

A restriction map must always be based on
the location of the beginning of the gene of interest.
False!  A restriction map can be established relative to the position of any cleavage site, most frequently to a site that occurs only once in the DNA of interest.
the orientation of the DNA fragment that codes for the gene of interest.
False!  A restriction map is often completed prior to the identification of the precise location of a gene.
the exact distance between restriction sites.
False!  The maps are generally established using gel electrophoresis, which provides only an estimate of DNA fragment size.
restriction enzymes that cleave within the DNA fragment of interest.
Correct!  Enzymes that do not cleave within a DNA fragment are often mentioned in the context of such a map, but do not help order the fragments generated by enzymes that cleave the DNA fragment.

Preparing a restriction map of a DNA fragment that includes two different restriction enzymes ResA and ResB involves
digesting the DNA separately with ResA and ResB and comparing the sizes of the resulting DNA fragments.
False!  This is only part of the process.
digesting the DNA simultaeneously with ResA and ResB and comparing the sizes of the resulting DNA fragments.
False!  This double-digestion must be compared with digestion by each of the enzymes alone.
digesting the DNA both separately with ResA and ResB and simultaeneously with ResA and ResB and comparing the sizes of the resulting DNA fragments.
Correct!  The relative positions of the restriction sites must be able to account for the sizes of the DNA fragments observed in each of the digestion conditions.
determining the nucleotide sequence of the DNA fragment, then using a computer program to locate the cleavage sites for ResA and ResB.
False!  This process is much more complicated and generates a very precise nucleotide sequence map.

DNA fragments generated by restriction enzymes are often examined by gel electrophoresis, a process based on the principle that
small DNA fragments move faster than large DNA fragments in an electric field.
False!  Because of a constant size-to-mass ratio, all DNA fragments move at about the same rate in an electric field.
small DNA fragments move slower than large DNA fragments in an electric field.
False!  Because of a constant size-to-mass ratio, all DNA fragments move at about the same rate in an electric field.
in an electric field, small DNA fragments move faster through a gel matrix than do large DNA fragments.
Correct!  The matrix of the gel forms a sieve that allows the smaller, more flexible fragments to move faster.
in an electric field, small DNA fragments move slower through a gel matrix than do large DNA fragments.
False!  The matrix of the gel forms a sieve that allows the smaller, more flexible fragments to move faster.

The major functional difference between agarose and polyacrylamide gels is
the higher pH at which the polyacrylamide gels must be run.
False!  Both types of gel perform well under a variety of pH conditions.
the higher temperatures at which the agarose gels can be run.
False!  High temperatures will melt agarose.
the greater size dimensions of agarose gels.
False!  Both types of gel exist in a wide range of size formats.
the smaller matrix pores of the polyacrylamide gel.
Correct!  The smaller pores of the polyacrylamide gel are capable of resolving differences of a single base pair in total length, making it more useful for examining smaller DNA fragments.

DNA fragments in an agarose or a polyacrylamide gel can be visualized by
direct examination of the gel under the correct wavelength of visible light.
False!  DNA in a gel is not generally visible under ordinary light.
direct examination of the gel under the correct wavelength of ultraviolet light.
False!  Although DNA does have an absorption maximum around 256nm, the amounts of DNA present in a gel are generally insufficient to allow direct detection.
staining the DNA in the gel with certain dye compounds, followed by direct examination under the correct wavelength of visible light.
Correct!  Several stains, such as methylene blue and silver stain, allow detection of DNA with visible light, although these stains are generally not as sensitive as other methods.
staining the DNA in the gel with intercalating compounds, followed by examination under the correct wavelength of ultraviolet light.
Correct!  Staining with ethidium bromide followed by visualization under ultraviolet light is a common, sensitive method of detecting DNA fragments in a gel.

The relationship between DNA fragment size and mobility in an agarose gel is best described as a
linear relationship between size and mobility.
False!  The relationship is much closer to logarithmic.
an approximately logarithimic relationship between size and mobility.
Correct!  This relationship does not always accurately describe mobility of very large and very small fragments in the gel.
strongly dependent on the concentration of agarose in the gel.
False!  Although large DNA fragments move more rapidly through gels with a low concentration of agarose, the relationship remains best approximated as a logarithmic between size and mobility.
independent of the voltage and current conditions used during electrophoresis.
False!  These parameters can strongly influence the relative mobilities of DNA, particularly circular DNA molecules.

The purpose of the presence of a DNA fragment size standard, such as bacteriophage lambda DNA digested with HindIII, is
to provide a set of DNA fragments of known sizes whose mobility can be measured and graphed to provide a standard curve.
Correct!  Since the conditions in each gel may be slightly different, such standards should always be present when estimation of the size of unknown DNA fragments is desired. a
practice encouraged by suppliers of DNA standards to ensure continued product sales.
False!  The standards need not be commercially supplied, but can be any DNA sample routinely used for reference.
to verify that each gel has performed as expected without error in gel preparation or electrophoresis conditions.
Correct!  Any variation in the expected performance of the DNA standard indicates some unanticipated problem. a
practice that should only be followed when the sizes of unknown DNA fragments on the gel must be determined.
False!  DNA standards are also used to verify that each gel has performed as expected.

The relative mobility of DNA in an agarose gel is most strongly dependent on the
percent GC composition of the DNA molecule.
False!  Differences in GC composition can affect mobility of fragments in polyacrylamige gels, but are rarely a significant influence in agarose gels.
the size of the DNA molecule.
False!  Conformation also plays a large role, as a convalently closed circular (supercoiled) DNA will move much faster than a relaxed or open circular DNA of the same size.
both the size and conformation of the DNA molecule.
Correct!  Both factors strongly influence mobility; a large supercoiled DNA can move faster than some smaller linear DNA fragments.
the pH of the electrophoresis buffer in the gel.
False!  Changes in buffer will generally affect all DNA fragments in a similar manner.

Gel electrophoresis reveals that a 4000 bp DNA fragment digested with EcoRI gives fragments of 1000 bp and 3000 bp and the same apparent fragments when digested with HindIII. The best description of the restriction map of this fragment would be that
the EcoRI and HindIII sites occur very near each other 1000 bp from the left end of the fragment.
False!  These sites cannot be positioned relative to each other without a double digest of the DNA fragment with both enzymes.
the EcoRI and HindIII sites occur very near each other 1000 bp from the right end of the fragment.
False!  These sites cannot be positioned relative to each other without a double digest of the DNA fragment with both enzymes.
nothing can be determined about the restriction map of this DNA fragment.
False!  It is correct to say that the restriction map of this fragment contains an EcoRI site 1000 bp from one end of the DNA fragment and a HindIII site 1000 bp from one end of the fragment.
the restriction map of this fragment contains an EcoRI site 1000 bp from one end of the DNA fragment and a HindIII site 1000 bp from one end of the fragment.
Correct!  Note that the position of these sites relative to each other cannot be determined without a double digest of the DNA fragment with both enzymes.
the restriction map of this fragment contains an EcoRI site 1000 bp from one end of the DNA fragment and a HindIII site 1000 bp from the other end of the fragment.
False!  These sites cannot be positioned relative to each other without a double digest of the DNA fragment with both enzymes.

Agarose gel electrophoresis reveals that a 2000 bp covalently closed circular DNA molecule is converted to a 2000 bp linear DNA band when digested with EcoRI but appears as a 1000 bp band when digested with HindIII. This might be adequately explained by
the presence of 2 HindIII located on opposite sides of the circular molecule, giving rise to two 1000 bp fragments that migrate as a single band.
Correct!  Agarose gels do not adequately resolve bands of very similar size.
the presence of many HindIII site, two of which generate a 1000 bp fragment, while the others generate many small fragments that are not revealed by the agarose gel.
Correct!  Small fragments are not always revealed due to weak staining intensity, ability to run faster than the tracking dye, and diffusion of the band as the electrophoresis procedes.
the use of an enzyme other the HindIII.
False!  Although this might have occurred, it does not account for the size estimate relative to the fragment observed with EcoRI.

As the same of the genome of an organism increases, the number of DNA fragments generated by digestion with restriction enzymes and detected by gel electrophoresis will generally
increase.
Correct!  However, the DNA fragments obtained from large genomes merge into a blur of many fragments of similar size with poor individual resolution.
decrease.
False!  The number of restriction fragments is roughly proportional to the size of the genome.
stay about the same regardless of the size of the genome.
False!  The number of restriction fragments is roughly proportional to the size of the genome.
will always allow resolution of all of the individual fragments that together comprise the genome.
False!  As the genome size increases, the number of restriction fragments increases until it becomes impossible to distinguish all of the individual DNA bands that comprise the total genome.

The preparation of detailed restriction maps can entail the digestion of a DNA sample under salt or pH conditions that are not optimal for a particular restriction enzyme, a practice that is relevant to the "star" activities of certain restriction endonucleases. These "star" activities can be described as
irrelevant because they do not interfere with accurate generation of restriction maps.
False!  Uncontrolled "star" activities can cause the disappearance of major DNA bands and the appearance of non- stoichiometric levels of minor bands.
present in enzymes that work in a wide range of digestion conditions and are resistant to digestion artefacts.
False!  "Star" activities increase the frequency of cleavage and greatly increase digestion artefacts.
the recognition and cleavage of nucleotide sequences larger and more specific than the normal recognition site.
False!  "Star" activities are less specific.
the recognition and cleavage of nucleotide sequences smaller and less specific than the normal recognition site.
Correct!  "Star" activities often allow an enzyme that normally recognizes a 6-bp cleavage site to recognize the central 4 bp, with greatly increased frequency of DNA cleavage.

The statistical frequency of the occurence of a particular restriction enzyme cleavage site that is 6 bases long can be estimated to be approximately
once every 4096 bases.
Correct!  The formula for calculating the estimated frequency is 1/4N where N=number of bases in recognition site.
once every 1024 bases.
False!  The formula for calculating the estimated frequency is 1/4N where N=number of bases in recognition site.
once every 256 bases.
False!  The formula for calculating the estimated frequency is 1/4N where N=number of bases in recognition site.
once every 24 bases.
False!  The formula for calculating the estimated frequency is 1/4N where N=number of bases in recognition site.

The restriction enzymes that are most useful for mapping very large DNA fragments are
enzymes with cleavage activity that is inhibited by DNA methylation.
False!  Most restriction enzymes fall into this category.
enzymes with large (greater than six bp) recognition sites.
Correct!  Large recognition sites result in the generation of only a few large DNA fragments that are relatively easy to place in the correct order.
enzymes with small (less than six bp) recognition sites.
False!  Small recognition sites result in the generation of too many small DNA fragments for adequate ordering of the fragments.
those enzymes that are commercially available.
False!  Although many enzymes are commercially available, some of those with the most specific cleavage sites, and hence the greatest utility in mapping large DNA fragments, are not.

The enzymes EcoRI (GAATTC) and HindIII (AAGCTT) both recognize a sequence containing two A's, two T's, one G, and one C, yet it is possible to isolate natural pieces of DNA that are thousands of base pairs long and are cleaved many times by one enzyme but not at all by the other. This might be explained by
the isolation of the DNA from a host that contains a methylase that modifies the DNA and prevents cleavage by one of the enzymes.
Correct!  This occurs frequently with DNA isolated from certain hosts, such as bacteria and plants.
the fact that biological restraints may exclude the use of certain nucleotide sequences in some DNA molecules.
Correct!  It has been observed that genes that are naturally able to transfer among a wide variety of bacterial hosts are frequently deficient in common 6-base restriction enzyme cleavage sites.
the observation that some nucleotide sequences may occur preferentially in certain DNA sequences.
Correct!  Satellite sequences, for example, are often composed of a short nucleotide sequence that (such as AAAGCTTT) is repeated hundreds of times in a DNA molecule, causing a clustering of any restriction site that is contained within the repeated sequence.

Features that are important to the use of Type II restriction enzymes for mapping of DNA sequences include
specificity of recognition and cleavage.
Correct!  Specificity is perhaps the key feature of these enzymes.
the short cohesive DNA termini generated by digestion.
False!  The nature of the terminal is generally of no consequence to routine restriction mapping.
relative ease of purification and stability of the proteins.
Correct!  This enables extensive commercial availability at reasonable cost.
the ability to reseal the termini to make new DNA molecules.
False!  This is a key feature of molecular cloning or recombinant DNA technology but not necessary for molecular mapping.

The wide variety of sequence recognition and cleavage specificity that is currently commercially available is a
result of the genetic engineering of enzyme specificity by the companies that sell restriction enzymes.
False!  The companies may engineer protein production levels, but generally work with naturally occuring restriction enzymes. a
result of the chemical or proteolytic modification of enzyme specificity by the companies that sell restriction enzymes.
False!  The most notable DNA enzyme exception to this statement is Klenow fragment of DNA polymerase I, which was at one time the result of proteolytic treatment of DNA polymerase I and is now produced from engineered DNA polymerase I genes.
due to the wide range of specificity observed in naturally occurring restriction enzymes.
Correct!  Virtually all commercially available restriction enzymes are derived from natural organisms, most frequently bacteria.