The Polymerase Chain Reaction

The Polymerase Chain Reaction, or PCR, absolutely requires
a single oligonucleotide synthesis primer and a DNA template.
False! PCR requires at least two opposing primers.
at least two oligonucleotide synthesis primers and a DNA template.
Correct!
a single oligonucleotide synthesis primer and a DNA or RNA template.
False! PCR of an RNA molecule requires the prerequisite synthesis of a cDNA template molecule.
at least two oligonucleotide synthesis primers and a DNA or RNA template.
False! PCR of an RNA molecule requires the prerequisite synthesis of a cDNA template molecule.

When PCR involves two different oligonucleotide synthesis primers, a successful amplification requires that the primers be oriented
in an opposing fashion to direct DNA synthesis towards each other.
Correct! The opposing primers are critical to the reaction.
so that DNA synthesis is directed in the same orientation from both primers.
False! This would result in linear synthesis of two different copies of only one template strand.
so that DNA synthesis is directed outwards or in opposite directions from the opposing primers.
False! The gap between the two primers would never be copied during the synthesis reaction.
in any desired manner, since primer orientation is not significant to PCR.
False! Primer orientation is critical to PCR.

Since the nucleotide sequence of the oligonucleotide PCR primers is always known before the reaction is started, the temperature at which a PCR reaction is performed
is not critical.
False! Low PCR temperatures can result in spurious priming and amplification of undesired DNA fragments.
is determined by the source of the polymerase used in the PCR reaction.
False! The different polymerases used in PCR reactions are typically active over very similar temperature ranges and are generally interchangeable.
can be calculated to attempt to minimize undesired amplification products.
Correct! These calculations are, however, only an estimate and reactions must often be empirically optimized for both temperature and ionic conditions.
can be used to manipulate the specificity of the mixture of amplification products.
Correct! Lower annealing temperatures allow less specific priming, which increases the number of different DNA fragments obtained from PCR of a mixed DNA template.

One of the great powers of PCR for the isolation of DNA fragments is that
the desired fragment can be readily detected when present at an extremely low level in a mixture of DNA molecules.
Correct! PCR is so sensitive that it can essentially detect and amplify a single DNA molecule.
no nucleotide sequence information about the desired DNA fragment must be known prior to PCR.
False! The oligonucleotide PCR primers must be designed to specifically anneal to and flank the desired or target DNA region, so some nucleotide sequence information about the oligonucleotide primer annealing sites must at least be known prior to primer design.
oligonucleotide PCR primers can be designed to add essentially any desired nucleotide sequences to the flanking regions of the amplified DNA.
Correct! Any 5' nucleotide sequence can be added to a PCR primer as long as the 3' region of the primer retains sufficient target sequence homology to anneal and initiate the synthesis reaction.
the amplification reaction is always so specific that it yields only the desired DNA fragment.
False! The product specificity of the reaction is dependent on the sequence of the primers, the complexity of the target DNA sample, and the temperature conditions of the reaction.

Gel electrophoresis detection of DNA fragments amplified by PCR differs from detection of DNA fragments amplified by standard restriction enzyme cloning methodology in that
the PCR amplification is so great that the fragments can be viewed directly without any staining applied.
False! Stains like ethidium bromide must still be used to view the amplified fragments.
PCR DNA samples always reveal a single DNA band for a desired gene, while the cloned DNA that corresponds to the same gene always reveal complex band patterns.
False! Gel electrophoresis can reveal PCR band patterns that are as complex, and at times more complex, than the cloned gene, due to the presence of spurious PCR products.
the PCR samples are free of the DNA vector bands that are always associated with cloned genes.
Correct! The PCR amplification process does not require the use of a vector, which is an important aspect of the traditional cloning process.
unlike traditionally cloned DNA fragments, the amplified fragments can easily be excised and recovered from the gel.
False! Both types of DNA fragments can be excised from a gel, although the level of contamination may be lower in the band obtained from the PCR.

The gel electrophoresis DNA patterns known as DNA fingerprints generated either by restriction enzymes or by PCR
are based on the examination of very different aspects of DNA structure.
False! Both methods really measure what might be called microsequence variation, slight differences in nucleotide sequence that can be used to distinguish two DNA molecules without complete sequence analysis of both molecules.
can always be distinguished from one another because of the simple band pattern of the PCR fingerprint.
False! The use of degenerate primers on a complex template may reveal a very complex PCR fingerprint, while restriction of a simple DNA molecule (such as a plasmid or viral DNA) may reveal a very simple DNA fingerprint.
are similar in that they provide a limited amount of information about the nucleotide sequences examined.
Correct! Band differences in restriction fingerprints are caused by the presence or absence of restriction cleavage sites, while PCR fingerprints generally measure the distances between primer annealing sites, yet neither method addresses the total nucleotide sequence of the DNA examined.
are very similar in their sensitivity to contaminating DNA.
False! Contaminating DNA can be present at fairly high levels before detection in a restriction enzyme fingerprint, while contaminating DNA can often be detected at very low levels in a PCR fingerprint.

The ability of hybridization analysis to detect a specific DNA molecule present at a low level in a mixed DNA sample is limited by
only the activity of the hybridization probe (the amount of probe necessary to detect a single molecule).
False! The complexity of the DNA sample is also an important consideration.
only the amount of DNA that can be loaded on the gel electrophoresis system used.
False! The activity of the probe, the complexity of the DNA sample, and the limits of the gel system are all important.
the amount of hybridization probe necessary to detect a single molecule combined with the amount of DNA that can be loaded on the gel electrophoresis system used.
Correct! Detection an also be limited by the gel electrophoresis sytem used.
only by the relative complexity of the mixed DNA sample.
False! Detection sensitivity is also affected by activity of the hybridization probe.

The ability of PCR to detect a specific DNA molecule present at a low level in a mixed DNA sample is limited by
no factors, since PCR is sensitive enough to detect a target molecule at infinite dilutions.
False! PCR is limited by the absolute requirement for the presence of the template molecule; a DNA sample that contains only one of the desired target templates can be divided into two PCR reactions, only one of which will yield the desired PCR product.
the activity of the hybridization probe (the amount of probe necessary to detect a single molecule) used to detect the PCR product.
False! PCR products are generally amplified to such a high level that they are easily detected by any one of several methods.
only the amount of DNA that can be loaded on the gel electrophoresis system used to detect the PCR product.
False! PCR products are generally amplified to such a high level that they are easily detected by gel electrophoresis combined with hybridization analysis to enhance specificity of resolution.
the relative frequency of the target molecule in the mixed DNA sample.
Correct! PCR is limited by the absolute requirement for the presence of the template molecule; a DNA sample that contains only one of the desired target templates can be divided into two PCR reactions, only one of which will yield the desired PCR product.

In comparison to the traditional DNA cloning/gene library screening approach, one of the great strengths of PCR for isolation of a desired gene is
the independence of any pre-existing sequence information about the desired gene.
False! The PCR primers must target the desired gene, either by design of the primers based on some sequence information pertinent to the desired gene or by association of primers with a desired genetic feature.
the greater speed of the PCR approach.
Correct! Cloning and screening that may have required months with conventional methods can often be accomplished in days with PCR strategies.
the greatly reduced cost of PCR in comparison to conventional cloning technology.
Correct! Although some might question differences in the technical costs of the PCR strategy for isolation of a gene, the reduced time requirement alone often generates significant labor cost savings.
that a single PCR reaction will usually provide amplified material for isolation of several unrelated genes.
False! This feature is a great advantage of a conventional gene library that contains many unrelated genes, while a PCR reaction typically contains only a subset of genes related by virtue of the common sequences of the annealing points of the PCR primers.

When a PCR examination reveals the identical DNA fingerprint patterns for two DNA samples, one obtained from a semen sample obtained from a rape/murder victim and the other from a blood sample obtained from a suspect observed with the victim prior to the crime, an appropriate conclusion would be that
the two DNA samples are sufficiently similar to yield the same PCR DNA fingerprint.
Correct! A PCR fingerprint examines only the relative sizes of the PCR products; two different DNA samples may yield essentially identical PCR fingerprints.
the two DNA samples are identical.
False! A PCR fingerprint examines only the relative sizes of the PCR products; two different DNA samples may yield essentially identical PCR fingerprints.
the two DNA samples are completely unrelated.
False! The two samples are sufficiently similar to yield the same PCR fingerprint, implying some genetic similarity.

When a PCR examination reveals two similar, yet clearly not identical DNA fingerprint patterns for two DNA samples, one obtained from a semen sample obtained from the body of a rape/murder victim buried in the woods for 6 months and the other from a blood sample obtained from a suspect who had confessed the crime and led authorities to the grave of the victim, an appropriate conclusion would be that
the suspect must be guilty, since the confession and knowledge of the grave should override the conflicting DNA fingerprint.
False! The contradictory DNA evidence might be a result of contamination or a degradation artefact.
the suspect cannot be guilty, since the PCR fingerprint proves that the semen sample was not from the suspect.
False! The semen sample was subject to both potential contamination and degradation prior to analysis, either of which could account for pattern differences.
the suspect might be guilty, but might not.
Correct! Knowledge of the location of the grave indicates only that the suspect knew of the location, but does not prove involvement in the rape/murder; the DNA differences might prove innocence, yet might also be a result of artefact, contamination, DNA degradation, or technical error.

When a gene fragment is PCR amplified from a single individual, then the amplified product cloned and several isolates sequenced, it is common to observe variation in the gene sequence at one or more locations throughout the region amplified. This variation might be explained by
the existence in the DNA sample of more than one allele of the amplified region.
Correct! Allelic sequence variation can be revealed by sequence analysis of several independent isolates.
the introduction of sequence errors during the amplification process.
Correct! Some polymerases used in PCR are inherently error-prone and introduce sequence changes, while some DNA sequences may be themselves error-prone during PCR.
technical errors made during the nucleotide sequence analysis.
Possible! With the increasing automation of nucleotide sequence analysis, reading errors become less likely.
the use of degenerate primers that themselves introduce errors.
False! The primers should not be considered as part of the gene sequence, since they were chemically synthesized and used to direct synthesis of an internal portion of the desired gene.

The use of "nested" primers during the PCR amplification of DNA from an mRNA population is a strategy that can be used to
increase the variety of cDNA molecules amplified.
False! This strategy increases the specificity of the PCR product.
increase the average size of cDNA molecules amplified.
False! The product size is generally smaller than might be obtained without nesting, but the product is more specific.
increase the specificity of the PCR reaction and minimize undesired amplification products.
Correct! This can be particularly valuable when amplifying isozymes or members of gene families.
increase the total yield of the PCR reaction.
False! The nested primers often decrease the total yield while increasing the product specificity.

The use of two sets of primers to amplify two adjacent and overlapping DNA fragments
can increase the average size of the resulting PCR product.
Correct! The initially amplified smaller fragments can anneal in the overlapping region to prime and extend one another, acting as "megaprimers" for the synthesis of a larger fragment.
will always increase the specificity of the PCR product.
False! Design of the internal, overlapping primers is critical to minimize the production of amplified products that are the result of the unintentional overlap and extension of otherwise unrelated sequences.
can introduce specific mutations within an amplified DNA fragment.
Correct! Mutations can be constructed in the internal, overlapping primers, thereby introducing specific internal changes in the PCR product.
Must always be performed in two separate reactions to first PCR the "left" and "right" portions of the desired molecule, followed by a reaction to extend and fuse the two half-products.
False! Such reactions can be performed in a single PCR by using limiting amounts of the overlapping internal primers and excess amounts of the flanking primers that amplify the desired final large product.

Information that can be used to help design PCR primers for amplification and isolation of a gene from an organism includes
the amino acid sequence of the protein encoded by the desired gene.
Correct! Since the codons that correspond to each of the amino acids are known, an amino acid sequence can be "reverse- translated" to generate nucleotide PCR primer sequences that might encode those regions of the desired protein.
the nucleotide sequence of a previously characterized and equivalent gene from a different organism.
Correct! This is particularly advantageous when the equivalent genes have been isolated from several different organsims and characterized and can be aligned to reveal regions with a high level of nucleotide sequence identity.
the nucleotide sequence of a previously characterized and closely related gene from the same or a different organism.
Correct! This is particularly advantageous when several related genes have been characterized and can be aligned to reveal regions with a high level of nucleotide sequence identity.
the knowledge that the transcript encoding the desired gene is poly-adenylated.
False! Since many transcripts are poly-adenylated, this does not help design gene-specific PCR primers.