The Polymerase Chain Reaction

The Polymerase Chain Reaction, or PCR, absolutely requires

a single oligonucleotide synthesis primer and a DNA template.

at least two oligonucleotide synthesis primers and a DNA template.

a single oligonucleotide synthesis primer and a DNA or RNA template.

at least two oligonucleotide synthesis primers and a DNA or RNA template.

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.

so that DNA synthesis is directed in the same orientation from both primers.

so that DNA synthesis is directed outwards or in opposite directions from the opposing primers.

in any desired manner, since primer orientation is not significant 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.

is determined by the source of the polymerase used in the PCR reaction.

can be calculated to attempt to minimize undesired amplification products.

can be used to manipulate the specificity of the mixture of amplification products.

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.

no nucleotide sequence information about the desired DNA fragment must be known prior to PCR.

oligonucleotide PCR primers can be designed to add essentially any desired nucleotide sequences to the flanking regions of the amplified DNA.

the amplification reaction is always so specific that it yields only the desired DNA fragment.

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.

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.

the PCR samples are free of the DNA vector bands that are always associated with cloned genes.

unlike traditionally cloned DNA fragments, the amplified fragments can easily be excised and recovered from the gel.

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.

can always be distinguished from one another because of the simple band pattern of the PCR fingerprint.

are similar in that they provide a limited amount of information about the nucleotide sequences examined.

are very similar in their sensitivity to contaminating DNA.

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).

only the amount of DNA that can be loaded on the gel electrophoresis system used.

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.

only by the relative complexity of the mixed DNA sample.

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.

the activity of the hybridization probe (the amount of probe necessary to detect a single molecule) used to detect the PCR product.

only the amount of DNA that can be loaded on the gel electrophoresis system used to detect the PCR product.

the relative frequency of the target molecule in the mixed DNA sample.

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.

the greater speed of the PCR approach.

the greatly reduced cost of PCR in comparison to conventional cloning technology.

that a single PCR reaction will usually provide amplified material for isolation of several unrelated genes.

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.

the two DNA samples are identical.

the two DNA samples are completely unrelated.

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.

the suspect cannot be guilty, since the PCR fingerprint proves that the semen sample was not from the suspect.

the suspect might be guilty, but might not.

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.

the introduction of sequence errors during the amplification process.

technical errors made during the nucleotide sequence analysis.

the use of degenerate primers that themselves introduce errors.

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.

increase the average size of cDNA molecules amplified.

increase the specificity of the PCR reaction and minimize undesired amplification products.

increase the total yield of the PCR reaction.

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.

will always increase the specificity of the PCR product.

can introduce specific mutations within an amplified DNA fragment.

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.

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.

the nucleotide sequence of a previously characterized and equivalent gene from a different organism.

the nucleotide sequence of a previously characterized and closely related gene from the same or a different organism.

the knowledge that the transcript encoding the desired gene is poly-adenylated.