The major eight components of a polymerase chain reaction are: DNA polymerase, buffer solution, primers, magnesium chloride, dNTPs (adenine, guanine, cytosine, thymine), the deoxynucleotide triphosphates, DNA template strand, water, and bovine serum albumin (BSA) (optional).

All of these answers are characteristics of multiplex PCR reactions. A multiplex reaction is meant to simultaneously amplify several different target regions of DNA utilizing several sets of primers. Incorporating several different primer sets is similar to performing multiple PCR reaction all in one tube. The advantage being that less reagents are used and the process is less time consuming. Because all of the primers are acting in one reaction tube they must be optimized to perform at similar annealing temperatures and under similar conditions.

The Initialization Step or Hot Start occurs prior to thermocycling in order to activate the DNA polymerase. This is a 1 to ten minute incubation at 95°C. The purpose of using a DNA polymerase that requires a hot start activation is to prevent non-specific amplification products.

Primers begin binding to the template strand during the annealing step. Extension of the DNA strand occurs later in the thermocycling process. Adenylation of the new strand by DNA polymerase occurs during a final elongation step after the thermocycling rounds. DNA double strands are broken apart in the denaturation step of thermocycling.

The steps of thermocycling involves denaturation, annealing and extension.

Denaturation is done at a high temperature generally around 94-98°C to break apart the hydrogen bonds of double stranded DNA into single strands.

Annealing is the second step of thermocycling completed at 50-65°C. This step must be done at a temperature low enough allowing primers to attach to the single strand, but high enough that hybridization is specific.

The extension or elongation step of thermocycling is used to allow the DNA polymerase to create the new complementary strand to the template DNA strand. Temperature depends on the DNA polymerase being used but generally ranges from 75-80°C.

Prior to the stages of thermocycling there is a often a hot start step to activate the polymerase if necessary. Following the stages of thermocycling is a final extension stage and final hold stage.

The most logical procedure for the student is to first use PCR to amplify the HBB gene from human genomic DNA with primers that have restriction sites at the ends (this will faciliate ligation to a restriction enzyme digested vector). Next, the expression vector should be digested using a restriction enzyme, and then the digested vector and the amplified HBB gene can be ligated together. The final product will be two segments of the original vector with the HBB gene spliced between them.

The primers are absolutely essential for the functionality of a PCR reaction. The heat-resistant DNA polymerases need the RNA primers to anneal before they can begin replicating DNA. Without the primers, there is no way for the DNA polymerase to function and, therefore, no product will be obtained.

Primers are essential for any DNA replication process and are synthesized by primase during in vivo replication. For PCR, primers targeted to specific genes are artificially created and introduced to the sample. Only when the primers are bound will DNA polymerase be recruited to the replication site.

PCR increases DNA product exponentially. 2 parents strands of DNA are split in the first cycle and amplified into 2 copies of DNA. The second cycles results in amplification of both of the first cycle products. The amount of strands doubles giving you 4 copies after the second cycle. The pattern continues and the amplificed product doubles with each cycle.

First cycle: 2 copies

Second cycle: 4 copies (22)

Third cycle: 8 copies (23)

Fourth cycle: 16 copies (24)

Fifth cycle: 32 copies (25 )

The extension or elongation step of thermocycling is used to allow the DNA polymerase to create the new complementary strand to the template DNA strand. Temperature depends on the DNA polymerase being used but generally ranges from 75-80°C.

Denaturation is done at a high temperature generally around 94-98°C to break apart the hydrogen bonds of double stranded DNA into single strands.

Annealing is the second step of thermocycling completed at 50-65°C. This step must be done at a temperature low enough allowing primers to attach to the single strand, but high enough that hybridization is specific.

Prior to the stages of thermocycling there is a often a hot start step to activate the polymerase if necessary. Following the stages of thermocycling is a final extension stage and final hold stage.

A thermocycler is used to complete a PCR amplification. The thermocycler can be set to cycle through the denaturation, annealing, and extension stages in a determined amount of cycles based on the reaction volume.

The purpose of a PCR reaction is to amplify a targeted DNA region into millions of copies. PCR helps to yield several copies of DNA in a few hours allowing analysis. Each PCR cycles approximately doubles the amount of DNA present in the reaction.