DNA Analysis - GRE Subject Test: Biology

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.

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 most suitable technique is PCR mutagenesis. The student will design primers that anneal imperfectly at the desired site of mutagenesis. These primers will contain the new 5-base pair sequence to be inserted in the new strand. One PCR will amplify a fragment containing the new mutant sequence and all upstream sequences. A second PCR will amplify a fragment containing the mutant sequence and all downstream sequences. Finally, a PCR will amplify one sequence from the two templates, using the original primers used to amplify the full-length fragment.

Polymerases are simply enzymes, and enzymes often denature at high temperatures. If a normal polymerase were used, it would become denatured during every high-temperature phase, which is required for breaking the double-stranded DNA into single-stranded templates. By using heat-stable polymerase, the enzyme will not denature, and there is no need to add new polymerase after every step of the cycle. The DNA strands will continue to be amplified by the heat-stable polymerase originally added.

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 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 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).

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.

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 )

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 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.

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.

Real time PCR is a technique that measures the amount of fluorescence that occurs with each cycle of PCR. The amount of fluorescence is directly proportional to the DNA concentration. The assay can also provide info such as the possible presence of inhibitors.

It is important to know how much DNA is present in a sample to optimize downstream processing.

DNA sequencing is used to determine the exact order of nucleotides that compose a given DNA molecule. This process can refer to any method that is used to determine the order of the nitrogenous bases in a strand of DNA.

DNA amplification is the duplication of regions of DNA to form multiple copies of a specific portion of the original region. DNA replication is the process of producing two identical replicas from one original DNA molecule. Gene flow is the movement of alleles from one population to another, owing to migration of individual organisms. Genetic recombination is the generation of new combinations of alleles on homologous chromosomes due to the exchange of DNA during crossing over.

Dideoxynucleotide triphosphates (ddNTPs) lack a 3' hydroxyl group, which is necessary for further polymerization past the ddNTP. In the absence of this 3' free hydroxyl group, ddNTPs cannot be linked to the next incoming nucleotide's 5' end. This halts DNA synthesis, and is the basis of Sanger (chain-termination) sequencing. Ordinary nucleotides do possess a 3' hydroxyl group, allowing them to polymerize further. Sanger sequencing takes advantage of this difference to terminate synthesis at various points.

The key to the chain termination method used in Sanger sequencing is the dideoxynucleotide. These modified bases, designated as ddNTP's rather than dNTP's, are missing their 3' hydroxyl group. This prevents further elongation of DNA strands being synthesized by a polymerase since DNA polymerases all require a 3' hydroxyl as their substrate. In the context of a whole sequencing reaction done before actual DNA sequencing, this creates strands of all possible lengths along your desired sequence.

cDNA clones are incredibly powerful tools that can be used to express eukaryotic proteins in prokaryotic cells. One of the primary reasons that this is possible is because both eukaryotes and prokaryotes contain very similar systems of translating mRNA transcripts. Another reason is because the cDNA clone does not contain any of the information that would normally be spliced (introns). Prokaryotes do not make the same post-transcriptional modifications that eukaryotic cells do, and must have introns removed by external means in order to create viable protein products.

Endonucleases cut DNA at restriction sites. These restriction sites are palindromic, meaning that they read the same forward as they do backwards.

The sequence 5'-CCTAGG-3' will have the complementary strand 3'-GGATCC-5'. Since this sequence is read the same forward as it is backwards, it is a palindromic sequence that can be potentially cleaved by an endonuclease.

5'-CCT|AGG-3'

3'-GGA|TCC-5'

The correct answer is bacteria. Both bacteria and archae utilize restriction enzymes as a defense mechanism to cut foreign intracellular DNA (specifically DNA of bacteriophage). Restriction enzymes are named based on the organism in which they were discovered - for example EcoRI is named after E.coli, and was the first restriction enzyme, found in the R strain.

The correct answer is five. Although you can only see three bands in your gel, a DNA fragment with four different restriction sites will be cut into five distinct pieces (if you're having trouble picturing this, drawing it out helps). In this case, some of the bands must be matching sizes, so they do not appear to be separate on your gel. For instance, there may be two 3kb bands, two 4kb bands, and one 5kb band.