What primers will be used to sequence your PCR product where are those sequences on your PCR product How did they get there?

What are the reasons why there needs to be more than 10 cycles in the PCR process? Name 2 reasons.

The ability to multiplex the PCR reactions used in STR analysis (many PCR amplifications occurringin the same tube at the same time) simplifies the DNA profiling technique a great deal. However,the analysis of the PCR products involves measuring only their size and their color, and this is allthe software needs to know in order to figure out which PCR products are from which locus. Howcan the analysis software tell all the various loci apart?

A possible forward primer to be designed is a primer that binds to the region of the OXA-M290 gene where the start codon is located, and the reverse primer is the primer that binds to the region of the gene where the stop codon is located.  In order to clone the PCR product generated using these primers into pET-28a, extra nucleotides must be added to the 5' ends of the primers. These extra nucleotides will contain the recognition sites for the restriction enzymes that are used for cloning. When the DNA produced by PCR using these primers is treated with the restriction enzymes, this will generate sticky ends at the ends of the PCR product. If pET-28a is digested with the same restriction enzymes, the sticky ends of the PCR product will bind to the sticky ends on the digested pET-28a, allowing the two DNA molecules to be connected together by DNA ligase. Which primer (forward or reverse) should contain the SacI recognition site, and which primer (forward or reverse) should contain the…

After you design the PCR primers, you run the PCR on fluid from the patient’s nasal swab. Next, you need to evaluate the results. PCR makes billions of copies of just one sequence in a sample. Since you know the sequence, you also know the length (number of nucleotides) of the region you copied. There are about 130 nucleotides in the N gene fragment. This is typically stated as 130 base pairs. How can you visualize DNA and estimate its size? Load the DNA sample into an agarose gel (similar in consistency to jello) and apply an electric current to the gel. The DNA is charged and will move through the gel. The longer the DNA fragment, the more slowly it moves. The DNA is visualized by adding a fluorescent dye to your sample that sticks to DNA. When you look at the gel under UV light, the DNA should glow. What is the charge of a DNA molecule? Based on #1, would you expect DNA to be drawn to the (+) or (-) electrode of the gel electrophoresis chamber?

How many molecules (target sequence copies) will be produced by 30 PCR cycles? Assume you start with only 1 copy of the target sequence (very unlikely)? Show your work!

Why do you detect mutations one at a time on PCR? Is there any way to detect multiple mutations in PCR at once?

We usually think of enzymes as being most active at around 37°C, yet in PCR the DNA polymerase is subjected to multiple exposures of relatively high temperatures and seems to function appropriately at 65–75°C. What is special about the DNA polymerase typically used in PCR?

What is the difference between PCR and real-time PCR, and how do you explain it?

A more modern molecular technique to RFLP fingerprinting is called Amplified Fragment Length Polymorphisms (AFLPs). In AFLP analysis, restriction enzymes are again used to digest genomic DNA into multiple fragments. Next, adapters complementary to restriction site overhangs are ligated to the fragments using an enzyme called DNA ligase. These adapters are complementary to primers used to amplify the fragments using the polymerase chain reaction (PCR). Can you think of any potential benefits of AFLP analysis over RFLP? Explain your reasoning.