6 Halo-Lic vector (Smal digested) (all) How would you tell the PCR reaction work
ID: 191062 • Letter: 6
Question
6 Halo-Lic vector (Smal digested) (all) How would you tell the PCR reaction works? How would you tell the Smal digestion reaction works? For linear DNAs, what is the relationship between the size and migration velocity of the DNAs? How will you visualize the DNAs in an agarose gel? (Note the specific dye and transilluminator used in the lab). For T4 DNA polymerase processing of the PCR product and linearized vector, it is important to understand how it works. What enzymatic activities of the T4 DNA polymerase are required? What is the role of each activity? Nucleotides are removed by T4 DNA polymerase from which end of the DNA? (5- or 3'-end?) What is the role of dATP (or dTTP) in the T4 DNA polymerase processing reaction? At which point does T4 DNA polymerase stop removing more nucleotide from the 3'-end? What is the mechanism? MacBook AiExplanation / Answer
1.
To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures, or separates into two pieces of single-stranded DNA. Next, an enzyme called "Taq polymerase" synthesizes - builds - two new strands of DNA, using the original strands as templates. This process results in the duplication of the original DNA, with each of the new molecules containing one old and one new strand of DNA. Then each of these strands can be used to create two new copies, and so on, and so on. The cycle of denaturing and synthesizing new DNA is repeated as many as 30 or 40 times, leading to more than one billion exact copies of the original DNA segment.
The entire cycling process of PCR is automated and can be completed in just a few hours. It is directed by a machine called a thermocycler, which is programmed to alter the temperature of the reaction every few minutes to allow DNA denaturing and synthesis.
2.
LIC is a cloning method that makes use of annealing of single-stranded complementary overhangs on the target vector and a PCR-generated insert of at least 12 bases. The commercial InfusionTM system (Clontech) is based on the same principle and requires a 15-base overlap region. Single-stranded overhangs can be generated by using T4 DNA polymerase and only one dNTP in the reaction mix, leading to an equilibrium of 3'->5'-exonuclease and 5'->3'-polymerase activity at the site of the first occurrence of this nucleotide.
3.Longer molecules migrate more slowly because they experience more resistance within the gel. Because the size of the molecule affects its mobility, smaller fragments end up nearer to the anode than longer ones in a given period.
4. The negative charge of its phosphate backbone moves the DNA towards the positively charged anode during electrophoresis. However, the migration of DNA molecules in solution, in the absence of a gel matrix, is independent of molecular weight during electrophoresis.[4][20] The gel matrix is therefore responsible for the separation of DNA by size during electrophoresis, Ethidium bromide which intercalates into circular DNA can change the charge, length, as well as the superhelicity of the DNA molecule, therefore its presence in gel during electrophoresis can affect its movement. For example, the positive charge of ethidium bromide can reduce the DNA movement by 15%.
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