Question

1. comapre and contrast the entire process of separating DNA using electrophoresis and separating proteins using elcterophoresis.

2. draw the diagram and explain what happens during the first round (cycle) of PCR. include all the materials that are used and insert where needed. include directional markers where necessary.

Answer 1

Major Differences between the gel electrophoresis of DNA and Protein

1. Gel electrophoresis of DNA is usually done by agarose gel electrophoresis.

            Gel electrophoresis is the standard lab procedure for separating DNA by size (e.g., length in base pairs) for visualization and purification. Electrophoresis uses an electrical field to move the negatively charged DNA through an agarose gel matrix toward a positive electrode. Shorter DNA fragments migrate through the gel more quickly than longer ones. Thus, you can determine the approximate length of a DNA fragment by running it on an agarose gel alongside a DNA ladder (a collection of DNA fragments of known lengths).

            In the case of DNA, the direction of migration, from negative to positive electrodes, is due to the naturally occurring negative charge carried by their sugar-phosphate backbone.

            Double-stranded DNA fragments naturally behave as long rods, so their migration through the gel is relative to their size or, for cyclic fragments, their radius of gyration. Circular DNA such as plasmids, however, may show multiple bands, the speed of migration may depend on whether it is relaxed or supercoiled. Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents that disrupt the hydrogen bonds, such as sodium hydroxide or formamide, are used to denature the nucleic acids and cause them to behave as long rods again.

            Proteins, unlike nucleic acids, can have varying charges and complex shapes, therefore they may not migrate into the polyacrylamide gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are usually denatured in the presence of a detergent such as sodium dodecyl sulfate (SDS) that coats the proteins with a negative charge. Generally, the amount of SDS bound is relative to the size of the protein (usually 1.4g SDS per gram of protein), so that the resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge-to-mass ratio. Since denatured proteins act like long rods instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape.

2.

A standard Polymerase Chain Reaction (PCR) is an in vitro method that allows a single, short region of a DNA molecule (single gene perhaps) to be copied multiple times by Taq Polymerase. From a single copy of DNA (the template), a researcher can create thousands of identical copies using a simple set of reagents and a basic heating and cooling (denaturing and annealing) cycle. The process became automated with the discovery of a heat resistant DNA polymerase from the thermophilic bacterium, Thermus aquaticus (Taq). Taq polymerase can withstand many heating and cooling cycles, which would denature DNA polymerases from other species.

            In addition to the template DNA and the Taq polymerase, PCR requires free nucleotides [dNTPs; adenine (A), cytosine (C), guanine (G), thymine (T)] in an equal molar ratio. It also requires two unique single stranded DNA oligonucleotide (oligo) primers, which anneal to the regions upstream (5’) and downstream (3’) of the DNA segment to be amplified. When these reagents are combined in an appropriate buffer, a series of heating (denaturing) and cooling (annealing) steps allow the Taq polymerase to copy the DNA in between the oligo primers. This molecular biology technique creates several micrograms of target DNA from just a few nanograms of template DNA through several cycles of denaturation, annealing, and synthesis. After the PCR is complete, the product can be verified based on size by gel electrophoresis.

Materials List:

Reagents for each 50µL PCR reaction:

• · Thin-walled PCR tube
• · Ice Bucket
• · 2 μL Template DNA (10 ng-500 ng)
• · 5 μl 10X Taq buffer with MgCl2
• · 1 μl dNTP mix (10 mM each nt)
• · 2.5 μL Forward Primer (10 μM stock)
• · 2.5 μL Reverse Primer (10 μM stock)
• · 36.8 μL Sterile dH2O
• · 0.2 μL Taq DNA Polymerase (5 units/μL)
• · PCR Machine
• · Agarose Gel

Basic Steps:

• · Initial Denaturation for 2 minutes at 94°C: This initiation step heats the double stranded DNA template strand to the point where the strands start denaturing and the hydrogen bonds are broken between the nucleotide base pairs. The initial denaturation step is commonly performed at 94–98°C.
• · Denature 30 seconds at 94°C: Continued denaturation of double stranded DNA.
• · Anneal primers for 30 seconds at 55°C: The forward and reverse primers are stable within this temperature range to anneal to each of the single stranded DNA template strands. The DNA polymerase is also stable enough to now bind to the primer DNA sequence.
• · Extend DNA for 1 minute at 72°C: The Taq polymerase has an optimal temperature around 70-75°C so this step enables the DNA polymerase to synthesize and elongate the new target DNA strand accurately and rapidly. There are now twice as many copies of your gene of interest as when you started.

Number of copies of DNA obtained after 'n' cycles = 2(n+1)