Question

-Understand the concept of (restriction enzyme produced) DNA fragment separation by gel electrophoresis.

-In gel electrophoresis, which DNA fragment (in terms of size) would migrate further from the sample well?

-Understand restriction enzyme(s) and recognize they leave blunt or sticky ends (ie. XbaI, SmaI, EcoRI, BamHI)?

-Understand the steps (process) of the Polymerase Chain Reaction and Thermal Cycling.

Please answer as much as you can and I will give a high rate

Answer 1

1. DNA digestion by using restriction enzymes are loaded onto gel electrophoresis. DNA is digested in separate reactions with one of up to three. A control 'uncut' sample is incubated without adding enzyme. Digested DNA samples are loaded into wells of an 0.8% agarose gel, and an electric field is applied causing negatively-charged DNA fragments to migrate toward the + electrode. The gel matrix acts as a sieve and allows smaller DNA molecules to migrate faster than larger molecules, so that the fragments are separated by size. The rate of migration is approximately proportional to the log of the molecular weight of the DNA molecule.

Enzymatic Unit = amount of enzyme necessary to digest 1 µg ds DNA to completion in 1 hr under standard conditions (typically 37°C, with optimal buffer). RE's typically are sold at a concentration of 5-20 U / µl, although high concentration versions may also be available.

2. DNA molecules migrate through the gel matrix at a rate that is inversely proportional to the log10 of their molecular weight (for convenience, expressed in base pairs). Larger molecules move more slowly because of greater frictional drag and because they worm their way through the pores in the gel less efficiently than small molecules.DNA molecules migrate at different rates, depending on the concentration of agarose in the gel. Higher percentage gels are better for resolving small fragments; lower percentage gels are better for resolving large fragments.

3. Xbal: Sticky ends, Smal: blunt ends,

EcoRI: recognises the sequence 5'GAATTC'3 - sticky ends

BamHI: recognises the sequence 5'GGATCC'3 - sticky ends

4. The polymerase chain reaction (PCR) was originally developed in 1983 by the American biochemist Kary Mullis. It is a technique used in the lab to make millions of copies of a particular section of DNA. It is a common tool used in medical and biological research labs.

Principle: Five components are required to set up a PCR:

• the DNA template to be copied
• primers, short stretches of DNA that initiate the PCR reaction
• DNA bases (A, C, G and T) are the building blocks of DNA and are needed to construct the new strand of DNA
• Taq polymerase
• buffer to ensure the right conditions for the reaction.

Three main steps:

1. Denaturing – when the double-stranded template DNA is heated to separate it into two single strands.
2. Annealing – when the temperature is lowered to enable the DNA primers to attach to the template DNA.
3. Extending – when the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme.
4. These three stages are repeated 20-40 times, doubling the number of DNA copies each time.
5. After PCR has been completed, electrophoresis can be used to check the quantity and size of the DNA fragments produced.