Hassun Faruqui, Abdullahi Ali & Zuhaib Dahir
Topic #4: Gel electrophoresis & Plasmids
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Gel electrophoresis is essentially the separation of charged molecules on the basis of size
by sorting through a gel meshwork. The desired gene becomes separated from the
unwanted fragments through this process. DNA is negatively charged, since each
nucleotide possesses a constituent phosphate group that carries a net charge of -1. The
molar mass in between each nucleotide are negligible because of the ratio of purines to
pyrimidines in a DNA molecule. This is why each nucleotide has the same mass to
charge ratio. Therefore the only difference between a large fragment of DNA and a small
fragment would be the number of nucleotides. This difference can be exploited
to separate DNA fragments according to size.
DNA that has been subjected to restriction endonuclease digestion will be cleaved into
fragments of different lengths. DNA fragments migrate through the gel depending on the
size of the fragment. The longer the fragment is, the more tedious the process of it getting
through the gel would be. The shorter the fragment is, the quicker it could get into the
gel. The longer the nucleotide chain, the longer it takes for migration.
Gel electrophoresis takes advantage of DNA’s negative charge. The gel itself is usually a
square of rectangular slab and consists of a buffer containing electrolytes and agarose, or
possibly polyacrylamide. The segments that are to be separated are mixed with a loading
dye containing glycerol. The loading dye allows visualization of the DNA solution.
Glycerol is a heavy molecule that causes the DNA to sink down. Now the gel is loaded
while it is submerged in a tray containing an electrolytic solution called the buffer. A
negative charge is placed at one end of the gel where the wells are, and a positive charge
is placed at the opposite end of the gel. The electrolyte solution conveys the current
through the gel. At the end, the negatively charged DNA will migrate toward the
positively charged electrode, with the shorter fragments migrating faster than the longer
fragments causing separation.
Plasmids:
* Bacteria provide necessary machinery for a gene to be expressed as a useful protein.
* Gene of insulin can be isolated and expressed by bacteria cells.
* Using genetic engineering technology insulin genes are inserted into bacteria cells.
* Benefits diabetic people who relied on insulin from animals.
* That could cause allergic reactions.
* Plasmids are small circler pieces if DNA that can exit and enter bacteria cells.
* It lacks a protein coat.
* Independent of the bacteria cell.
Hassun Faruqui, Abdullahi Ali & Zuhaib Dahir
* DNA in Plasma can be replicated using enzymes and ribosomes.
* Bacteria cells benefit from Plasma.
* Protects bacteria by carrying genes for resistance to toxic heavy metals (mercury).
* Enables bacteria to break down herbicides.
* Relationship between Bacteria and Plasma is endosymbiotic (both benefit).
Useful links:
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http://learn.genetics.utah.edu/content/labs/gel/