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Gel Electrophoresis (DNA Fingerprinting)

Biotechnology Digital Assignment
by

Vicky Wong

on 1 April 2014

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Transcript of Gel Electrophoresis (DNA Fingerprinting)

- widely used laboratory method to separate mixtures of DNA, RNA, or proteins according to charge, molecular size and shape
What is Gel Electrophoresis?
- used in forensics, genetics, biochemistry, immunology and molecular biology

- to determine who a person's parents or siblings are
eg. It can be used to identify the parents of babies who were switched at birth.

- Used in forensic science to solve crimes
eg. Blood, skin, or other tissue left at the scene of a crime can be analyzed to prove whether the suspect was present or not at the scene

- to identify a body
eg. Useful if the body is badly decomposed or if only body parts are available due to a natural disaster, battle, etc...

- To get a DNA fingerprint so that you can look for evolutionary relationships among organisms

- To test for genes associated with a particular disease.
History of Gel Electrophoresis
1930s - first reports of the use of sucrose for gel electrophoresis
1955 - introduction of starch gels
1959 - introduction of acrylamide gels (Raymond and Weintraub); accurate control of parameters
such as pore size and stability
1964 - disc gel electrophoresis (Ornstein and Davis)
1969 - introduction of denaturing agents especially SDS separation of protein subunit (Weber and Osborn)
* DNA electrophoresis was first performed in the 1970s*
How does Gel Electrophoresis Work?
- Uses a permeable gel (agarose and electrolyte)
with a row of holes on one side
How Does Gel Electrophoresis Work?
(Continued)
How Does Gel Electrophoresis Work?
(Continued)
- Once the gel has run, it is photographed under UV light

- Ethidium Bromide binds to DNA and fluoresces when exposed to ultraviolet light

- DNA fragments will produce many different "bands" or lines as it moves across the gel

- Once the bands have begun to approach the other side of the gel, the current is turned off
How do you Compare the Bands?
(DNA FINGERPRINTING)
GEL ELECTROPHORESIS
What is Gel Electrophoresis used for?
http://learn.genetics.utah.edu/content/labs/gel/
- provides information to the molecular weights and charge of proteins, subunit structures of proteins, and the purity of a particular protein preparation
- Arne Tiselius (1902-1971), a Swedish physical chemist won the 1948 Nobel Prize in chemistry for his research on electrophoresis
- credited with the invention of electrophoresis and is known as the father of electrophoresis
- "A technique for separating protein molecules of varying sizes in a mixture by moving them through a block of gel, as of agarose or polyacrylamide, by means of an electric field, with smaller molecules moving faster and therefore farther than larger ones." (Dictionary.com)
- Different samples of DNA are broken into individual strands using restriction enzymes that cut the DNA at specific, known locations

- DNA is mixed with a dye or radioisotope to see the location when in the gel (Ethidium bromide)

-DNA samples are placed into the holes using a pipette
* agarose is a polysaccharide polymer extracted from seaweed/agarose (used for longer segments)
- buffer system used to carry the current
and protect the samples during electrophoresis
* Tris Borate EDTA (Ethylenediaminetetraacetic acid)
* Tris Phosphate EDTA
* Tris Acetate EDTA
* polyacrylamide is a polymer formed from acrylamide subunits (used for shorter fragments)
How do you Know the Size of the Bands?
- One well in a gel is always left for markers, a mixture of DNA fragments of known size which separate out into a ladder.

- We can estimate the size of the fragments we are investigating by comparing how far they have migrated with the migration of fragments of known size
Conclusion
- Important to get a better understanding of genes

- Find cures to diseases, classify species, and find ways to improve the life of organisms

- knowing how to sequence DNA, we can know where in the genome or gene the mutation has occurred
- An electrical current is applied to the gel slab and causes one side of the gel to have a positive charge and the other side a negative charge

- DNA will travel from the negative end to the positive end, due to the negatively charged phosphate groups that make up the backbone of the DNA

- Larger strands travel slower than the shorter strands, therefore the shorter strands travel farther
- Bands can be used to compare genetic similarities like to determine parent/sibling, disease, and other genetic information
Example:
CRIME SCENE
* bp (base pair)/ kbp (kilo-base pair) is a unit of measurement of DNA or RNA length used in genetic
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