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Gel Electrophoresis (DNA Fingerprinting)
Transcript of Gel Electrophoresis (DNA Fingerprinting)
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?
How Does Gel Electrophoresis Work?
- 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?
What is Gel Electrophoresis used for?
- 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
- 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
* bp (base pair)/ kbp (kilo-base pair) is a unit of measurement of DNA or RNA length used in genetic