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Electrophoresis

Gel electrophoresis is used to separate charged molecules of different sizes.

This is done by passing them through a gel (like jello) in an electrical field.

The gel acts as a support for the separation of the molecules of different sizes.

The gel is usually composed of a jelly-like material called agarose which is made from seaweed.

We can study molecules such as DNA or protein – this procedure will separate them by size (“lengths”).

Wells, or holes, are created in the gel. These will hold the DNA or proteins to be separated.

The gel contains very small holes. These regulate the speed which molecules move through it, based on the size. Smaller molecules move more easily through the small holes in the gel.

As a result, large fragments of DNA lag behind small fragments, thus allowing the experimenter to separate these molecules based on their size.

Sometimes molecular weight markers are used along with the specimen, so we can know the size of the DNA fragment which has been separated.

Different individuals or organisms form different banding patterns in the plate when their DNA has been separated.

DNA is cut into pieces for separation for electrophoresis by restriction enzymes. These enzymes were originally discovered in bacteria – they were used by the bacteria to defend themselves from invasion by other bacteria and viruses!

Procedure: gel electrophoresis

1. DNA fragments are placed in wells, at one end of a porous gel (kind of like gelatin)

2. Restriction enzymes cut the DNA into fragments.

3. An electric voltage is applied to the gel. One end of the gel becomes positive, the other end becomes negative.

4. The negatively charged DNA moves toward the positive end of the gel.

-> The smaller pieces move faster and further.

-> The larger pieces move slower, and less far.

5. We turn off the voltage. The DNA stops moving.

-> Based on size, the DNA fragments make a pattern of bands on the gel.

-> These bands can then be compared with other samples of DNA.

6. We add a stain (like food dye) – now we can see little bands of DNA. These bands let us compare the genomes of different organisms, or of different individuals.

{ image from http://24.media.tumblr.com/tumblr_m4ns7fPCrw1qkssp4o1_500.gif }

How can DNA move through this seemingly solid gel?

“A polyacrylamide gel is not solid but is made of a laberynth of tunnels through a meshwork of fibers.”

agar gel electrophoresis

agar gel electrophoresis second

http://www.bio.davidson.edu/genomics/method/SDSPAGE/SDSPAGE.html

GEL electrophoresis

Step-by-step animation of gel electrophoresis

http://www.dnalc.org/resources/animations/gelelectrophoresis.html

http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html

Use of DNA profiling for paternity, and forensic investigations

Paternity Investigation:
 Trying to determine the biological parents of a child.
 DNA fragments in a child come from the mother and father.
 A band present in the child must come either from the mother or from the father
 Comparing male 1 with the child, then male 2 with the child.

Interpretation:
 The bands on the child’s fragments are either found on the mother or from male 1.
 Male 1 therefore is this father of this child.
 None of the Male 2 bands appear in the child
paternity_vs_crime_scene_med

Forensic Investigation:
 A specimen of DNA is taken from the victim, or the crime scene.

 DNA samples are taken from 3 suspects.
 The bands are compared. We must first eliminate the victim’s DNA from the specimens!
Interpretation:
 The bands on the specimen are matched by the bands on the Suspect 1.
 This means that Suspect 1 was present at the crime scene.The law will still require to prove a crime was committed, and then that Suspect 1 committed the crime.

* * * * * * * * * * * * * * * * * * * * * * * * * * * * * ** * * * *

Online electrophoresis lab

Protocols are here:
http://www.scienceteacherprogram.org/biology/NLee05.html

The app is here:
http://learn.genetics.utah.edu/
Virtual gel electrophoresis

PBS NOVA Create a DNA fingerprint
http://www.pbs.org/wgbh/nova/education/body/create-dna-fingerprint.html

Mcdougal Littell WoW Biolab
https://www.classzone.com/books/hs/ca/sc/bio_07/virtual_labs/virtualLabs.html

Standards addressed:
New York State Math, Science, and Technology Learning Standards met by this lesson:
Standard 1: Analysis, Inquiry and Design – Students will use mathematical analysis, scientific inquiry, and engineering design, as appropriate, to pose questions, seek answers, and develop solutions
Standard 2: Information Systems – Students will access, generate, process, and transfer information using appropriate technologies
Standard 4: Science – Students will understand and apply scientific concepts, principles, and theories pertaining to the physical setting and living environment and recognize the historical development of ideas in science
Standard 5: Technology – Students will apply technological knowledge and skills to design, construct, use, and evaluate products and systems to satisfy human and environmental needs
Standard: Interdisciplinary Problem Solving – Students will apply the knowledge and thinking skills of mathematics, science, and technology to address real-life problems and make informed decisions

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