In
our example, we used EcoRI. The pink type is the location of the gene. For simplicity,
the diagram is just showing a few bases. The gene for insulin is of course much
longer. The pink arrows indicate where EcoRI would cut the DNA.General intro
Biotechnology is an ever growing field that utilizes biological materials to solve existing problems or improve life as we know it. This field includes genetic manipulation of plants to improve their health, vigor and disease resistance, the production of biological products, like insulin in an inexpensive and safe way, and of course its application to forensic sciences. In this laboratory you will extract and spool DNA from liver cells and perform you own 'forensic' DNA analysis.
Basics
DNA is a very large polymer that belongs to the nucleic acid family of macromolecules. It is composed of individual monomer subunits called nucleotides. The four nucleotides found in DNA are thymine, adenine, guanine and cytosine. Each of these individual nucleotides is made up of a sugar, deoxyribose, a phosphate group and a nitrogen containing base. Nucleotides are covalently bound end to end to form a long strand of bases. DNA is actually a double stranded molecule. That means that 1 intact DNA molecule is made up of 2 strands running anti-parallel. We are all familiar with DNA as a double stranded helix. What determines how these two strands combine to form the double stranded helix? The bases adenine, thymine, guanine and cytosine obey the base pairing rules, i.e., adenine always forms hydrogen bonds across the helix to thymine and guanine always forms hydrogen bonds across the helix to cytosine, so A=T and G=C.
DNA is the molecule of life. Within the sequence of bases found in your chromosomes is all of the information needed to not only determine your species, but all of the information that makes you uniquely you. You inherited your genome from your mother and your father. So unless you are an identical twin, your combination of genes is completely unique. This is the basis for its use in forensics. Your DNA is unique to you....
The discovery and use of restriction endonucleases are key to our manipulation of DNA. Restriction endonucleases (restriction - cut, limit; endo - within; nuclease - enzyme that acts on nucleic acids) were first isolated in the early 1960's by Werner Arber. It had been known for some time that some bacteria, like E. coli had enzymes that could chop up the DNA of viruses that infected them. Some of these enzymes make random cuts in the DNA, but other restriction enzymes cut at very specific spots and leave hanging bases creating sticky ends. Some of the restriction endonucleases make blunt cuts across the DNA strands.
The table below lists three different restriction enzymes, their site of action and the result of their cutting action. Enzymes names indicate the source of the enzyme. For example, EcoR1 stands for E. coli restriction enzyme 1. HindIII was originally extracted from Haemophilus influenzae.
Table 1
| Enzyme | Site of Cut | Resulting Pieces |
| EcoR1 | GAATTC CTTAAG |
G + AATTC CTTAA + G |
| HindIII | AAGCTT TTCGAA |
AAGCT + T T + TCGAA |
| BamHI | GGATCC CCTAGG |
G + GATCC CCTAG + G |
The ability to cut DNA at specific spots allows genetic manipulation and recombination of specific segments of DNA to create new genetic combinations. This recombination process is essentially how insulin was bioengineered.
The gene for insulin was
cut out using a restriction enzyme. In
our example, we used EcoRI. The pink type is the location of the gene. For simplicity,
the diagram is just showing a few bases. The gene for insulin is of course much
longer. The pink arrows indicate where EcoRI would cut the DNA.
The same enzyme was used
to cut open the bacterial chromosome.
Again the pink arrows indicate where the enzyme would cut through the DNA strand.
EcoRI leaves 'sticky ends',
i.e. unpaired nucleotides. Both the bacterial chromosome (green type) and the
insulin gene have sticky ends. 
The insulin gene and bacterial
chromosome were mixed and the sticky ends combined.
Different strands of DNA treated with the same endonuclease will produce sticky
ends with complimentary base pairs.These complementary bases, line up and rejoin
to produce a double strand of DNA. Now the bacterial chromosome contains the
human gene for insulin. The bacteria can produce this essential human hormone.
Why is this such an important advance for medicine? Prior to the production
of insulin by bacteria, all insulin was extracted from pig pancreases. The supply
of insulin depended on the number of pigs slaughtered for food. If pig production
went down, so did the supply of insulin. A second problem solved with genetically
engineered insulin is that pigs as mammals can transmit disease to humans. Diabetics
at one time ran the risk of contracting some viral disease from their insulin
injections.
Each of us has a unique series of bases that make up our DNA. The restriction enzymes cut along a specified sequence and will generate fragments of DNA that are unique for each one of us. This fact is the basis of DNA fingerprinting and its use in forensic sciences. In lab this week we will be using DNA from suspects and the victim of a crime. You will be provided with DNA that has already been treated with restriction enzymes.You will prepare an agarose gel and then perform an electrophoresis experiment. Your instructor will assist in the staining of your gel. You will analyze and report your results online.
Agarose gel electrophoresis is a technique that separates molecules based on size and charge in an electric field. Basically, DNA fragments are added to a jello-like slab of agarose and then exposed to an electric field. DNA is a negatively charged molecule. Restriction enzymes will cut the DNA into fragments of various lengths. These negatively charged fragments will move through the gel away from the negative electrode. How far and how fast they move in the gel is determined by the size and charge of the fragment. Smaller fragments will move farther away from the origin. Larger fragments move more slowly and will remain closer to the origin.
Link to animation of gel electrophoresis.
Activities:
1. Open the accompanying .pdf file. Print this file twice. This file contains example sequences of 2 different strands of DNA. Each strand is uniquely colored. Cut out each strand of DNA. You should have 2 strands of black-print DNA, and 2 strands of red-print DNA. Use Table 1 to determine where EcoR1 would cut the red-print strand of DNA. Label each fragment with EcoR1. Now cut the strand in the appropriate locations. Use Table 1 to determine where Hind III would cut the other red-print strand of DNA. Label each fragment with HindII. Now cut the strand in the appropriate locations. Repeat this procedure with the black-print strands of DNA.
How many fragments were generated from the red-print DNA with EcoR1?
How many fragments were generated from the black-print DNA with EcoR1?
How many fragments were generated from the red-print DNA with Hind III?
How many fragments were generated from the black-print DNA with Hind III?
Tape your fragments onto a sheet of paper. Divide the page into columns. Label the columns, Red - EcoR1, Red-Hind III, Black-EcoR1, and Black-Hind III. Arrange the fragments generated from each enzyme under each column heading. Place the largest fragments at the top of the column. The next largest fragment should be placed below that fragment and so on until all of the fragments are arranged in the column from largest to smallest. When you are satisfied that you have them placed correctly in the columns, tape them down so that the largest fragments are at the top of the sheet and the smallest are at the bottom. You must turn in this sheet when you come to class.
2. Complete and submit online the pre-lab quiz.
Links to web.
safety - hazards
orientation
foundation
activity