1. Biotechnological Tools

2. What are we doing here?!?! One of the major advances in genetic research is the usage of recombinant DNA. Recombinant DNA refers to the practice of placing DNA (genes) from one organism into the genome of a second organism in hopes that the second organism will make use of the genes and make the proteins for which the genes encode. Basically, you take a desired gene from one thing and stick it into another so it too will be able to make the desired protein from the inserted gene. To understand and perform any biotech work you have to know the tools and the procedures needed in order to get the job done. So let’s take a look…

3. Tools of the Trade Restriction Endonucleases AKA – Restriction Enzymes These enzymes cut DNA at specific sequences called recognition sites. The cuts at the recognition sites can be in the form of either blunt ends or sticky ends (with sticky ends being the more useful of the two). Restriction enzymes naturally occur in bacteria as a defense against viral infection. The virus would inject its DNA into the bacterial cell only to be cut into many pieces and be rendered useless.

4. Sticky vs. Blunt Ends There are two possibilities when a restriction enzyme cuts through the DNA. 1.Sticky Ends – Staggered ends on a DNA molecule with short, single-stranded overhangs. 2.Blunt Ends – A straight cut, down through the DNA that results in a flat pair of bases on the ends of the DNA. Sticky ends are preferred in the lab because the single-stranded overhangs are complementary to each other and can be stuck back together with other sticky ends made from the same restriction enzyme. Blunts ends can match up with any other blunt end so you don’t get that exact match between the two pieces of DNA you are trying to put together.

5. Sticky Ends vs. Blunt Ends

6. Methylases & DNA Ligase Methylases are enzymes that add a methyl group (–CH 3 ) to a nucleotide in the recognition site of the DNA. This extra methyl on the nucleotide changes the shape of the recognition site and the restriction enzyme is unable to cut it because of the change in shape. Methylases are also naturally found within bacteria – it is how they protect their own DNA from their restriction enzymes. DNA Ligase reforms the phosphodiester bonds between adjacent nucleotides when you are trying to connect the foreign DNA fragment and the host cells DNA.

7. Gel Electrophoresis Gel electrophoresis is a technology that separates molecules based on charge and size by sorting them in a gel meshwork. The gel (meshwork) is like the rubber band tunnel at McDonald’s. You know the one – that thin, long, trap with those thick black bands running through it this way and that. You have to really squirm your way through it to get to the other side. Well…The smaller you are the easier it is to fly through it. And vice-versa of course. The charge on the molecule dictates the direction the molecule moves within the gel. Opposite charges attract so positive molecules move to the negative electrode while negative molecules move to the positive electrode.

8. Tasing the DNA DNA has a negative charge – all those phosphates in the backbone! We use that property to make the DNA move. An electric current is passed through the gel and charged particles (like DNA) will move in response to that electric current. Your DNA samples are loaded into wells (small pits) within the gel. The wells for DNA are near the negative electrode so they will move to the positive electrode – opposites attract! When the switch is thrown - the DNA fragments move. The smaller the fragment, the further it moves within the gel. After the gel has been run, it is treated with ethidium bromide that clings to rungs of the DNA and causes it to fluoresce (glow) under a UV light.

9. Gel Electrophoresis

10. Plasmids Plasmids are small, circular pieces of DNA that can exit and enter bacterial cells. They contain “bonus” DNA in that they can have genes in them that allow the bacterial cell to become resistant to some of the things that would normally kill it. These genes are known as resistance genes. We can insert foreign DNA into plasmids and put them into bacterial cells for them to use. We use the resistance genes to show us whether or not a plasmid has successfully adopted a plasmid that contains the foreign DNA. The foreign DNA interrupts one of the resistance genes on the plasmid and the bacteria that has that plasmid loses its ability to be resistant to whatever substance it provide the resistance against.

11. Plasmid Pics

12. Transformation The introduction of foreign DNA into a bacterial cell is known as transformation. Transformation requires a vector (or delivery vehicle) that will bring the foreign inside the bacterial cells. Plasmids or viruses are normal vectors for transformation. Any cell that has been successfully transformed and that now contains the foreign DNA is known as a competent cell.

13. To Sum It All Up… These are the basic tools needed to perform any experiments or work in a biotech lab. Future lectures will deal with some of the techniques and processes that are employed in these same labs.

14. FIN