DNA Technologies – Southern Blotting & ELISA

1. DNA Technologies – Southern Blotting & ELISA By C. Kohn, Waterford WI

2. Review • If we wanted to read a gene or even our entire genome base by base (letter by letter), we would use the Sanger Method • The Sanger Method used ddNTPs to stop the same sequence at different places; the ddNTPs would give a color to the last base added

3. Review • If we wanted to compare someone’s DNA fingerprint, we would use PCR Electrophoresis and Restriction Fragment Analysis • We would cut out a gene, replicate it billions of times, and then cut it up with a restriction fragment • We would then pull the cut up fragments through a gel and compare banding patterns

4. Overview • The Sanger Method is useful if we want to read large amounts of DNA and know the exact coding sequence of bases • However, the Sanger method is not cheap nor is it as quick. It takes a lot of sophisticated equipment and highly trained individuals to run this test • PCR Electrophoresis and Restriction Fragment Analysis are quick, cheap tests that most people with a little training could perform. • However, the results aren’t as precise as the Sanger Method; we can’t read the actual DNA • We can only compare different kinds of DNA

5. Southern Blotting • The Southern Blotting test enables us to detect if a specific gene exists on a PCR Electrophoresis DNA fingerprint. • For example, if you wanted to know if you were a carrier for a recessive genetic disease, you could run the PCR Electrophoresis on the section of your DNA that would contain the gene for this disease. • You could then run a Southern Blot on the gel to see if the gene for the disease is actually there

6. Southern Blotting • Southern Blotting allows us to determine if a sample of DNA has a specific gene or mutation without having to go through the difficulty or expense of running the Sanger Method Test. • It provides us the information of the Sanger Method at the cost and ease of PCR

7. Southern Blotting Steps Southern Blotting has a few steps. • 1. Run PCR Electrophoresis (amplification, addition of a restriction fragment, gel electrophoresis) • 2. Transfer the DNA to a membrane • 3. Add a probe with the complementary sequence for the gene you are looking for • (e.g. GATCA for CTAGT) • 4. If the gene your are looking for is present, the probe will bind to it and create a signal (e.g. it may glow with bioluminescence if the gene is there)

10. Genes and Disease • Faulty genes regularly cause disease. • A genetic test can easily determine if an individual or animal is a carrier of a genetic disease • This can be especially valuable if the disease is recessive, or if the individual carries but does not express the disease. • (e.g. if they are Rr for a recessive disease, they would carry it but not have the symptoms of it). • Southern Blotting enables us to see if an individual has genes for any kind of genetic disorder.

11. But what if… • However, not all diseases are genetic. • Some diseases are caused by organisms outside of the body. • An infectious disease is a disease passed on to us or an animal by another organism. • For example, you can get a rhinovirus (cold virus) from another individual. This has nothing to do with your DNA. • Would PCR & Southern Blotting be useful in this case? • No, because genetic tests only look at DNA.

12. ELISA • ELISA is a test that checks for proteins instead of DNA. • Proteins are made from information encoded in DNA, but proteins and DNA are two separate things • ELISA detects antibodies in blood • Antibodies are proteins used to ‘label’ things in the blood. • For example, your blood as antibodies that tell your body whether or not you have type A, B, O, or AB blood. • Antibodies are proteins, not genes or DNA

13. Antibodies  Molecular Post-Its • Antibodies help your body to recognize the antigens for other substances, including diseases. • For example, chicken pox has chicken pox antigens. • Your body would produce chicken pox antibodies to identify the chicken pox antigens. • Antigen is short for “antibody generator”

14. Antibodies & Antigens • Antibodies and Antigens are like locks and keys • With a lock, only one kind of key will fit • The same is true for antigens and antibodies • The shape of an antibody is specific to the antigen it binds to • An antibody will only bind to one kind of antigen • If an individual has a disease, their blood should produce antibodies specific to that disease. • These antibodies will tell us if you have the disease or not

16. ELISA Testing – A Test for Antibodies • ELISA works by coating wells (or depressions) in a dish with the antigen proteins for a specific disease. • A sample of blood is added to the wells of the dish • If the disease is there, they should have those antibodies in their blood • These antibodies will bind to the antigen in the well. • A second round of antigens are added, and these second antigens are dyed a specific color. • If the protein is there, the coloredantigen will change the color of the well.

17. ELISA – Detection of Specific Proteins • 1. Add an antigen for a specific disease • 2. Add blood w/ antibody proteins for the antigen • 3. Add a second, colored antigen for the same disease • 4. If the antibody protein for a disease is there, the well will change color.

18. Summary • Southern Blotting is a test for genetic disease. • It works by absorbing DNA on an electrophoresis gel onto a membrane • The membrane is then treated with a probe that will bind only to a specific DNA sequence (such as a gene for a genetic disease) • If the gene is present, it will ‘light up’ • ELISA is a test for infectious disease • ELISA works by coating wells in a dish with an antigen for a disease • If an individual has the disease, they will have antibodies in their blood for the disease • These antibodies will bind to the antigen; a second antigen will cause a color change in the well if they have the disease.