Presentation on theme: "DNA Technologies – Southern Blotting & ELISA By C. Kohn, Waterford WI."— Presentation transcript:
DNA Technologies – Southern Blotting & ELISA By C. Kohn, Waterford WI
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
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
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
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
Southern Blotting In a nutshell, Southern Blotting allows us to determine if a sample of DNA has a specific gene 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
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)
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.
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
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
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”
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
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 colored antigen will change the color of the well.
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.
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.