|
|
We sue two types of controls in DNA analysis. We start running both from the very beginning, through PCR, onto to gel electrophoresis and through to sequencing. If either control results in an incorrect identification, we have to start the ENTIRE process over and all our results are considered invalid.
Bushmeat Processing: Using aseptic techniques, the bushmeat samples are cut into approximately 1 cc sections. They labeled and stored in ethanol at -20 degrees Celsius. Special care is taken to ensure no cross-contamination or human contamination occurs. Samples are then carried back or shipped to the WKU biotechnology Center. -----> This has already been done. |
Digestion liquefies the tissue in such a way that keeps the DNA intact for extraction. We use special "kits" as pictured to streamline the process. Extraction requires more steps to break through the cell and organelle membranes to free the DNA. Once extraction is complete, the DNA sample is tested to ensure an adequate amount of intact DNA was extracted from the sample. -----> This process has already been done with our meat samples. In Lab 10, you will practice extracting DNA from your own cheek cells. |
PCR makes copies of a DNA fragment from one original copy. The goal is to amplify a specific region, the target DNA or gene of interest (GoI), depending on the type or goal of research. The PCR cocktail includes the following ingredients: the DNA sample, primers (short sequences of RNA or DNA that start replication), dNTPs (free nucleotides), taq polymerase (a heat stable form of DNA polymerase derived from bacteria) and a buffer solution. There are three steps to PCR in which the temperature is cycled (in the thermo"cyler"). You need to know the steps and what happens in each! The total number of resulting DNA strands is (the number of original strands) X 2^n, where n = the number of PCR cycles. -----> In Lab 10, you will be given PCR products from our meat samples in lab and asked make some calculations. |
Agarose gel electrophoresis is a method used to separate DNA strands by size, and to determine the size of the separated strands by comparison to strands of known length. Your PCR products are deposited in the top of the gel. Using electricity, the DNA (with a negative charge) is pushed through the gel towards the positive electrode. As your gel "runs," the DNA is separated by size. The DNA strands show up as bands under UV light and you can read the results. Your products can be compared with the ladder or marker, which has standard sized DNA fragments of KNOWN length used for comparison. In this way, you can know the exact length of your DNA samples. -----> You will MAKE & RUN an agorose gel in Lab 11 to make sure our PCR product contains the cytochrome b gene. |
Once we know we have amplified (copied) the right gene we are ready to sequence the gene. We expect the sequence (the order of As, Ts, Cs and Gs) within the cytochrome b gene to be different for different species. Samples are placed into a sequencer apparatus which can detect the order of nucleotide bases in our sample. The sequence is then cleaned and edited, |
6) BLASTing: The National Institute of Health (NIH) and National Center for Biotechnology Information (NCBI) hosts a database called GenBank, which houses all known DNA sequences. Once the sequences of our samples are ready, they are pasted into a search tool (called a BLAST) which matches them to the correct species! -----> You will be provided the sequence of successful samples in Lab 11 and asked to determine the species of origin in lab. |
We have no idea!!!! In fact we might not have ANY. The calculations you completed in Exercise I are the best case scenario. The truth is PCR often fails. There are many reasons! Too much enzyme, low quality DNA, poor primers, low primer concentrations, incorrect PCR cycling, and so on! We need to SEE our DNA to know it exists and to confirm it is the right length! We do that through a technique called gel elctrophoresis.
Had we met in lab this week, you all would get your PCR products back from the freezer. Just tiny little tubes with very little solution at the bottom. No way to tell if we have any DNA, much less if it is the right stuff! So, in lab, you would have made, loaded and run a gel to ensure all all samples amplified, the C+ and C- did what they were supposed to do, and you would have checked to make sure all our samples amplified the right gene (CYTb), by checking the length! Procedure: Part 1
|
|
Procedure: Confirm the actual species of origin for our samples and compare it to the putative species (what is was sold as).
|
|
Procedure
Think back to where we started. If you need to go back to the Pre-Lab for Lab 10, or explore the bushmeat section of our research librabry. Read the artcile in the sidebar. It was the first phase of the reseach we are now conitnuing in BIOL 121. Think about the questions below and complete the Lab Notebook Guide.
|