DNA Technology: Gel Electrophoresis

DNA Technology Gel Electrophoresis: Gels are used in order to measure the size of different macromolecules (DNA, protein, RNA, etc.) and is simply like a maze that the substances must migrate through Longer things will not go as far as smaller things A ladder can be used in order to find out relative sizes between the samples placed into the gel In order to see them, you use a marker/die, and for DNA it is common to use EtBr (Ethidium Bromide) and fits itself in between the DNA and base pairs (picture shown on the right) Polymerase Chain Reaction (PCR): PCR allows for a shit ton of copies of that particular DNA fragment with which you are interested in The steps include: 1. Denaturation of the DNA at 96°C 2. Annealing of the primer to the DNA at 55°C 3. Primer extension via DNA polymerase at 72°C (not just any DNA pol, you need one that is from a heat tolerant microorganism known as Thermus aquaticus and its polymerase is known as the Taq polymerase which is a DNA Pol I) The chain reaction portion comes from the fact you get exponential growth from two strands going to four, then eight, sixteen, etc. The polymerase may not know where to stop originally, but then when it reads the other way it will, and eventually it will approach the correct length DNA Libraries & Generating cDNA: A DNA Library basically allows one to type in any protein into it and have its DNA sequence spit back out cDNA is single stranded made from an RNA sequence (typically using reverse transcriptase); in order to made it dsDNA you need DNA Polymerase Remember, it is not necessarily possible to come up with an mRNA sequence from an amino acid sequence because multiple codons code for the same amino acid DNA Cloning & Recombinant DNA: DNA Cloning is simply making identical copies of a piece of DNA Restriction enzymes allow us to make cuts at certain DNA sequences Using the cut out DNA you can place it into a plasmid (circular DNA that is made to work in the organism) “Sticky ends” can help with pasting the cut piece of DNA into the plasmid, DNA ligase also helps to connect the two backbones together The model organism we use in order to place these plasmids into is E. coli Heat shock works to sort of “open up” the plasma membrane to take up the recombinant DNA (artificial DNA formed through multiple methods) The vector plasmid must have some sort of antibiotic resistance in order for you to know that the bacteria has that specific plasmid Hybridization (Microarray): Up regulated means that there is a higher amount of mRNA and therefore protein being manufactured within the cell Down regulated means that there is a lower amount of mRNA and therefore protein being manufactured within the cell Microarray: basically a well of complementary mRNA that can help show how concentrated it is within the cell in order to determine if it is upregulated or downregulated within the cell (typically attach a fluorescent tag to it) Expressing Cloned Genes: Remember, that cDNA only contains exons there are NO introns Remember, since bacteria have the antibiotic resistance and are exposed to the antibiotic through their immediate surroundings, any surviving bacteria must therefore have the gene of interest Southern Blot: Southern Blot: allows for the visualization of a specific DNA of interest and can help to see if a gene is present in a longer strand of DNA 1. Take the DNA and cleave it, resulting in multiple smaller strands of DNA 2. Run the smaller DNA fragments on a gel (helps separate the fragments based on size/charge) 3. Denature the DNA by increasing the pH (making it more basic) 4. Transfer the gel onto a filter (basically the image transfers onto the filter quite nicely) 5. Expose the filter to radio-labeled DNA (compliment of our DNA of interest) and basically anneals to this gene of interest 6. Expose the filter to radiation which results in signal on the film wherever the radio-labeled DNA is found Also, remember SNOW DROP for different gel electrophoresis techniques DNA Sequencing: 3 main steps to DNA sequencing: 1. Take the DNA of interest and PCR it in order to get a lot of copies 2. You want to add in regular deoxyribose nucleotides but also dideoxy nucleotides (ddNTP) which only have a H on the 3’ carbon, which does not allow for the strand to continue to grow (you can tag these ddNTPs with different fluorescent tags in order to see which is the last nucleotide added to that particular sequence) 3. Run it on gel electrophoresis in order to tell the sequence of nucleotides in that particular DNA fragment of interest Gene Expression & Function: If we have some sort of idea as to what a particular gene does, we can knock out the gene and see if the cell can still perform that function even when that particular gene is no longer present One can also do Reverse Genetics which is where you take a look at a particular gene, sequence it, and look for other genes with similar sequences. If you know what the similar sequence does, then you likely have a pretty good idea of what that gene might do Applications of DNA Technology: In medicine, it is important in the creation of insulin and growth hormone (can basically grow these proteins in E. coli) or even vaccines Another important application is used in forensics (can even take mitochondrial DNA) and using PCR can identify a suspect Another is even agriculture which is where scientists can make plants resistant to certain herbicides and pesticides, they can even delay ripening of the crop in order to make sure that it gets from the farm to the store on time Safety & Ethics of DNA Technologies: Technically you could place cancer genes into a bacterial cell and infect an individual with it Is it ethical to fix a known mutation even if it may generate an even worse one in the process? Or is it ethical to make someone smarter or even more athletic? These are issues that arise with the idea of genetic modification There are even problems with privacy issues as well (such as the government tracking you) and can even lead to discrimination (such as insurance company raising its rates for individuals it knows has the breast cancer gene) Additional Notes: restriction enzymes are meant to destroy unprotected DNA which is usually foreign (hosts protect DNA through methylation). Viruses do NOT have restriction enzymes Also, Sanger Sequencing shows the complementary strand, so you need to reverse the sequence in order find the original one (also read it from down → up for 5’ → 3’ for the complementary strand) Palindromic Sequences are ones where a DNA strand that reverse compliments itself such as: 5’ GGATCC 3’ 3’ CCTAGG 5’ If no antibiotic is present, bacteria grows immensely fast meaning that it will completely overrun the petri dish