Lecture 12 Biotechnology and Human Diseases 1 Describe the utility of restriction endonucleases in the analysis of DNA What is unique for the sequences recognized by restriction endonucleases Restriction endonucleases also called restriction enzymes help us to obtain fragments of DNA DNA is too large to study without being broken down Restriction endonucleases along with DNA ligase are key tools to form recombinant DNA molecules These restriction enzymes are able to cut several dsDNA using TaqL to create sticky cohesive or blunt ends DNA ligase then forms a bond between two sticky ends to form recombinant DNA Ligase ligates two fragments with the same sticky end Restriction endonucleases also recognize palindromes which is a DNA strand in which the top and bottom strands are read the same 2 Understand the rationale and the application of reverse transcriptase in recombinant DNA biology mRNA cDNA Reverse transcriptase uses RNA to synthesize DNA This process goes against the central sigma and can produce unique segments of DNA because it comes from mRNA Reverse transcriptase has three activities 1 RNA Dependent by DNA Polymerase 5 3 where the RNA template is copied 2 Ribonuclease H 5 3 by exonuclease where the RNA is degraded 3 DNA dependent DNA Polymerase 5 3 where the double stranded cDNA is synthesized This process forms something called cDNA complimentary from mRNA cDNA does not contain introns because mRNA has no introns and is the template cDNA also lacks the regulatory regions of a gene promoter promoter proximal elements and enhancers because mRNA does not have these either This means that the reverse transcriptase produces a large amount of exons as there are no introns An example of this is insulin synthesis Insulin comes from the pancreas It is made by taking proinsulin mRNA and converting it to proinsulin cDNA via reverse transcriptase This is then joined into a plasmid to make a transformed bacterium which produces insulin 3 Understand the principles and applications of Northern blot Southern blot Western blot These blot techniques allow us to detect DNA RNA and proteins In each of them a probe is used First gel electrophoresis is done by laying down nitrocellulose paper on agarose gel an alkali solution and stacking paper towels on top Next remove the nitrocellulose paper and place it in the buffer w probe Northern Blot Detects RNA with a single strand DNA probe The RNA does not need to be denatured as it is already just one strand Southern Blot Detects DNA with a single strand DNA probe The DNA must be denatured for the probe to work Western Blot Detects Proteins with an antibody probe The proteins must be separated by gel electrophoresis This probe is an antibody probe that recognizes antigens in proteins 4 Understand the principles of PCR reaction and its applications and the unique feature of Taq DNA polymerase PCR polymerase Chain reaction This is a way to replicate DNA in vitro in a test tube DNA replication relies on a primer enzymes nucleotides and an available 3 PCR has three main steps 1 Denature the DNA into separate strands by heat Unwinding DNA is a denaturing process H bonds are breaking here 2 Anneal the primers to the flanking region of single stranded DNA 3 Extend these primers using DNA polymerase In PCR the polymerases used must be heat stable to avoid them breaking down Because in this reaction heat is repeatedly applied to denature the DNA And during cooling cycles the primers will anneal to the proper sequence When heat is again applied polymerase expands from the primer THis process of heating and denaturing allows for rapid creation of DNA Taq DNA polymerase is this heat stable polymerase It was actually discovered in hot springs geysers PCR can be used to identify molecular structures to sequence things and to genetically engineer things There was even a covid test based on these principles 5 Understand the techniques used for the detection of mRNA RT PCR Southern blot microarray RT PCR real time PCR This is a technique that allows us to monitor the progress of a PCR reaction in real time It is also called qRT PCR and qPCR This process works by detecting a fluorescent compound with a dye in a reaction that binds to DNA to monitor its progress There is more fluorescence as the reaction proceeds This occurs because the PCR product accumulates more and more and the fluorescent reporter molecules bind to the double stranded DNA Northern blot allows us to find the amount of RNA using a DNA probe and gel electrophoresis Microarray a technique that helps us identify thousands of genes at once This is done using complements of single stranded nucelotides These are different colors that represent different cells and help us to identify lots of mRNA at once Normal cells turn into mRNA and then into cDNA and are labeled with a green color Cancer cells are broken into mRNA and then cDNA and are labeled with a red color Yellow spots mean that both cells produced a message 6 Explain the use of SDS gel electrophoresis SDS PAGE in the analysis of proteins 7 Understand the techniques used for the detection proteins western blot and ELISA 8 Understand the biotechnologies that are being used in the diagnosis treatment and prevention of Covid 19 9 What is genome editing