PCR and Electrophoresis

PCR Why replicate DNA sections? Insulin Favourable DNA Forensics for analysis PCR = Polymerase chain reaction PCR A technique that amplifies DNA allowing you to increase the amount of DNA from a small sample. It's important as it has many applications: forensics, genetic diseases, monitoring disease spread. How does it work? Artificial replication of DNA Cyclic reaction Needs: DNA samples DNA polymerase Excess A, T, G, C Primer - 3/4 base pairs long. Starts a bit of the double-strand, Allows DNA polymerase to work as doesn't work on single strands. Thermal cycler  3 stages: Denaturing - Heat DNA sample to 94-96 degrees to break hydrogen bonds. The whole strand unzips in denaturing. Annealing - Cool to 68 degrees. This allows the primer to hydrogen bonds creating 1st new double-stranded DNA section.  Extending - Heat to 72 degrees, DNA polymerase binds to end and catalysts addition of nucleotides. Uses of PCR: Tissue typing  Detection of oncogenes (cancer genes) Detecting mutations, e.g. Down syndrome  Identifying viral infections Monitoring spread of infectious disease Forensic science Research Detect ancient DNA Cloning Identifying transgenic plants  

Questions 1. Why are primers needed in PCR but not natural replication?  In PCR, DNA completely unzips. DNA polymerase only works on double strands, not single. A primer is 3/4 bases long and attaches to the single strand to start a double so polymerase can work. In natural it unzips slowly so there is always a double strand. 2. Why does DNA polymerase need to be extracted from T. aquaticus, from hot springs? Otherwise DNA polymerase would denature at high temperatures due to the high temperatures. T. aquaticus = Taq polymerase.  

Electrophoresis Separates different size fragments of DNA using an electric current. Uses: DNA profiling Paternity disputes  Analysing disease Equipment Electrophoresis tank Agarose gel Power supply Buffer solution Loading dye Digested DNA How does it work? DNA cut into pieces Gel placed in tank and the tank is filled with buffer. Loading dye mixed with DNA, placed into well in jelly. Electric current is passed through the gel DNA carries a negative charge, as phosphate groups are negatives  Fragments migrate to anode (+) Lighest fragments move fastest. Do it for a set amount of time.

Additional Information What is the human genome? It is all the genetic material an organism contains. Genes only make up 2% of human DNA, these are called exons. Introns are large non-coding regions of DNA which are removed from messenger RNA before it is translated into a polypeptide chain.