Biotechnology Notes - DNA Sequencing & PCR

Original biotechnology broad area of biology which uses living organisms to develop and produce various products DNA sequencing Various methods, all of which determine the exact order of the base pairs in a segment of DNA. Polymerase chain reaction (PCR) technique which makes many copies of a...

Original biotechnology broad area of biology which uses living organisms to develop and produce various products DNA sequencing Various methods, all of which determine the exact order of the base pairs in a segment of DNA. Polymerase chain reaction (PCR) technique which makes many copies of a specific section of DNA; used to amplify the presence of a gene of interest so there is more to work with in a lab setting Ingredients of a PCR mixture DNA to be copied, free nucleotides (dNTPs), taq polymerase, primers, buffer PCR primers Two different short DNA fragments which match the two opposite ends of the region of DNA that needs to be copied, one on each strand taq polymerase DNA polymerase which can make new DNA at higher temperatures than animal polymerases; polymerase used in PCR Steps of PCR 1\. Denaturation\ 2. Annealing\ 3. Extension denaturation (PCR) heat the components to 96C to separate the two DNA strands of a double helix (by splitting apart base pair hydrogen bonds) annealing (PCR) Cool reaction to 55-65C so primers can bind to the now single-stranded template DNA extension (PCR) Raise reaction temperature to 72C where taq polymerase is most active and thus begins synthesizing new DNA starting at the primers Typical number of PCR cycles repeat the sequence of temperature changes 25-35 times to amplify the specific DNA section only to millions of copies restriction enzyme Enzyme that cuts DNA at a specific sequence of nucleotides restriction site A specific sequence on a DNA strand that is recognized as a cut site by a restriction enzyme; sequences of DNA being cut are often palindromic when comparing the top strand and bottom strand (see picture for this card) sticky ends Single stranded ends of DNA left after cutting with restriction enzymes blunt ends Restriction fragments with no overlapping ends DNA Ligase enzyme which can connect the backbones of DNA fragments; used in biotechnology to reconnect DNA pieces together after both pieces have been cut by the same restriction enzyme plasmids small circular DNA molecules in prokaryotes that replicate separately from the main chromosome gel electrophoresis method of separating a mixture of different DNA fragments or proteins by running electrical charge through a gel; The electricity will move the molecules through the gel, but the smaller fragments move faster and so the molecules are separated by size DNA ladder sample of protein or DNA fragments of known size which is inserted into an electrophoresis gel so that once it has run, the unknown fragments can be compared to the ladder to determine relative size Know how to do plasmid mapping - fragments to map, map to fragments, and both in the context of a gel