Biotechnology Flashcards
Purpose of polymerase chain reaction
Amplify small amounts of DNA to quickly produce thousands of identical copies
How is polymerase chain reaction used
Biotechnologies such as electrophoresis (allows size of PCR fragments to be determined), DNA sequencing and recombinant DNA, Forensics to amplify small amounts of DNA from a crime scene, Evolution to amplify fossil samples and observe migration patterns, Detect hereditary diseases, Tissue typing for organ transplants, Earlier detection of infectious diseases
Denaturing
Two strands are separated by heating DNA to around 96 degrees to break the bonds between strands, Taq polymerase (comes from bacterium Thermus aquaticus found in hot springs) is used as DNA polymerase is destroyed at a high temperature
Annealing
Temperature is cooled to 55-65 degrees, Primer is attached/anneals to each strand of DNA to initiate replication by Taq polymerase, Primer- short piece of ssDNA (known sequence) binds to template strand on a specific part of DNA strand to act as a starting point
Elongation (extension)
Temperature is heated up to around 73 degrees, Taq polymerase binds to the primer and starts to bring free DNA nucleotides (one at a time) to the template strand to synthesis DNA that is complementary to the template strand, Results in DNA amount to be doubled
Purpose of gel electrophoresis
Separate fragments of DNA according to size to create a banding patter known as an individual’s DNA profile
Uses of gel electrophoresis
Compare DNA of different individuals, create a DNA profile/fingerprint, identify suspects, paternity testing and identifying genes
DNA ladder
Fragments separated with known sizes (synthetically created and bought)
Buffer solution
Allows for movement of current
Gel electrophoresis steps
Restriction enzymes are used to cut DNA into fragments creating different length nucleotide fragments, the same restriction enzyme is used for all samples and it cuts the DNA at a particular sequence, DNA sample is placed in the agarose gel that contains pores within the gel matrix, Electrical current is passed through the negatively charged DNA to the positive electrode, A banding pattern is created as different length fragments move at different rates, smaller fragments travel faster and thus end up further whilst longer strands are slower and stay closer to the top, DNA fragments are stained with a dye or made radioactive so it’s possible to detect their location
DNA sequencing
Determination of the precise order of nucleotides in a sample of DNA
Sanger method
Determines which part of the DNA sequence has an adenine, thymine, guanine or cytosine
Sanger method ingredients (5)
DNA, Primers- has a nucleotide sequence that is complementary to the end of strand which allows DNA polymerase to begin replication, DNA polymerase, Nucleotides, Dideoxy nucleotides- synthetic nucleotides that lack the OH group meaning the elongation process is stopped as the hydroxyl group (OH) allows the following nucleotide to bond it
Sanger method steps
DNA is amplified using PCR, DNA is denatured to separate the strands, Primer attaches to the beginning of the area to be copied, Equal amounts of DNA are distributed between 4 test tubes, DNA polymerase added to each test tube, All 4 DNA nucleotides are added to each test tube, Dideoxynucleotides are added to each test tube which stop the elongation/sequencing process forming different length fragments are formed, Electrophoresis carried out to order the fragments
