Chp 4

• DNA amplification increases the amount of the target DNA sample so that it is large enough to be used or analysed
• DNA manipulation uses polymerase chain reaction to create a large quantity of DNA identical to the trace sample
• polymerases are enzymes that catalyse the formation of polymers by linking nucleotides
• DNA polymerase is used in PCR and DNA sequencing multiple copies of the target DNA
• microsatellite = short repeated sequence of nucleotides found at a locus on a chromosome
• DNA sequencing = any process used to determine the order of the four nucleotide bases in DNA
• taq polyermerase = DNA polymerase that has heat resistant properties and is most commonly used in PCR
• reverse transcriptase = DNA polymerase synthesises single-stranded DNA using single-stranded RNA as a template, used to make complementary DNA from modefied mRNA
• RNA polymerase = enzymes that synthesise RNA from DNA during transcription
• PCR = polymerase chain reaction
• each cycle of PCR doubles the amount of sample DNA
• PCR mixture contains:

  • DNA, target DNA to be amplified
  • free nucleotides to build new DNA strands
  • heat resistant DNA polymerase, elongate new DNA strands
  • two DNA primers complementary to the target DNA ends
• PCR step one:

  • Denaturation - sample is heated to 95 degrees which breaks hydrogen bonds to create single-stranded DNA
• PCR step two:

  • Annealing - temperature is reduced to 50-60 degrees which allows primers to anneal (bind) to complementary sequences at each end of the target DNA sequence
• PCR step three:

  • Extension - temperature is increased to 72 degrees, taq polymerase attaches to primers and moves along each strand, adding free nucleotides to form double-stranded DNA
• gel electrophoresis is a technique used for separating fragments of nucleic acids or to study protein molecules
• DNA is negatively charged due to the phosphate group in each nucleotide
• DNA profiling is a technique that compares and identifies individuals based in their unique DNA sequence
• polymorphisms = inherited variations
• DNA profiling uses differences between a number of polymorphic sections to identify individuals
• DNA sample - provides a template to produce copies of in PCR
• primers - to bind to the single-stranded DNA and to provide a point in whihc DNA synthesis can be initiated and designate the sequence to be copied
• mix buffer - to provide a suitable chemical envrionment for the activity of taq polymerase by maintaining the approriate pH and providing any required salts
• PCR tube - to provide a vessel for the PCR reaction to occur, the tube will contain the DNA sample, polymerase, primers, nucleotides and buffer
• separate DNA, RNA or protein fragments within an agarose or polyacrylamide gel
• the information match DNA fragments from other samples, such as in DNA profiling or to isolate a particular fragment for further use in other techniques
• plasmids = small circular DNA molecules in bacterial cells
• bacterial transformation = genes can be inserted into plasmids and be incorporated into bacterial cells
• endonucleases = restriction enzymes
• vectors = carriers
• restriction enzymes are a large group of naturally occuring enzymes in bacteria
• restriction enzymes cut foreign DNA into smaller fragments destroying it
• bacterial cells block restriction enzymes from cutting their DNA
• each restriction enzyme targets a sequence of bases
• two types of rstriction enzymes

  • sticky end
  • blunt end
• sticky end restrictione enzymes leave DNA fragments with overhaning ends that have exposed bases
• exposed bases are able to form complementary base pairs with nucleotides of other DNA molecules
• the base pairing ability of sticky ends allows DNA from different species to join, formign recombinant DNA molecules
• blunt end restriction enzymes leave clean cut ends by cutting the sugar-phosphate backbome on both strands of the DNA molecule at the same location
• polymorphisms = smal variations in DNA sequences