Outline the process of DNA profiling for the identification and determining genetic relationships between organisms: Obtain DNA cells (from a cell sample e.g. skin, hair, blood, semen)/ Cut DNA using restriction enzymes (endonucleases – naturally occurring enzymes in bacteria, which the bacteria uses to cut viral DNA which invades them)/ Multiply copies of DNA (amplify DNA) through PCR (polymerase chain reaction)/ Separate DNA through Gel electrophoresis: Use agarose gel, Load DNA with a loading dye into wells, Buffer solution conducts a current which is applied/ Visualise DNA: Stain e.g. ethidium bromide or Southern blotting e.g. radioactive/fluorescent probes/ Compare/analyse: Analyse samples by comparing position of bands, width of bands and number of bands – if these match, samples share DNA/genetics
Describe the method to extract DNA: Open cells: Add 'lysis' (to separate) buffer, detergent which disrupts cell membranes and Proteinase K (enzyme) which cuts DNA from histones/ Separate DNA from cellular debris: Add salt solution which makes proteins and cell debris clump together, run through centrifuge/ Isolate pure DNA sample: Take solution of Eppendorf tube, add isopropyl alcohol (DNA is insoluble in alcohol), run through centrifuge to obtain a mass of DNA/ Left with purified DNA sample
Explain the process of cutting DNA: Restriction enzymes cut DNA, they can cut DNA in very specific places, often related to the specific DNA bases. This is very useful in biotechnology/ Need to use same type of restriction enzyme in all DNA samples to compare them/ Using the same enzymes should mean the DNA is being cut in the same identification points. Unless they're clones, those pieces may be differently sized as DNA has some differences/ During electrophoresis, some fragments of DNA move quicker or slower and thus spread out to create DNA bands which can be compared
Amplifying DNA using polymerase chain reaction (PCR)
STAGE 1 – REACTION MIXTURE: Free nucleotides/ DNA primer (short section of DNA with fluorescent marker)/ Taq polymerase (type of DNA polymerase that withstands high temperatures) STAGE 2 – DENATURATION: Heat to 95oC/ Causes strands to separate/ Makes 2 strands of DNA STAGE 3 – Annealing: Lower temperature to 55oC/ Primers anneal (attach) STAGE 4 – Extension: Heat to 70oC/ Taq polymerase form complementary base pairs between exposed DNA and free nucleotides STAGE 5 – REPEAT: Repeat several times to replicate DNA
Describe the process of gel electrophoresis:(1) Used to determine relatedness between species which help scientists to better classify organisms (2) Helps in genetic fingerprinting e.g. crime scene victim vs. criminal (3) Can take results and isolate genes of interest through southern blotting (4) For gel electrophoresis there are several different stains that can be used. Ethidium bromide is one such stain. The stain we used to stain DNA in the gels you made is called toluidine blue.(5) Set up electrophoresis tank. Contains an agarose gel (polysaccharide polymer) which lets DNA molecules travel through. Electrodes are attached to either end, connected to a power supply (36 volts) (6) One end of the gel has wells in it, this is where DNA is placed into (7) End of the tank where the wells are placed needs to be negatively charged (closest to cathode) and the opposite end needs to be positively charged (closest to anode) to allow DNA to travel from cathode to anode (8) Immerse gel in buffer solution (contains ions which conduct a current) (9) Load DNA sample (DNA solution, restriction enzymes and loading dye) into wells and run the gel (10) DNA is slightly negatively charged (phosphate contribute negative charge), so DNA sample will be repelled by the cathode and move toward the anode (11) DNA fragments will move through the gel. Different sized pieces of DNA will spread throughout the gel e.g. larger, longer STRs which are made up of longer strings of DNA letters are heavier than shorter STRs and thus will move more slowly (12) DNA bands are created this pattern of bands is a genetic profile/fingerprint. Gel is stained to reveal these
Describe the process of Southern Blotting and hybrdisation: Double stranded DNA is separated into single strands/ Southern Blotting*: Membrane incubated with labelled DNA probes (radioactive phosphorus or fluorescent marker)/ The membrane is placed directly onto the gel and a wad of dry absorbent paper is placed on top/ This acts as a wick to draw buffer solution up through the gel, carrying DNA fragments onto the membrane/ During this process, the fragments maintain their positions relative to each other and are denatured into single strands, exposing the base sequences.