DNA Fingerprinting DNA Fingerprinting DNA Fingerprinting technique for identifying individuals based on their unique DNA Such as identifying crime scene suspects Individuals have significant differences in base pair sequencing called polymorphisms of noncoding regions DNA Fingerprinting Involves four procedures 1 Isolate genomic DNA 2 Amplify polymorphic region s 3 Cut DNA into smaller fragments 4 Visualize DNA fragments and determine their sizes Isolate Genomic DNA DNA can be obtained from almost any tissue or biological Hair saliva blood semen urine teeth bone However DNA obtained from samples is typically limited in fluid amount Amplify Polymorphic Regions Polymerase Chain Reaction PCR is an important research technique used to increase the quantity of DNA Amplify Polymorphic Regions One PCR cycle DNA strands separated by heat denature DNA primers bind to template strands annealing DNA polymerase synthesizes new strands using free dNTPs nucleotide monomers extension Amplify Polymorphic Regions Amount of DNA doubles with each cycle making rate of synthesis exponential Cut DNA into Smaller Fragments Some bacteria protect themselves from viruses by Creating a restriction endonucleases that recognize a specific sequence of base pairs and cut any DNA at theses sites Cut DNA into Smaller Fragments We can use these restriction endonucleases To cut up replicated polymorphisms Creating various fragments of different lengths Leads to fragments of different sizes Cut DNA into Smaller Fragments Two examples are EcoR1 and Sal1 Visualize DNA Fragments Restriction digestion from two different individuals will result in different Number of DNA fragments Sizes of DNA fragments To visualize the DNA fragments for comparison Agarose gel electrophoresis Visualize DNA Fragments Agarose is a polysaccharide like agar or pectin Dissolves in boiling water and then gels as it cools A comb is placed in the liquid agarose so when gel solidifies produces a series of wells Used to load DNA into gel Visualize DNA Fragments DNA sample is loaded with a loading buffer containing dyes Electric current is applied across the gel DNA is negatively charged due to PO4 Migrates from the negative black electrode to the positive red electrode Visualize DNA Fragments Rate of migration of DNA through agarose depends on the size of DNA Smaller DNA fragments move more quickly Larger DNA fragments move more slowly Visualize DNA Fragments To visualize DNA fragments Ethidium bromide Intercalates between the bases of double stranded DNA When bound to DNA it fluoresces under UV light Very sensitive Ethidium bromide is a mutagen Visualize DNA Fragments Once visible you can compare different sizes of DNA fragments between individuals Often use DNA marker ladder Contains known lengths base pairs of DNA fragments DNA marker 1kb ladder Visualize DNA Fragments Gel Electrophoresis Overview Ladder Sample 1 Sample 2 Sample 3 1 Add ladder and DNA samples to wells 2 Run electrical current 3 Visualize under UV light 4 Compare fragments Estimate size of each fragment using ladder 1000 500 100 50 Gel Electrophoresis UV light box is used to visualize separated DNA fragments of the gel Well 1 Ladder 2 CS 3 S1 4 S2 5 S3 6 S4 7 S5 8 S6 Get Started Instructor loads DNA ladder into first well 1 2 Spin down samples in bubblefuge Needs counter weight 3 Add loading dye to samples 2 ul Black mark on strip indicates crime scene 4 Spin down samples in bubblefuge again 5 Load the wells of your gel 10 ul 6 Turn on power supply and run at 120V for 30 minutes 7 Turn off power supply 8 Carefully remove gel into petri dish 9 Teacher will image gel have a camera phone ready for pictures Needs counter weight Clean Up Used gels microtubes with DNA samples and parafilm go in the trash Turn off equipment If you lab is the last of the day pour running buffer down the sink Wipe down benchtop