DNA Analysis February 7 2014

DNA collection & handling What are the most common sources of DNA evidence? Blood and bodily fluids Tissue samples, esp skin Hair root sometimes Investigators collect blood or buccal swabs (inner cheek cells) for reference samples. DNA storage Blood and bodily fluids should be frozen Tissue samples should be dried, then frozen

DNA Extraction Before analysis, DNA must be extracted from cells. Soaps are used to dissolve the cell and nuclear membrane. Enzymes are used to break down the proteins that are bound to DNA. Salt is used to stabilize the DNA Alcohol is used to precipitate the DNA DNA extraction from banana – remember 9th grade?

DNA Amplification Often investigators find very little DNA at a crime scene, so it must be amplified, or copied, before analysis. DNA is amplified using a process called polymerase chain reaction (PCR).

PCR Steps Heat is used to separate the two strands of DNA. The solution is cooled and primers – short pieces of nucleotides that can begin the replication process -- are added. Taq polymerase, an enzyme that will attach individual nucleotides to the growing strands is added. Once the chain is complete, the process is repeated. The amount of DNA doubles each cycle. In three hours, one million copies can be made. 1 2 3 4 Watch me!

What are the steps to processing DNA that we have discussed so far? DNA collection – collecting evidence DNA extraction – extracting DNA from cells PCR – amplifying (copying) the DNA These steps would be done in any DNA forensic investigation. The next step is to analyze the DNA. There are many different methods of doing this.

DNA fingerprinting (RFLP analysis) DNA samples are cut into small pieces using restriction enzymes. Restriction enzymes recognize specific sequences of bases and cut the DNA at that point. Example: EcoRi recognizes the sequence GAATTC and cuts between the G and the A. G A A T T C C T T A A G This cuts the DNA into fragments of different lengths. Different people will have different lengths of fragments, based on the sequence of their DNA. Restriction Fragment Length Polymorphism

DNA fingerprinting (RFLP) DNA samples are cut into small pieces using restriction enzymes. Restriction enzymes recognize specific sequences of bases and cut the DNA at that point. Next, the fragments are sorted by size using gel electrophoresis. How does gel electrophoresis separate fragments by size? DNA is negatively charged, so it moves through the gel towards the positive electrode. Small pieces diffuse faster than large pieces, so the fragments end up forming distinct bands according to length, with small ones closer to the + end and large ones closer to the sample wells.

DNA fingerprinting (RFLP) DNA samples are cut into small pieces using restriction enzymes. Restriction enzymes recognize specific sequences of bases and cut the DNA at that point. Next, the fragments are sorted by size using gel electrophoresis. DNA fragments are visualized in some way (such as radioactive probes). The number and location of different-sized fragments of DNA can then be analyzed. Watch me!

DNA fingerprinting (RFLP) Shown here are fragments of DNA that have been cut with EcoRi, then separated according to length with electrophoresis. Where is the positive end of the gel and where is the negative, and how can you tell? Which fragment is which?

DNA fingerprinting (RFLP) Shown here are strands of DNA from two different people. The restriction sites for a particular enzyme are marked with horizontal lines.

DNA fingerprinting (RFLP)

DNA fingerprinting (RFLP)

RFLP activity – make a strand of DNA Work in groups of 2-3 Get a sheet of yellow lined paper. Cut it into four pieces length-wise. Tape the pieces together so that you have one long strip of paper. Write bases (A, C, G, T), one on each line down the length of the paper. Add the complementary bases next to them. This is your DNA strand.

RFLP activity – Use Restriction Enzymes You will now cut your piece of DNA into pieces using the simulated restriction enzyme TWI. This enzyme recognizes the sequence “A T” and cuts between the A and the T. A T C G T A G G G C Note that two strands are read in opposite direction. This is because DNA is antiparallel. Example: Two restriction sites

RFLP activity – Electrophoresis You will now simulate electrophoresis by sorting your strands of DNA according to size. Count how many lines long each piece of DNA is. We will make a class ‘gel’ on the board. Pick one lane and shade in the boxes that represent the lengths of your fragments. Size Group 1 Group 2 70-104 65-70 55-65 50-55 45-50 40-45 35-40 25-30 20-25 15-20 10-15 5-10 0-5