BREAKING NEWS! • Cheese was stolen from the Queen of England! • The cheese was on a table in the sitting room, & half of it had been eaten by a thief! • Officer Li Gase dusted for fingerprints & found none – the maid claimed that they had been wiped clean earlier. • The half-eaten cheese was sent to the lab for further tests.
How can we find out who stole the cheese? • What kinds of tests can we do in the lab? • We will need some training in forensics…
Remember! • The chemical structure of everyone’s DNA is the same! • The only difference between people (or any other organism) is the order of the base pairs (A, T, G, & C). There are so many millions of base pairs in each person’s DNA that every person has a different sequence!
DNA Fingerprinting • Using these sequences, every person could be identified solely by the sequence of their base pairs. • Scientists are able to determine whether 2 DNA samples are from the same person, related people, or non-related people! • Scientists use a small number of sequences of DNA that are known to vary among individuals a great deal, & analyze those to get a certain probability of a match!
1st Step – PCR • Polymerase Chain Reaction: used to make millions of exact copies of DNA from a biological sample This allows us to run tests on the DNA without using cells, even if we start with just a small amount of the DNA sample!
PCR – Polymerase Chain Reaction • The DNA sample is incubated in a test tube with a special DNA Polymerase, a supply of nucleotides, & short pieces of single-stranded DNA primers.
PCR – Polymerase Chain Reaction • A primer is added to the beginning of the isolated desired gene. • DNA is heated to break the hydrogen bonds between the nitrogen bases. • DNA is cooled to allow the primers to hydrogen bond. DNA Polymerase attaches & replicates both strands. • Copies are made at an exponential rate of only the desired gene! Click
Scientists have used PCR to amplify DNA from a variety of sources. • Fragments of ancient DNA from a 40,000 year old frozen wooly mammoth • DNA from a single embryonic cell for rapid prenatal diagnosis of genetic disorders • DNA of viral genes from cells infected with difficult-to-detect viruses like HIV • DNA from a tiny amount of blood or semen found at the scenes of violent crimes
2nd Step – Restriction Enzymes • Restriction Enzymes: proteins found in bacteria that cut both strands of DNA only at specific sequences
Restriction Enzymes There are hundreds of restriction enzymes, & each cuts DNA at a specific sequence!
The enzyme HaeIII cuts DNA wherever it encounters this sequence: 5‘-GGCC-3‘ 3‘-CCGG-5‘ • The cut is made between the G and C Here, we now have 2 fragments (pieces) of DNA!
The Sma I enzyme recognizes and cuts (between the C and G) at this sequence: 5’-CCCGGG- 3’ 3’ -GGGCCC- 5’
Some REs leave DNA pieces with staggered ends called “sticky” ends. • This is because they have nucleotides that are exposed & can easily join back together with a complementary DNA strand.
How many DNA fragments (pieces) do we get when we cut this DNA sample? • 2 • 1 • 8 • 4 4 fragments!
What sequence does this enzyme recognize and cut? A. 5’-GGCC-3’ 3’-CCGG-5’ B. 5’-CCGG-3’ 3’-GGCC-5’ C. 5’-GCCG-3’ 3’-CGGC-5’ 5’-CCGG-3’ 3’-GGCC-5’
Why are restriction enzymes used in DNA Fingerprinting? • To break the DNA up so we only have to test 1 of the fragments • To get revenge on the criminals by chopping up the DNA • Each person has a unique DNA sequence, so using restriction enzymes on different people would result in a different number of DNA fragments • All of the above
Two different DNA samples cut with the same enzyme, results in different fragments!
So far in our DNA Fingerprinting, we have: • Made copies of the DNA samples with PCR • Cut the DNA samples into fragments using restriction enzymes • Remember!The ultimate goal of DNA Fingerprinting is to compare DNA sequences! • So now what?
3rd Step – Gel Electrophoresis • Used to separate DNA fragments by size • The cut DNA is now fragments of different lengths. When added to the gel, an electrical current will separate the DNA fragments! • The small fragments travel further, while the larger fragments don’t travel as far.
Gel Electrophoresis • Made with a rectangular piece of gel, with spots at the top to insert the DNA samples • The bottom of the gel is positively (+) charged. • DNA is negatively (-) charged, so when the electrical current is turned on, the DNA fragments will move to the bottom of the gel!
DNA fragments are added to the top of the gel. Electric current is turned on. DNA fragments separate according to size. Remember! The smaller fragments will travel further!
How many fragments was DNA Sample “A” cut into? • 3 • 4 • 2 • None Sample A was cut into 4 fragments! You can see them separated here.
Which fragment is the largest? • Fragment #1 • Fragment #2 • Fragment #3 • All the same size Fragment #1 Fragment #2 Fragment #1 is the largest! It did not move very far down the gel. Fragment #3
If DNA sample “A” was the DNA found on the stolen cheese, which suspect can we prove guilty? • Suspect with DNA “B” • Suspect with DNA “C” • Suspect with DNA “D” • Not enough information Suspect with DNA “C”! Notice how it matches perfectly with sample “A”!
Applications of DNA Fingerprinting • Forensics: solving crimes by analyzing the DNA • Which suspect has DNA that matches the DNA from the crime scene?
Applications of DNA Fingerprinting • Paternity Tests: the child will have similar DNA sequences to mom AND dad • If Mary is the mother, who is the father? Bob or Larry? How do you know?
Applications of DNA Fingerprinting • Evolutionary Studies: shows how closely related species are to each other • Which of these organisms are most closely related to each other?
Forensics 101 • You’ve now completed a crash course in forensics! • We still haven’t found out who stole the Queen’s cheese… How could we use the knowledge we gained today to investigate?