1. PCR and Gel Electrophoresis The Polymerase Chain Reaction –Denaturation, Annealing, Elongation Gel Electrophoresis Application in forensic science

2. The Polymerase Chain Reaction A process that is used to make a huge number of copies of a DNA sequence in a laboratory, quickly and without the need for a host organism PCR is DNA replication of a specific region of the genome, outside of the nucleus of a cell The whole process takes place in a microfuge tube (a small test tube) and consists of three steps: denaturation, annealing, and elongation.

3. Step 1 - Denaturation Cycle 1 The double-stranded DNA molecule is denatured, (separated), into its two single strands. This occurs when the DNA strand is heated to 94 to 96 °C for 20 to 40s, causing the hydrogen bonds holding the two strands together to break.

4. Step 2 - Annealing Cool the mixture to 50–65 °C to allow a primer to anneal (attach) to the 3’ end of the target sequences on both template strands.

5. Step 3 - Elongation Elongation: Heat to 72°C for DNA polymerase (Taq polymerase) to extend the primers using free-floating nucleotide precursors to make complementary copies of each template strand. –Taq polymerase is a special DNA polymerase found in the bacteria. It can function in these temps without being denatured. Note, the source nucleotides is huge and never runs out.

6. Cycle 3 Repeat the same steps again Notice that now we are creating segments with pure target DNA sequence (yellow) and nothing extra Once this repeats 20 times, most of the segments will be pure target DNA

8. Polymerase Chain Reaction (PCR)

9. Summary Within 20 cycles, the PCR reaction delivers over one million copies of the DNA target sequence. Since each cycle, regulated simply by increasing and decreasing the temperature, takes only about 5 min, millions of copies of a DNA sequence can be synthesized in less than 2 h!

10. Summary As we are amplifying our DNA segment using additional base pairs, only a very small amount of a biological sample is required. There is enough DNA in a hair follicle or a tiny blood spatter to perform PCR. Once the DNA of interest has been amplified using PCR, it is analyzed using different methods

11. Gel Electrophoresis Once the targeted DNA sequence has been amplified by PCR, it needs to be separated and purified from the rest of the contents in the test tube. A process called gel electrophoresis, uses the physical and chemical properties of DNA to separate the fragments.

12. Gel electrophoresis separates nucleic acids and proteins by how fast they move through a gel made of Agarose (a polysaccharide from seaweed) Negatively charged DNA fragments travel through the gel, away from a negative electrode at the starting end and toward a positive electrode at the destination end The smaller the DNA fragment, the faster it moves through the gel

13. Step 1

14. Step 2 A molecular marker is a DNA fragment of known size that is used to determine the lengths of unknown fragments by comparison (a standard) Multiple markers of different known sizes are used

15. Step 3 Apply an electric current to the gel. DNA fragments are negatively charged, so they migrate toward the positive pole. Shorter DNA fragments migrate faster than longer DNA fragments.

16. Step 4 To be able to see the DNA fragments once the process is complete, the electrophoresis gel is stained

17. Why? Using gel electrophoresis, researchers can compare the size of the DNA fragments from an unknown sample to the size of the DNA fragments from a known sample They can then reach conclusions about the identity of the unknown sample. These conclusions may include: –the identity of the tissues of an endangered species –the identity of the perpetrator of a crime –the father in a paternity test on the Maury Povich Show.

18. How? When we cut the DNA via PCR, we choose target sequences on the DNA that are variable among people. –No two people will have the same number of bases in those areas –Example Target Sequence# of bases for Susan# of bases for Jim A100115 B5677

19. We then compare our results with our known DNA (e.g. Evidence) Those who are a match will have identical results and those who are related will have similar results Therefore we can conclude that Susan is guilty because her DNA matches that left at the scene Target Sequence# of bases for Susan # of bases for JimKnown # bases left at the crime scene A100115100 B567756

20. Who is the daddy?

21. However, its looks more like this. Who is guilty???

22. Restriction Enzymes

23. Discovery In 1962, Werner Arber, a Swiss biochemist, provided the first evidence for the existence of "molecular scissors" that could cut DNA. He showed that E. coli bacteria have an enzymatic “immune system” that recognizes and destroys foreign DNA, and modifies native DNA to prevent self-destruction.

24. Molecular Scissors By the early 1970s these enzymes started to be identified and purified. It was shown that each species of bacteria had its own population of a SPECIFIC restriction enzyme. Each enzyme recognized its own specific sequence of DNA bases. It is at this sequence that the DNA was cut.

25. Uses in Biotechnology Restriction enzymes are isolated from bacteria for use in biotechnology research. The function of restriction enzymes in bacterial cells is to cut apart foreign DNA molecules (i.e. from bacteriophages). Restriction enzymes are named from the bacterium from which it was discovered. For example: EcoRI

26. EcoRI E—the first letter of the Genus name of the bacterium, Escherichia. co—the first two letters of the species name of the bacterium, coli. R—a particular strain (type) of this bacterium, strain RY13. I—the particular order among several produced by this strain.

27. Restriction Enzyme Recognition Sequences Restriction enzymes cut DNA in areas of specific base pair sequences, called restriction sites. What do the following phrases have in common?: –Doc, note I dissent: a fast never prevents a fatness. I diet on cod. –Dog DNA and God Restriction enzymes usually recognize palindromes in the nucleotide sequences

28. Restriction Enzyme EcoRI Eco RI recognizes the sequence 5’….GAATTC….. A cut is made between the G and the A on each strand. This restriction enzyme leaves the nucleotides 5’AATT overhanging. These are known as “sticky ends” because hydrogen bonds are available to “stick” to a complimentary 3’TTAA Note: Restriction enzymes don’t stop with one cut! They continue to cut at every recognition sequence on a DNA strand. Restriction Enzyme Cut from EcoRI

29. Restriction Sites as “Molecular Signposts” Using two, or more different restriction enzymes on a DNA fragment enables those restriction sites to be mapped onto that DNA fragment. Scientists create a “Restriction Map”, showing the locations of cleavage sites for many different enzymes.

30. Restriction enzymes, DNA, and Electrophoresis DNA normally comes in “Genome sized” lengths (usually several million bp in length.) These are the “elephants” in the race through the agarose and can’t enter the gel matrix when they are this big. Restriction enzymes made possible the cutting of DNA into smaller fragments together with their separation and visualization by agarose gel electrophoresis.

31. Your Turn: Restriction Enzyme mapping challenge- Exercise 1 and 2

32. Eco Eco Digest Eco cuts to yield two DNA fragments

33. Eco Eco Bgl Bgl Or Bgl Digest Bgl also cuts to yield two DNA fragments. But where is the Bgl site in relation to the Eco site?

34. Eco Bgl Eco Bgl Double Digest Shows it must be: A restriction digest with both Eco and Bgl enzymes provides the answer.

35. Plasmid Mapping

36. CRISPR/Cas9 System Dr. Jennifer Doudna- UC Berkeley Dr. Emmanuelle Charpentier-Germany Science 2012.