Lab 8: Amplification of the tPA Locus using the Polymerase Chain Reaction (PCR)

1. Lab 8:Amplification of the tPA Locus using the Polymerase Chain Reaction (PCR)

2. Warm Up You have 10 minutes: • Read 8.1 in the lab manual • On the page write a short summary of the reading • Write one question you have

3. Why are we doing this? To use a very powerful technique to amplify buccal cell DNA and determine student genotypes!

4. Three Steps to DNA Testing • Extracting the DNA from the cells • Amplification of the DNA using PCR • Analysis of the DNA using gel electrophoresis

5. Where do we find DNA in eukaryotic cells? • Nucleus • Mitochondria and Chloroplasts

6. Extracting your DNA 1. Obtain numbered chelex tube (record the number in your notebook) 2. Use a sterile pipette tip to scrape the inside of both cheeks 3. Add cheek cells to chelex tube 4. Boil (lyse cells and destroy nuclease) 10 minutes 5. Centrifuge for 5 minutes 6. Transfer 20uL of DNA to microtube tube AVOID chelex beads 5 ul PCR tube Heat Centrifuge Transfer 20 ul supernatent

7. What is PCR? • PCR is a technique used to produce millions of copies of a selected gene segment or locus of DNA. • The PCR technique was invented by Dr. Kary Mullis in 1983. He was awarded the Nobel Prize in Chemistry in 1993.

8. In criminal forensics, PCR is used to amplify DNA evidence from small samples that may have been left at a crime scene. PCR can be used to amplify DNA for genetic disease screening. Where is PCR used?

9. How does PCR work? Ingredients 1. First we add all our ingredients into our PCR tube: Template DNA – Which contains the the DNA fragment to be amplified Primers – are 2 short single stranded polynucleotides that flankthe sequence to be amplified • Forward • Reverse Nucleotides – the building blocks for new DNA strands dATP, dCTP, dGTP, dTTP Taq DNA polymerase is a temperature resistant enzyme which builds DNA strands. Taq was isolated from thebacteriumThermusaquaticus, which normally lives in hot springs in temperatures around 100° C. Taq is stable under the extreme temperature conditions of PCR. Magnesium chloride - enzyme cofactor help Taq Polymerase work efficiently Buffer – a solution which maintains the pH and provides a suitable chemical environment for PCR

10. How Does PCR Work? 2. Then we put or PCR tube in a thermal cycler, or PCR machine The PCR process usually consists of a series of twenty to thirty-five cycles. Each cycle consists of three steps. Step 1: Denaturing temperature is raised to 94-96°C to break hydrogen bonds Step 2: Annealing temperature is lowered to 56°C to allow primers to attach to the target sequence Step 3: Elongation or Extension temperature is raised 72°C Taq polymerase binds and adds nucleotides to build new DNA strands

11. Building new DNA Fragments

12. What do we use? DNA sample (5 µL) Master mix (20 µL/reaction): 2.5 µL 10x PCR buffer w/o MgCl2 OR 3.25 µL 10x PCR buffer w/ MgCl2 0.5 µL dNTP’s (10 mM) 2.5 µL Forward primer (4pM/ µL) 2.5 µL Reverse primer (4pM/ µL) 0.15 µL Taqpolymerase 11.1 µL ddH2O Note: if you are using 10x PCR buffer w/o MgCl2, you will need to add 0.75 µL MgCl2 (50 mM)

13. Chromosome 8 The region we will be amplifying is located in an intron (non Translated region), of the tPA gene located on Chromosome 8.

14. Chromosome 8 and the tPA Gene The diagram indicates the intron we will be targeting for PCR. The intron that we will be targeting for amplification is dimorphic, which means the locus has two forms. • one form carries a 300 bp DNA fragment known as an Alu element • the second form of the locus does not carry this fragment.

15. The tPA Gene • The locus (ALU) we will amplify is located in the tissue Plasminogen Activator (tPA) gene. • This gene is on chromosome 8. • The gene codes for a protein that is involved with dissolving blood clots. • tPA is a protein given to heart attack victims to reduce the incidence of strokes.

16. What are ALU elements? • Alu elements are short, around 300bp, DNA fragments that are distributed throughout our genome. • Estimated that we may carry over 1,000,000 copies of this fragment.

17. Gel Electrophoresis • Gel electrophoresis is a method that uses an electrical current and a gel matrix to separate molecules like DNA and proteins according to their size and electrical charge. STEPS • Put samples into wells • Run electrical current

18. Set Up Your Lab Notebook Title Date Purpose Method (Flowgram 8.2) Note where DNA is in each step. Observations for today

19. When Finished Do NOT take off any of your safety equipment unless your lab table is completely doneAND you have completelycleaned-up your lab equipmentand bleached your table. • Read 8.3 • Flowgram for making gels and 8.4 (Start at step 3) • Read and annotate Protein Structure and Gene Regulation Packet WASH HANDS

20. Where is your DNA? 5 ul PCR tube Heat Centrifuge Transfer 20 ul supernatent

21. Making a 50 mL 2% Gel • Weigh 1 g of agarose and add to your flask • Add 50 mL 1x SB buffer • Microwave until it has all dissolved • Pour into gel box with combs

22. How to Study • Start early • Be an active studier • Study with others • Overstudy

23. Create 2 note cards for yourself • The three steps of DNA Testing • PCR Side One • Title and picture Side Two • Definition • Example • In your own words

24. Possible Genotypes and Expected Results: • Marker • 2. Homozygous Alu + (400bp sequence) • 3. Homozygous Alu – • (100bp sequence) • 4. Heterozygous • (400bp sequence and 100bp sequence)

25. Gel Electrophoresis • Gel electrophoresis is a method that uses an electrical current and a gel matrix to separate molecules like DNA and proteins according to their size and electrical charge. Virtual Gel Electrophoresis

26. Loading and Running Gels: • Carefully remove comb from gel, put dams down on both ends of the gel tray • Place gel tray into gel box with buffer ensuring that the wells are closest to the black electrode • Load samples • Once everyone has loaded their sample plug red electrode to red and black electrode to black on power supply, make sure the power is turned off on power supply before connecting electrodes! • Adjust voltage to 125-135 volts allow gel to run for about 10 minutes or until you see the dyes separate. • After gel run is complete, turn off power supply and unplug electrodes

27. Loading and Running Gels continued • Your gel is now ready to be stained and photographed • Answer questions at the end of Lab 8

28. Gel Loading Techniques

29. Gel Loading Techniques

30. WHILE YOU ARE WAITING • Write your observations -Make a table to show which samples are in which lanes e.g. Lane 1-Marker • Skip 3 whole pages and start a new lab 2 -Title & Date (October 29, 2012) -Purpose -Method

31. WHEN YOU ARE FINISHED • RESULTS -Tape in image -Title image Figure 1: Photo of Alu elements of the tPA gene for student samples -Paragraph description of results in words (2 bands or one) referencing Figure 1 • CONCLUSION -Description explaining results • POST LAB QUESTIONS

32. Pre Lab Readiness • Genetics is the study of heredity: How biological information is transferred from one generation to the next as well as how that information is expressed within anorganism. • DNA Replication is the process of making an identical copy of a section of duplex (double-stranded) DNA, using existing DNA as a template for the synthesis of new DNA strands. In humans and other eukaryotes, replication occurs in the cell nucleus. • (Animation:http://www.johnkyrk.com/DNAreplication.html) • Genes are units of information about specific traits. They are passed from parents to offspring. Each gene has a specific location on a chromosome. • Genotype is the genetic constitution (the genome) of an individual or an organism.

33. Pre Lab Readiness (continued) • Phenotype is the observable physical or biochemical characteristics of an individual or organism. • Alleles are alternative forms of a gene. If two alleles of a pair are the same, it is a homozygous condition. If the two alleles are different, this is called a heterozygous condition. • Polymerase Chain Reaction (PCR) is an in vitro process that yields millions of copies of desired DNA through repeated cycling of a reaction involving the DNA polymerase enzyme. • Thermalcycler is a laboratory apparatus used to amplify segments of DNA via the polymerase chain reaction (PCR) process[. The device has a thermal block with holes where tubes holding the PCR reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps.

34. 10-6 micro µ 10-9 nano n 10-12 pico p

35. Lab 8 terms • Buccal Cells are cells from the inner cheek lining. • Chelex beads bind divalent magnesium ions (Mg++) which serve as cofactors for nucleases that will degrade DNA. • Nuclease a family of enzymes that will degrade nucleic acids (DNA). • Amplification an increase in the number of copies of a specific DNA fragment. • Intron segment of a gene that does not code for protein. Introns are transcribed into mRNA but are removed before being translated into protein. • Exon segment of a gene that encodes regions of protein