2. The Search for a Jumping Gene: Module based on a kit from Bio-Rad Laboratories, Inc. Adapted by Dan Murray from a presentation by: Stan Hitomi - Monte Vista High School, Danville, CA. Kirk Brown - Tracy High School, Tracy, CA. Use PCR to Determine Your Molecular Genotype

3. Outline What is PCR? PCR Mechanism Transposable Elements PV92 PCR Experimental Protocol Expected Results

4. What is PCR?

5. PCR stands for Polymerase Chain ReactionPCR stands for Polymerase Chain Reaction The technique was invented by Kary Mullis in 1983. Awarded the Nobel Prize in Chemistry in 1993.The technique was invented by Kary Mullis in 1983. Awarded the Nobel Prize in Chemistry in 1993.

6. What is PCR? DNA replication gone crazy in a tube!DNA replication gone crazy in a tube! Only a small region of the DNA actually gets replicatedOnly a small region of the DNA actually gets replicated

7. What’s in a PCR Reaction? Source DNA – contains target DNA region to be copiedSource DNA – contains target DNA region to be copied Primers – short single stranded DNAs complementary to ends of target DNA regionPrimers – short single stranded DNAs complementary to ends of target DNA region Nucleotides – dATP, dCTP, dGTP, dTTP; the building blocks of DNA strandsNucleotides – dATP, dCTP, dGTP, dTTP; the building blocks of DNA strands Taq DNA polymerase – temperature resistant enzyme which builds DNA strandsTaq DNA polymerase – temperature resistant enzyme which builds DNA strands

8. PCR Mechanism

9. How does PCR work? Mix PCR reaction reagentsMix PCR reaction reagents Cycle through various temperaturesCycle through various temperatures 94 o C – Melting 94 o C – Melting ~55 o C – A nnealing~55 o C – A nnealing 72 o C – Extension 72 o C – Extension Repeat Repeat

10. Melting: 94ºC Heat separates strands 3’ 5’ 3’ 94 o C 5’ 3’ 5’

11. Annealing: ~55ºC Primers bind template 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ ~55 o C

12. Extension: 72ºC Taq polymerase builds strands 72 o C 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’

13. Desired-length PCR product made starting in third cycle Cycle 1 Cycle 2 Cycle 3 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’3’ 3’3’ 3’

14. Transposable Elements

15. What are Transposable Elements? Segments of DNA which have the ability to move to or be copied to other regions of the genomeSegments of DNA which have the ability to move to or be copied to other regions of the genome Replicate Element Donor molecule Recipient molecule Generally, they are thought of as “Selfish DNA”; they usually neither benefit nor harm their hostGenerally, they are thought of as “Selfish DNA”; they usually neither benefit nor harm their host Discovered by Barbara McClintock (1940s-50s) while working with maizeDiscovered by Barbara McClintock (1940s-50s) while working with maize

16. Alu Elements Alu elements are a type of transposable elementAlu elements are a type of transposable element Alu elements are 300bp in lengthAlu elements are 300bp in length Found only in primates (including humans)Found only in primates (including humans) Approx. 500,000 Alu copies per haploid genome, representing about 5% of the genome!Approx. 500,000 Alu copies per haploid genome, representing about 5% of the genome! Named for the Alu I restriction site within the elementNamed for the Alu I restriction site within the element

17. Alu at PV92 PV92 is a locus on human chromosome 16PV92 is a locus on human chromosome 16 An Alu insertion occurred there within the last 1,000,000 yearsAn Alu insertion occurred there within the last 1,000,000 years Possible Genotypes at PV92: +/+ +/- -/-Possible Genotypes at PV92: +/+ +/- -/- Used in population genetics, paternity analysis, and forensicsUsed in population genetics, paternity analysis, and forensics Not associated with any phenotypeNot associated with any phenotype

18. PV92 PCR

19. The Target DNA Chromosome 16 PV92 Locus Alu Element

20. Some Got It, Some Don’t PV92 with Alu PV92 without Alu PCR

21. Experimental Protocol

22. The Procedures in a Nutshell Isolate DNA from cheek cellsIsolate DNA from cheek cells Build PCR reactionBuild PCR reaction Do PCR in thermal cyclerDo PCR in thermal cycler Analyze using agarose electrophoresisAnalyze using agarose electrophoresis

23. Isolation of Cheek Cell DNA Collect cheek cells and place in InstaGene Matrix Matrix binds cellular Mg 2+ Agitate Disperses cells in Matrix

24. Isolation of Cheek Cell DNA 56°C 100°C Loosens connective tissue and inactivates nucleases Ruptures cell membranes and denatures proteins

25. Isolation of Cheek Cell DNA Gets rid of InstaGene Matrix Target DNA now ready!

26. PCR reaction now ready to go! Assemble PCR Reaction Add PCR Master Mix containing: Nucleotides Primers Reaction buffer Electrophoresis dyes Taq polymerase

27. Controls will show the three possible outcomes Amplify DNA with PCR

28. Stain gels and analyze. Load, Run, and Stain Gel

29. Expected Results

30. Primer binding sites PV92 Amplification Specifics Region to be amplified Now assume an Alu element has inserted at that locus Alu element Region to be amplified No Alu: With Alu:

31. PV92 Amplification Specifics Region to be amplified 641bp300bp941bp No Alu: With Alu:

32. 941 bp 641 bp -/- +/- +/+ Actual Results

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