Lab. 8 & 9

1. Lab. 8 & 9 Polymerase Chain Reaction (PCR)

2. Objectives: 1.Introduce the students to the preparation of the PCR reaction. 2.Examine the PCR products on agarose gel electrophoresis. 3.Explain some of PCR applications.

3. Introduction PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro. It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993.

4. PCR is used to amplify specific regions of a DNA strand (the DNA target) in vitro. This can be a single gene, a part of a gene, or a non-coding sequence

5. What is PCR? :( Polymerase Chain Reaction)Why “Polymerase”? It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.

6. Why “Chain”? It is called “chain” because the products of the first reaction become substrates of the following one, and so on.

7. The “Reaction” Components 1) Target DNA - contains the sequence to be amplified. 2) Pair of Primers - oligonucleotides that define the sequence to be amplified. 3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks. 4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction. 5) Mg++ ions - cofactor of the enzyme. 6) Buffer solution - maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme.

8. Once assembled, the reaction is placed in a thermal cycler, an instrument that subjects the reaction to a series of different temperatures for set amounts of time. This series of temperature and time adjustments is referred to as one cycle of amplification. Each PCR cycle theoretically doubles the amount of targeted sequence (amplicon) in the reaction.

9. The Reaction PCR tube thermocycler

10. PCR Procedures :steps • Each cycle of PCR includes steps for DNA template : 1. Denaturation :94°C 15 sec_2 min 2. Primer annealing:40–60°C 15 sec_60 sec 3. Primer extension:70–74°C 1–2 minutes

11. Denature (heat to 94oC) Lower temperature to 55oC Anneal with primers Increase temperature to 72oC DNA polymerase + dNTPs

12. First step: Denaturation : Heating denatures the target DNA by heating it to 94°C for 15 seconds to 2 minutes. The two intertwined strands of DNA separate from one another, producing the necessarysingle-strandedDNA template for replication by the thermostable DNA polymerase.

13. ٍsecond step:primer annealing: The temperature is reduced to approximately 40–60°C. At this temperature, primers can form stable associations (anneal)with the denatured target DNA and serve as primers for the DNA polymerase. This step lasts approximately 15–60 seconds.

14. Third step: primer extension the synthesis of new DNA begins as the reaction temperature is raised to the optimum for the DNA polymerase. For most thermostable DNA polymerases, this temperature is in the range of 70–74°C. The extension step lasts approximately 1–2 minutes.

15. The next cyclebegins with a return to 94°C for denaturation , annealling then extention and so on. At 30 cycles there are 1,073,741,764 target copies (~1×109). After 30–40 cycles, the amplified product may then be analyzed for size, quantity, sequence, etc., or used in further experimental procedures. *Number of amplicon=2^n

16. Denature (heat to 94oC) Lower temperature to 55oC Anneal with primers Increase temperature to 72oC DNA polymerase + dNTPs

21. Thermusaquaticus : • is a species of bacterium that can tolerate high temperatures one of several thermophilic bacteria. • It is the source of the heat-resistant enzyme Taq DNA Polymerase, one of the most important enzymes in molecular biology because of its use in the PCR. • Taq produces an enzyme called DNA polymerase, that amplifies the DNA from the primers by the polymerase chain reaction, in the presence of Mg.

22. Applications of PCR Classification of organisms Genotyping Molecular archaeology Mutagenesis Mutation detection Sequencing Cancer research Detection of pathogens DNA fingerprinting Drug discovery Genetic matching Genetic engineering Pre-natal diagnosis

23. MOLECULARIDENTIFICATION:

24. Detection Of Pathogens Molecular Identification:

25. Detection Of Pathogens Molecular Identification:

27. Prenatal Diagnosis 644 bp 440 bp 204 bp Molecular Identification: • Chorionic Villus • Amniotic Fluid Molecular analysis of a family with an autosomal recessive disease.

29. Chorionic villi are tiny, finger-shaped growths found in the placenta. The genetic material in chorionic villus cells is the same as what is in fetal cells.

30. forensic testing

31. M S1 S2 V E M M S1 S2 V E M M S1 S2 V E M Which suspect—S1 or S2—was at the crime scene? (V = victim, E = crime scene evidence, M = molecular weight standard)

33. Summary blood, chorionic villus, amniotic fluid, semen, hair root, saliva 68,719,476,736 copies Gel Analysis, Restriction ,Sequencing

34. Reagent Sterile deionized water 10X Taq buffer 2 mM dNTP mix Primer I Primer II Taq DNA Polymerase 25 mM MgCl2 Template DNA

35. PCR Thermocycler Example of PCR program: 1- 94C for 5 minutes x 1 cycle 2- 94C for 1 minute 3- 55C for 1 minute 4- 72C for 1 minute 5- Repeat step 2 x 40 cycles 6- 72C for 7 minutes x 1 cycle 7- hold at 4C until ready to load onto gel