Basic Polymerase Chain Reaction for Bachelor Degree

This slide contain basic chemistry of PCR process with easy explanation and analogy

1. Polymerase Chain Reaction (PCR): Basic to Application Leonardo Tejo Gunawan,S.Si.,M.Biotek Hanyang Biomedical Research Institute Lab Division of StemCell Biology and Regenerative Medicine 2024.05.25

2. “Our world is built on Biology and once we begin to understand it, it then becomes Technology ” Ryan Bethencourt, American Scientist and Bio-Hacker INTRODUCTION

3. Old and Modern Biotechnology 1990-2003 Yeast utilization Wine, Beer Cheese

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5. Quick Review A PLASMID is a small circular DNA molecule found in bacteria and some other microscopic organisms. Plasmids are physically separate from chromosomal DNA and replicate independently. They typically have a small number of genes — notably, some associated with antibiotic resistance — and can be passed from one cell to another.

6. Quick Review Purin : Adenine. Guanine Pirimidine : Thymine, Cytosine Hidrogen Bond In general, DNA direction is from 5’ to 3’ Because the nucleotide chain structure is from C number 5 to C number 3 1 nucleotide consist of 1 N base, 1 sugar molecule, 1 phosphate group The entire DNA in an organism is called the GENOME 1 nucleotide purine base bonded to 1 nucleotide pyrimidine base is called 1 base pair (bp)

7. Quick Review Purin : Adenine. Guanine Pirimidine : Thymine, Cytosine In general, DNA direction is from 5’ to 3’ Because the nucleotide chain structure is from C number 5 to C number 3 1 nucleotide consist of 1 N base, 1 sugar molecule, 1 phosphate group The entire DNA in an organism is called the GENOME 1 nucleotide purine base bonded to 1 nucleotide pyrimidine base is called 1 base pair (bp)

8. Quick Review Gene is a region in DNA Genome that encodes a function and considered the basic unit of inheritance -> FENOTIPE Genes will be transcribed to form an mRNA which will produce amino acids

9. Basic of Polymerase Chain Reaction

10. Polymerase Chain Reaction (PCR) PCR Technology simplified this complicated process Basically, PCR is an in vitro embodiment of the DNA replication process that occurs in cells

11. Polymerase Chain Reaction (PCR) Kary Mullis (1993 Nobel Prize) Ideally, the yield result from PCR is 2 n Taq Polymerase -> Thermus aquaticus Pfu Polymerase -> Pyrococcus furiosus M-MLV Reverse Transcriptase A technology used for amplifying a millions of copies ( in vitro ) of a specific DNA sequence in approximately two hours

12. Polymerase Chain Reaction (PCR)

13. Polymerase Chain Reaction (PCR)

14. No. PCR Component Description 1. DNA template As template that will be amplified 2. PCR buffer Stabilized the pH 3. Cation (Mg2+) divalent As enzyme cofactor by catalyze the reaction 4. dNTP To create complementary base 5 . DNA polymerase To elongate and join the dNTP 6. Nuclease free water As solvent 7. Primer a strand of nucleic acid that serves as a starting point for DNA synthesis -- specifically design according to gene that want to amplify PCR Components

15. PCR Components : DNA Template

16. One set primer contains 2 type: - Forward primer - Reverse primer PCR Components

17. 1 st Generation PCR

18. Hot Start

19. Classic PCR End-point analysis of amplification product (30 cycles)

20. Electrophoresis Electrophoresis is a method whereby charged molecules in solution (DNA/RNA/proteins) migrate in response to an electrical field DNA have negative charge because it has O- on phosphate group DNA move from negative pole to positive pole

21. No. Component Function 1. Agarose To make a gel 2. Running buffer TAE ( Tris b ase -Acetic a cid-EDTA) TBE ( Tris b ase -Boric a cid-EDTA) As buffer to maintain the pH and as a solvent 3 Loading dye Sucrose: to make DNA stay in the bottom EDTA: to stop enzymatic reaction Bromophenol blue: as a dye to see the DNA position during the run 4 DNA binding dyes DNA fluorescence dye Visible under UV light 5. DNA marker As ruler, contain fragment DNA with specific size Electrophoresis Component

22. 1. Make agarose gel 2. Loading samples 3. Run the gel 4. Visualize under UV light Electrophoresis Workflow

23. Fluorescence Dye used in PCR Ethidium Bromide (EtBr) Gelred

24. 2 nd Generation Real-Time PCR ( qPCR )

25. Probes

26. Real-Time PCR: Probes

27. Real-Time PCR: Probes

28. qPCR /Real-time PCR Measures the accumulation of DNA product after each round of PCR amplification. Quantifying PCR Log Target DNA Cycle #

29. Absolute Determines input quantity of a nucleic acid target Requires a standard curve Relative Determines fold differences in input target nucleic acid quantities between samples Performed with or without a standard curve Typically used for gene expression analysis with a target gene normalized to a reference gene 14500 copies or 10ug/ml Types of quantification

30. 10 8 Absolute quantification

31. 10 6 10 8 Absolute quantification

32. 10 4 10 8 10 6 Absolute quantification

33. 10 8 10 6 10 2 10 4 Absolute quantification

34. 10 8 10 6 10 2 10 4 T Absolute quantification

35. Absolute quantification

36. 10 8 10 6 10 2 10 4 T Absolute quantification

37. 10 5 copies/well Absolute quantification

38. 3 rd Generation Digital PCR ( dPCR )

39. DIGITAL PCR is an advanced molecular technique for the precise and absolute quantification of nucleic acids (DNA or RNA). Unlike traditional PCR, which provides relative quantification, dPCR offers a digital readout of the number of target molecules in a sample by partitioning the sample into many individual reactions, each containing zero or one target molecule. Principles : Partitioning: The sample is divided into thousands to millions of individual, nanoliter -sized reactions, such that each partition ideally contains either zero or one target molecule. PCR Amplification: Each partition undergoes PCR amplification independently. Detection: Partitions are analyzed post-PCR to determine which ones contain amplified products, providing a count of positive reactions. Quantification: The number of positive partitions is used to calculate the absolute quantity of the target nucleic acid using Poisson statistics .

40. The Power of Partitioning into Droplets

41. DIGITAL PCR Droplet Digital PCR ( ddPCR ): The sample is mixed with an oil emulsion to create thousands to millions of droplets, each serving as an individual reaction chamber . .

42. DIGITAL PCR Chip-based Digital PCR: The sample is loaded into a microfluidic chip containing thousands of tiny wells

43. DIGITAL PCR Advantages: High Precision and Sensitivity: Accurate quantification of low-abundance targets and rare mutations. Absolute Quantification: Provides direct count of target molecules without the need for standard curves. Robustness: Less affected by inhibitors compared to traditional PCR. Partition Independence: Each partition acts as an independent PCR reaction, reducing the impact of sample heterogeneity . Limitations: Cost: Higher cost compared to traditional qPCR due to specialized equipment and reagents. Complexity: Requires precise sample handling and partitioning techniques. Throughput: Lower throughput compared to high-throughput qPCR platforms, although newer dPCR systems are improving in this regard. Data Analysis: Requires sophisticated software and statistical analysis for quantification.

44. PCR Application Pathogen Detection and Identification Food Fraud Halal Authenticity SNP Genotyping Mutation Detection Drug Discovery Cloning (Recombinant Products) Mutagenesis Gene Expression Studies Pre-Natal Diagnosis DNA Fingerprinting Gene Therapy Etc...

45. PCR Application

46. Gene Cloning PCR to A. tumefaciens

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