polymerase Chain Reaction(PCR)

It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.


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

1. (PCR) Ajay Subedi Janamaitri foundation institute of health sciences,Balaju,Kathmandu POLYMERASE CHAIN REACTION 1

2. It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993 . It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase. It is called “chain” because the products of the first reaction become substrates of the following one, and so on. 2

3. Why PCR? Simple Powerful sensitive specific reliable 3. Fast 3

4. Types of PCR 1 . Amplified fragment length polymorphism (AFLP) PCR 2. Allele-specific PCR 3. Alu PCR 4. Assembly PCR 5. Asymmetric PCR 6. COLD PCR 7. Colony PCR 8. Conventional PCR 9. Digital PCR ( dPCR ) 10. Fast cycling PCR 11. High Fidelity PCR 12. High-Resolution Melt (HRM) PCR 13. Hot start PCR 14. In-situ PCR 15. Intersequence specific (ISS) PCR 16. Inverse PCR 17. LATE (Linear-After-The-Exponential) PCR 18. Ligation mediated PCR 19. Long-Range PCR 20. Methylation-specific PCR (MSP) 21. Miniprimer PCR 22. Multiplex PCR 23. Nanoparticle-Assisted PCR ( nanoPCR ) 24. Nested PCR 25. Overlap extension PCR (OE-PCR) 26. Real-Time PCR (Quantitative PCR ( qPCR )) 27. Repetitive sequence-based PCR 28. Reverse Transcriptase PCR (RT-PCR) 29. Reverse-Transcriptase Real-Time PCR (RT- qPCR ) 30. RNase H-dependent PCR 31. Single Specific Primer PCR 32. Single Specific Primer-PCR (SSP-PCR) 33. Solid Phase PCR 34. Suicide PCR 35. Thermal asymmetric interlaced PCR (TAIL-PCR) 36. Touch down PCR 37. Variable Number of Tandem Repeats (VNTR) PCR 4

5. Principle The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Artificial process which imitates natural DNA replication 5

6. Materials required for PCR PCR tubes Tube holder Pipettes Ice box Centrifuge PCR machine UV transilluminator 6

7. Components of PCR DNA template - the sample DNA that contains the target sequence. At the beginning of the reaction, high temperature is applied to the original double-stranded DNA molecule to separate the strands from each other. Usually target sequence is 100bp-1500bp length . DNA polymerase - a type of enzyme that synthesizes new strands of DNA complementary to the target sequence. The first and most commonly used of these enzymes is Taq DNA polymerase ( from Thermis aquaticus ), whereas Pfu DNA polymerase ( from Pyrococcus furiosus ) is used widely because of its higher accuracy when copying DNA. Although these enzymes are subtly different, they both have two capabilities that make them suitable for PCR : 1) they can generate new strands of DNA using a DNA template and primers, and 2) they are heat resistant. 7

8. Taq polymerase A thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus Taq's optimum temperature for activity is 75–80°C Can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72°C 8

9. Primers - short pieces of single-stranded DNA that are complementary to the target sequence. The polymerase begins synthesizing new DNA from the end of the primer. - Primers are oligonucleotides with 15-20 bases in length -Two primers forward and reverse primer . -Annealing temperature depends upon primer sequence (~ 50% GC content) -Avoid primer complementary (primer- dimer formation) -The last 3 nucleotides at the 3` end is the substrate for DNA polymerase - G or C -The primers must not base pair with each other or with themselves or form hairpins. 9

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11. Nucleotides ( dNTPs or deoxynucleotide triphosphates) - single units of the bases A, T, G, and C, which are essentially "building blocks" for new DNA strands . Mg or Mn ions- Mg preferred, provides stable conditions Buffer solution -suitable chemical environment for optimum activity and stability of DNA polymerase 11

12. Very simple Lab procedure To perform PCR, the extracted sample (which contains target DNA template) is added to a tube containing primers, free nucleotides ( dNTPs ), and Taq polymerase. The PCR mixture is placed in a PCR machine. PCR machine increases and decreases the temperature of the PCR mixture in automatic, programmed steps which generates copies of the target sequence exponentially. 12

13. Thermocycling protocol annealing 95 ºC 95 ºC 55-60 ºC 72 ºC 4 ºC 5 min 1 min 30 sec 1 min ∞ Hold Initial denaturation of DNA 1X 35-40X 1X extension denaturation 13

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17. Initial Denaturation Heating separates the double stranded DNA Denaturation heating the reaction to a temperature of 94–96 °C for 5 mins depending on the GC content of the template performed only once at the beginning of the reaction Heat Cool 17

18. Denaturation First regular cycling event and consists of heating the reaction to 94–98 °C for 30–60 seconds. Causes melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules. 18

19. Annealing  (primer binding) A nnealing of the primers to the single-stranded DNA template. Typical annealing temperature is between 50 and 55 ° C, for 30-60 seconds optimal temperature depends on the primer sequence and length Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. The polymerase binds to the primer-template hybrid and begins DNA formation. 19

20. Extension (synthesis of new DNA ) DNA polymerase duplicates DNA by synthesizing a new DNA strand complementary to the DNA template strand (72C)-optimal temperature for Taq DNA polymerase D uring this step dNTPs that are complementary to the template in 5' to 3' direction are added 20

21. Extension Temperature Target sequence Length Time ≤ 500bp 30 seconds 500 - 1500bp 60 seconds >1500bp 90 seconds 21

22. Final extension This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle Its objective is to ensure that any remaining single-stranded DNA is fully extended. 22

23. Final Hold  This step at 4°C for an indefinite time may be employed for short-term storage of the reaction. 23

24. PCR Amplification Exponential Amplification of template DNA 24

25. Detection of PCR products Agarose Gel electrophoresis easiest and commonest way to separate and analyze DNA on the basis of molecular weight have lower resolving power for DNA than acrylamide gels, but they have greater range of separation, and are therefore usually used for DNA fragments of 50-20,000 bp in size t he lower the concentration of the gel, the larger the pore size, and the larger the DNA that can be sieved -technique consist of 3 basic steps preparation of agarose Electrophoresis of DNA fragments Visualization of DNA fragments 25

26. Preparation of Agarose Gel Insoluble in water and buffer at RT but dissolves on boiling On cooling undergoes polymerization (sugar polymers crosslink with each other causing solution to gel) Density or pore size determined by the concentration of agarose 0.5 -2% agarose use, usually 1% preferred Buffer- 1x TBE( tris -borate-EDTA) or TAE 26

27. Electrophoresis of DNA Technique used to separate charged molecules DNA is negatively charged at neutral PH DNA migrates to anode when electric field is applied across the gel Migration of DNA depends upon -molecular size of DNA - agarose concentration -applied current -conformation of DNA 27

28. Progress of gel is monitored by observing the migration of visible dye-tracking dye Commonly used are Xylene cynol and Bromophenol blue 50 volts for 1-2 hrs 28

29. Visualization of DNA DNA is not visible in gel Stained with specific dye such as ethidium bromide -1 µl of 10 mg/ml Visualize under UV light-DNA floresces * ethidium bromide must be handled carefully as it is mutagen and carcinogen 29

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31. PCR phases Exponential If 100% efficiency – exact doubling of products. Specific and precise Linear High variability. Reaction components are being consumed and PCR products are starting to degrade. Plateau End-point analysis. The reaction has stopped and if left for long – degradation of PCR products. 31

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33. 33 REAL TIME PCR (RT-PCR)

34. Lets see a short animation video on Lab procedure of RT- PCR for diagnosis of COVID-19. 34

35. Real time PCR In order to amplify small amounts of DNA, the same methodology is used as in conventional PCR using a DNA template, at least one pair of specific primers, deoxyribonucleotides , a suitable buffer solution and a thermo-stable DNA polymerase.

36. Principal: Quantitative PCR is carried out in a thermal cycler with the capacity to illuminate each sample with a beam of light of a specified wavelength and detect the fluorescence emitted by the excited fluorophore . The thermal cycler is also able to rapidly heat and chill samples, thereby taking advantage of the physicochemical properties of the nucleic acids and DNA polymerase.

37. A substance marked with a fluorophore is added to this mixture in a thermal cycler that contains sensors for measuring the fluorescence of the flurophore after it has been excited at the required wavelength allowing the generation rate to be measured for one or more specific products. 38

38. This allows the rate of generation of the amplified product to be measured at each PCR cycle. The data thus generated can be analysed by computer software to calculate relative gene expression (or mRNA copy number) in several samples. This measurement is made after each amplification cycle, and this is the reason why this method is called real time PCR (that is, immediate or simultaneous PCR)  not at its end, as in conventional PCR . In the case of RNA quantitation , the template is complementary DNA ( cDNA ), which is obtained by reverse transcription of ribonucleic acid(RNA). In this instance the technique used is quantitative RT-PCR or Q-RT-PCR.

39. Two common methods for the detection of products in quantitative PCR are: - Non-specific fluorecent dyes that intercalate with any double-stranded DNA - Sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence to quantify messenger RNA (mRNA) and non-coding RNA in cells or tissues.

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41. Advantages Real-Time chemistries allow for the detection of PCR amplification during the early phases of the reaction. Measuring the kinetics of the reaction in the early phases of PCR provides a distinct advantage over traditional PCR detection. Traditional methods use Agarose gels for detection of PCR amplification at the final phase or end-point of the PCR reaction Sensitive assay, highly quantitative, and highly reproducible Can detect as few as 5 molecules Good Excellent dynamic range, linear over several orders of magnitude Useful for diagnostic purposes

42. Disadvantages of Real time PCR: Expensive Can pick up RNA carryover or contaminating RNA leading to false positives Need a skillful person

43. Medical applications OF PCR   genetic testing , where a sample of DNA is analyzed for the presence of  genetic disease   mutations . Prospective parents can be tested for being  genetic carriers , or their children might be tested for actually being affected by a  disease .   Prenatal testing  can be obtained by  amniocentesis , chorionic villus sampling , or even by the analysis of rare fetal cells circulating in the mother's bloodstream. PCR analysis is also essential to  preimplantation genetic diagnosis , where individual cells of a developing embryo are tested for mutations. 45

44. Medical applications: PCR can also be used as part of a sensitive test for  tissue typing , vital to  organ transplantation .   blood type     Oncogenes . 46

45. Characterization and detection of infectious disease organisms have been revolutionized by PCR: The  human immunodeficiency virus   tuberculosis , The spread of a  disease  organism  through populations The sub-types of an organism that were responsible for  earlier epidemics  can also be determined by PCR analysis. 47

46. Forensic applications In its most discriminating form,  genetic fingerprinting  can uniquely discriminate any one person from the entire population of the  world .   DNA database  of earlier evidence or convicts. Simpler versions of these tests are often used to rapidly rule out suspects during a criminal investigation. Evidence from decades-old crimes can be tested, confirming  the people originally convicted.   parental testing , where an individual is matched with their close relatives. 48

47. In Molecular diagnosis Viral load and identification of virus Bacterial infection: early detection specific targeting and better treatment detect Mycobacterium tuberculosis Legionella pneumophila , Listeria monocytogenes , Neisseria gonorrhoea. Antibiotic resistance : Staphylococcus aureus, Staphylococcus epidermidis , Helicobacter pylori , Enterococcus faecalis Enterococcus faecium