000_My_Real_PCR Presentation_ @ JUTH_on_ThursDay_20.10.22.pptx

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1. Aliyu Yahaya Ibrahim Waziri Stephen Godiya Shuaibu Abubakar Sadiq Halima Aliyu Ibrahim {1} Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 POLYMERASE CHAIN REACTION PRINCIPLES, INSTRUMENTATION AND APPLICATIONS OF PCR THERMAL CYCLER JOS UNIVERSITY TEACHING HOSPITAL (JUTH) @ Present By

2. OUTLINE Introduction Definitions Principles Types of PCR Modification of PCR Instrumentation of PCR Test Procedures Applications of PCR Differences between PCR and some L aboratory A nalyzers Merits of PCR O ver some L aboratory A nalyzers and it’s L imitations Conclusion Recommendations References {2} Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022

3. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {3} INTRODUCTION This technique was first invented in 1983 by an American Scientist and Biochemist known as Kerry B. Mullis . This procedure was entirely carried out Biochemically (i.e) in-vitro and patented in 1985. In 1993, H e shared the Nobel Prize in Chemistry with his friend ‘ Michael Smith ’ and was First Published at Cetus Corporation and Development Publication Company . ( John, 2017)

4. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {4} DEFINITIONS PCR is a powerful in-vitro technique that is designed to permit S elective A mplification of a S pecific T arget DNA S equence with in a H eterogeneous C ollection, based on the replication of a double-stranded DNA template, which made it possible to generate million copies of DNA fragments of interest. This reaction derives it’s name from one of it’s key components, known as “ DNA P olymerase, ” and is set in a motion called “ C hain R eaction” in which the products of these steps serve as templates for exponential amplification. H ence the name : ( Belkum , 1998)

5. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {5} CONT’D .…. [ Polymerase Chain Reaction ]…. P = P olymerase C = C hain R = R eaction. Which is simply known as PCR ( Belkum , 1998) DNA RNA SIMILARITIES

6. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {6} 1 ST PRINCIPLE OF PCR The technique is based on thermal cycling process that exposes the reactions to separate cycles of heating and cooling stages, which are broken down in to three ( 3 ) phases, to permit different temperature dependent reactions in the processes. Th ese are: Stage 1 st : Denaturation : Optimum Temperature = 95 o C Stage 2 nd : Hybridization : Optimum Temperature = 54 o C Stage 3 rd : Elongation : Optimum Temperature = 72 o C ( John, 2017)

7. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {7} DYNAMICS OF THERMOCYCLER ( John, 2017)

8. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {8} This technique is also based on the enzymatic replication of DNA , in which a short segment of  DNA  is amplified using primer mediated enzymes. DNA Polymerase synthesizes new strands of DNA complementary to the template DNA . The DNA polymerase add nucleotide to the pre-existing 3 ’ hydroxyl (OH) group to kick-start the sequencing . Therefore, primers are required as more nucleotides are added to the 3 ’ end of the DNA polymerase. ( John, 2017) 2 ND PRINCIPLE OF PCR

9. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {9} TYPES OF PCR Conventional PCR Long-range PCR Nested PCR Assemble PCR Droplet PCR Methylated-Specific PCR Reverse Transcriptase PCR Gradient PCR Hot-Start PCR In situ PCR Multiplex PCR Immuno PCR In Silico PCR Asymmetric PCR ( Belkum , 1998)

10. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {10} MODIFICATIONS IN PCR Conventional PCR This is the simplest and original version , which utilizes only one Tag. Polymerase with out any further modification. ( Belkum , 1998) FURTHER MODIFIED PCR Gradient PCR This was one of the modified PCR from the native version, which is capable of creating optimization of different temperature gradients and checking the template amplification efficiency . Long -Range PCR This uses different mixtures of the Tag. Polymerase to form a very long range of sequence. As the name implies LR- PCR. Hot Start PCR This uses heat to denature some unwanted antibodies that are capable of inactivating the Tag. DNA polymerase during the sequencing processes .

11. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {11} The basic technology behind this M olecular P hoto- T yping M achine is that it provides a thermal controlled environment for the B iomolecules process. It has a thermal block with holes where tubes holding the PCR reaction mixtures can be inserted, then raises and lowers the temperature of the block in a discrete pre-programmed steps. It is an important instrument needed in all the laboratories, especially in the fields of M olecular B iology, B iotechnology and G enetic E ngineering. ( Pelt-Verkuil, 2008) INSTRUMENTATION OF THERMAL CYCLER Behind the technology The basic idea of a thermal cycler is that it provides a thermally controlled environment for PCR samples. A thermal cycler usually contains a heating block with holes or depressions in it that receive sample tubes (though other types of sample vessels are now possible also; see below). For the PCR reactions to work properly, the block must change temperature at specific times, and spend specific durations of time at specific temperatures. The researcher programs the temperature cycling information into the thermal cycler either by computer or via a console on the instrument, or uses a preprogrammed routine built into the machine.

12. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {12} Heat Plate Chamber Lid and Lock Upper Heat Plate Sample Block and Vail Digital Display Screen Tempt and Time Buttons Function Keys Power OFF Switch Program Start Button Key Pad Power ON Switch Heat Release Vent. ( Pelt-Verkuil, 2008) DIAGRAMATIC VIEW CON’T…INSTRUMENTATION Heat Release Vent Power ON Switch Power OFF Switch Program Start Button Temperature and Time Adjusting Buttons Digital Display Screen Chamber Lid Silver

13. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 { 13} TEST PROCEDURES (A )- B iochemical C omponents and R eagents R equired To carryout this experiment , a DNA A mplifier is needed, in which all the basis Biochemical components and their molecular reagents required for the process are introduced, then incubated at various temperatures . ( Pelt-Verkuil, 2008) T ag DNA ( T arget and Template DNA ) DNA P olymerase ( H eat S table ) P rimers : 2-O ligonucleotide P rimers N ucleotides ( d NTP s ) : d ATP , d GTP , d CTP , and d TTP B uffer: = (PH 8.3 – 8.8) for suitable chemical environment. M agnesium C hloride ( M g C l 2+ )

14. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 { 14} EXAMPLE OF TEST METHOD ( B )- MATERIAL Pipette 1uL of the sample into the PCR tube Add 5ul of the Buffer to maintain the PH at ( 8.3 – 8.8) Add 5uL of d NTP Add 3 uL M g C l ++ Add 1uL of the Primer Add 1uL of DNA Polymerase Gently mix Transfer the tube to the PCR M achine for analysis Micro Pipette and T ips , PCR Tubes / W ells, Reagents Thermal cycler Machine. PROCEDURE ( Pelt-Verkuil, 2008)

15. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 { 15} VISUAL PRESENTATION OF TEST PROCEDURS ( John, 2017)

16. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 { 16} Original DNA Template and it’s Basic Components Required for Sequencing. Initialization This is the 1 st phase of the reaction. It starts by heating the mixture at (80 – 95) C. At this temperature, the hydrogen bonds holding together the two (2) polynucleotides of double helix are broken. So the target DNA strands become denatured in to single stranded molecules. Denaturation PCR STEPS IN DNA SEQUENCING ( Pelt-Verkuil, 2008)

17. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 { 17} This is the 2 nd phase known as hybridization, in which the temperature is reduced to (50- 60) C. This allows primers to attach to their annealing positions. The tempt. must be low enough to enable hybridization between primers and the DNA template to prevent formation of mismatched hybrids. Renaturation This is the 3 rd phase known as Extension, in which the tempt. Is raised again to (60 - 75) C. The DAN polynucleotides synthesized are long and have identical 5’ ends but random 3‘ ends. Elongation ( Pelt-Verkuil, 2008)

18. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 { 18} This process continues till the required C ycles are attained and which M ultiple C opies of DNA are synthesized. Multiple C opies of DNA S ynthesis At the end of the reaction, the mixture is subjected to A G el E lectrophoresis for C loning and further S equencing of the PCR products, in which t he ladder is a mixture of fragments with known size to compare with the PCR fragments . Gel E lectrophoresis ( Pelt-Verkuil, 2008)

19. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 { 19} ( Pelt-Verkuil, 2008) FORENSIC SCIENCE

20. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {20} PICTORIAL REPRESENTATION Denaturation OF ( Pelt-Verkuil, 2008)

21. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {21} (David and Turlotte 1998) VISUAL PRESENTATION OF DNA SEQUENCING ( John, 2017)

22. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {22} APPLICATIONS OF PCR Diagnosis of I nherited D isease D isorder. * Diagnosis of N on-Inherited D isease D isorder . Gene C loning , G enotyping and G enome T esting ( G enetics). Amplification and I dentification of M icrobial DNA . Detection of G ene E xpression and V ariation in A llele. Diagnoses of Infectious D iseases . D NA Finger Print (Forensic Studies ). Mutagenesis S tudy and B reeding . Detection of F ood P athogens . Research, E tc . ( Pelt-Verkuil, 2008)

23. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {23} Molecular Amplifier ( PCR) Machine The technique is based on thermal cycling process that exposes the reactions to separate cycles of heating and cooling stages. This works based on amplifying of Genetic Materials. E.g: DNA or RNA fragments using specific enzymic process. Ability to detect inanimate agents & dynamic range of quantification. ( Belkum , 1998) Spectros , Lumines and ELISA . Analyzers All these Instruments have the same working principles. That is, they produce light & measure its intensity Spectrophotometrically. They work based on antigen – anti-body reaction detection on blood specimens. Quantification ability are specified and their ranges of quantification are limited. DIFF. BETWEEN PCR AND SOME LAB. ANALYZERS

24. What is it ? It is an integrated device that maintains identical well -to- well conditions to monitor micro reaction tubes. A lso known as Real- time analyzer with quantitative amplification ability . Illuminates and collects a wide range of optical signals. Enables exquisite control of thermal conditions. Provides an open platform for all chemistries. Features a very fast data acquisition rate. THE NEXT GENERATION PCR (REAL -TIME PCR) (NeXt Gen. Rotor 6000) THE LEADING PCR {24} ( Pelt- Verkuil , 2008) Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022

25. Sa mples spin continually during heating and cooling.  400 RPM G-force keeps reagent at base of tube.  R emoves bubbles and condensation  W ill not pellet components No variation during continuous movement .  Well-to-Well thermal , optical and Color High-speed data collection  All samples read in one revolution (0.15 sec) 400 RPM >10 G WORKING PRINCIPLES How does the Rotor format work? Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 ( Belkum A., 1998) ( Belkum , 1998) {25} Rotor-Gene 6000 Enzymatic Reactions. Color Mechanism. Optical Mechanism. Heating Mechanism. Cooling Mechanism.

26. Reaction Chamber PMT Detector Assembly LED Light Source Assembly Tubes in Rotor Spin Past Optics Lens Detection Filters Spindle/Motor Assembly Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {26} ( Kadri , 2019) CONT’D Optics mechanism

27. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {27} THE NEXT GENERATION PCR NeXt Gen. Roter PCR ( Kadri , 2019) LED L ight Source (Rotates for each Channel ) R otor S pins Tubes at 400 rpm L ens F ilter set (Rotates for each Channel ) S ensitive PMT (Photomultiplier ) Detector 3 D Animation CONT’D Colour M echanism

28. Centrifugal fan drives air around chamber Chamber vent seals to contain air Note: holes in the rotor allow free airflow Heater elements switch on Heating Mechanism {28} Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 ( Kadri , 2019)

29. Cool air in Centrifugal fan Drives air into chamber Centrifugal fan drives air around chamber Chamber vent opens expelling hot air Cooling mechanism Heater elements switch off Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {29} ( Kadri , 2019)

30. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {30} MERITS OVER OTHER LABORATORY ANALYZERS 1. Uses less patient sample and Produce good assay parameters, thus has high analytical Simplicity, Sensitivity Specificity and reliability. 2. Results are obtained more quickly within (3hrs) and has closed system to avoid risk of contamination. 3. Usually no radioactive emission and has dynamic range of quantification 4. Detect point of mutation, smaller / less common organisms such as viruses 5. Better Precision in determining sizes of alleles essential for some disorders 6. Lower turnaround time, highly promising because it uses internal probes that are capable of providing additional information and specification, thus it gives the probability of later sophisticated studies, like Total Genome Sequencing, Cloning. Etc.... ( Pelt-Verkuil, 2008)

31. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {31} LIMITATIONS The DNA polymerase used in the PCR reaction is error prone and can lead to mutations in the fragment that is generated. The specificity of the generated PCR product might be altered by the nonspecific binding of the primers to other similar sequence on the template DNA 3. In designing primers to generate a PCR product, some prior sequence information are usually needed. 4. False reaction due to cross contaminations and Unspecific amplification, Tag. Polymerase is expensive and most be known. 5. Required capacity building: highly trained personnel and laboratory infrastructure. ( Pelt-Verkuil, 2008)

32. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {32} CONCLUSION The future of PCR appears promising and new versions of these classic PCR have drastically shortened the time required to reach diagnosis and reduced the number of false P ositive and N egative results mistakenly generated by other laboratory analyzers.

33. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {33} RECOMMENDATIONS Because of its Ease of use , S peed , S ensitivity , Specificity and wide range of use with great spectrum of Research and D iagnostic applications , it should be recommended in this facility. Staff and Students should be Train and Retrain in other to master not only the Operating Procedures, but also its Instrumentation.

34. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 {34} REFERENCES Belkum A, Primer Design Tips & Tools. Thermo Fisher Scientific , Inc. 2015. John P. A, PCR Optimization : Reaction Conditions and Components. Applied Biosystems . 2017. Pg . 222 : 9, 823. Pelt-Verkuil E, Brief comparison between in-vivo DNA replication and in-vitro PCR Amplification . Principles and Technical Aspects of PCR Amplification . Netherlands: Springer. 2008 . Pg. 9 - 15. Commercial kits are readily available and offer low-risks, faster than traditional protocols with high-quality DNA recovered ( Hassanzadeh et al., 2016). Polymerase Chain Reaction (PCR) is an everyday and indispensable scientific technique use to amplify DNA fragments to generates millions of copies of a particular DNA in a few hours ( Garibyan & Avashia , 2013;Kadri, 2019;Kuzdraliński et al., 2017). In PCR, a small amount of DNA is added to a solution containing essential components such as DNA polymerase, primers, and nucleotides and heated in a thermal cycler to separate DNA strands, and once cooled, the DNA polymerase creates the copy of individual DNA strand. ..

35. Compiled By: Foreign Graduates, Medical Laboratory Department (Chemical Pathology Unit) Jos University Teaching Hospital (JUTH)_ 2022 THANKS THERMOCYCLER MACHINE {35}