PCR ,PCR INSTRUMENTATION,STUDYS FOR GENE REGULATION.pptx

A brief on PCR,PCR INSTRUMENTATION,STUDYS FOR GENE REGULATION.

1. PCR ,PCR INSTRUMENTATION & STUDYS FOR GENE REGULATION Submitted to: Dr. ANAND KUMAR TENGLI, ASSOCIATE PROF . , Dept. of pharmaceutical chemistry, JSSCP – MYSORE. 1 Submitted by: N.Surendra chowdary M.Pharm, 1 st Year Pharmaceutical Analysis. JSS College Of Pharmacy, Mysuru

2. Contents : INTRODUCTION TO PCR Types of PCR Components of PCR Steps involved in PCR The principle of real-time PCR Instrumentation of REAL TIME PCR STUDYS FOR GENE REGULATION Prokaryotic Gene Regulation Eukaryotic gene regulation

3. INTRODUCTION TO PCR: DEFINITION : PCR(polymerase chain reaction) is an exponentially progressing synthesis of the defined target DNA sequences in vitro First disc overed by Kary Mullis in 1983 , for which he received the Nobel Prize in Chemistry in 1993. Why “Polymerase”? It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase. Why “Chain”? It is called “chain” because the products of the first reaction become substrates of the following one, and so on.

4. Types of PCR: RT-PCR: Basic PCR technique will only amplify the DNA templates , but to amplify RNA the RT-PCR technique is used . To apply PCR to the study of RNA ,the RNA sample must first be reverse transcribed to cDNA to provide the necessary DNA template for the thermostable polymerase . This process is called reverse transcription hence the name called RT-PCR. HOT-START PCR : Thermostable DNA polymerases have activity i,e less than 25% at low temperatures . So that PCR primers can anneal to template sequences that are not perfectly complimentary. So to avoid the formation of undesired amplification products the reaction is heated to temperatures >60 degree centigrade before polymerization begins. LONG PCR : Basic PCR works well when smaller fragments are amplified , amplification efficiency decreases as the amplicon size increase over 5Kb and leads to formation of truncated products which are not suitable substrates for additional cycles of amplification . So we use two thermostable DNA polymerases to increases the yield of substrates for additional amplification. QUANTITATIVE REAL-TIME PCR : It is a fluorescent version of PCR , where an increase in fluorescence detected at each cycle correlates with the increase of amplicons which helps in visualization of the reaction as it progresses . In this technique fluorescently labelled oligonucleotide probes or primers or fluorescent DNA binding dyes are used.

5. Components of PCR: 1. Template DNA (e.g., plasmid DNA, genomic DNA)-contains the sequence to be amplified. 2. Forward and reverse PCR primers- oligonucleotides that define the sequence to be amplified. 3. MgCl2 (25 m M)- cofactor of the enzyme 4. dNTPs (a mixture of 2.5 m M dATP , dCTP , dGTP , and dTTP)- DNA building blocks. 5. PCR buffer: 500 m M KCl , 100 mM Tris-HCl, pH 8.3, 25°C- maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme 6. Thermal stable DNA polymerase (e.g., Taq DNA polymerase)- enzyme that catalyzes the reaction 7. PCR additives/cosolvents (optional; e.g., betaine, glycerol, DMSO, formamide, bovine serum albumin, ammonium sulfate, polyethylene glycol, gelatin, Tween-20, Triton X-100,β- mercaptoethanol , or tetramethylammonium chloride).

6. Steps involved in PCR : 1.Setting Up PCR The common volume of a PCR is 10, 25, 50, or 100 μL . All of the reaction components can be mixed in together in a 0.5-mL PCR tube in any sequence except for the DNA polymerase, which should be added last . It is recommended to mix all the components right before PCR cycling. For each PCR, the following components are mixed together: 1. Template DNA 2. Primers 3. Mg2+ 4. dNTP 5. PCR buffer 6. DNA polymerase

7. 2. PCR Cycling Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 92 to 95°C for 2 to 5 min. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules. Most PCR will reach sufficient amplification after 20 to 40 cycles of strand denaturation at 90 to 98°C for 10 sec to 1 min Annealing step: The reaction temperature is lowered to 55 to 70°C for 30 s to 1 min allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm( Melting temp.) of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA formation.

8. Extension/elongation step: The temperature at this step depends on the DNA polymerase used; a temperature of 72 to 74°C for 1 min per kilobase of expected PCR product is used . At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended. Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.

9. DNA polymerase

10. THE PRINCIPLE OF REAL-TIME PCR(QPCR): The amount of the nucleic acid present into the sample is quantified using the fluorescent dye or using the fluorescent-labeled oligos.” When a dye or probe binds with the target template, it releases a fluorochrome which resultantly emits fluorescence for the detector to detect. The detector captures a signal as a positive template amplification.

11. I. DNA binding dye: The dye has its own fluorescence. Once the dye binds to the double-stranded DNA the fluorescence emitted by the dye increases 100 to 1000 fold than the original signal. However, the original dye fluorescence is taken as the baseline (as a reference) for the detection. Two common dyes employed are the SYBR green and Eva Green , notedly, the technique is used in the validation of other assays such as DNA microarray. The result of the experiment depends on the specificity of the primers used in the PCR reaction. Because even though the primers remain bound non-specifically, the DNA binding dye binds to the non-specific sequence and gives the fluorescent signals. Therefore the chance of the non-specific detection is high in the SYBR green dye-based method.

12. Graphical representation of how dye binds with the DNA.

13. II. Probe-based detection method: In the probe-based detection method, two different types of single and short-sequence-specific probes are utilized; 1)A linear probe and 2)molecular beacons 3) scorpion probe. 1.Linear probe: Linear probes are the TaqMan probe , which relies on the activity of Taq DNA polymerase. The probes structurally consist of labeled short single-stranded sequence-specific DNA molecules that are radio or fluorescent-labeled. Here the probe is labeled with the fluorescent dye described as a reporter molecule (TAMRA and  Black Hole)  , situated at the 3’ end. The other 5’ end has the quencher dye (FAM) which is in close proximity to the reporter dye and quenches the fluorescence of the reporter dye. Now, in the probe base method, not only the probe but the Taq DNA polymerase plays an important role. The Taq DNA polymerase used in the real-time PCR has the 5’ to 3’ exonuclease activity, which removes the probe by extending the DNA. Once the probe dissociates the reporter molecules emitted fluorescent light. Because, if the DNA (the sequence of our interest) is amplified, the reporter molecule is unquenched and releases the fluorescence. The amount of fluorescence released during each run is directly proportional to the amount of DNA amplified during the reaction.

14. The main advantage of the probe-based method is that we can use multiple probes for multiple template DNA sequences. This means we can amplify multiple templates in a single reaction efficiently. Image of the process of probe hybridization.

15. 2.Molecular beacons: The molecular beacon remains in a hairpin structure in which  the central loop is complementary to the target sequences. One end of the hairpin loop has the quencher dye and one end has the reporter fluorescent dye. M olecular beacon facilitate helps in preventing non-specific binding during the reaction which is commonly observed while using linear probes. Because structurally, the complementary sequences present on both ends of the hairpin loop-like structure helps to prevent non-specificity.

16. when the two ends of the hairpin stem are in close proximity with each other, the reporter molecule remains quenched and cannot generate fluorescence. But when it binds to the complementary sequence, the two ends of the hairpin separate from each other, the quencher blocks, the reported dye is released and emits the fluorescence. The detector records emission. The process of how a molecular beacon probe binds with the template.

17. Scorpion probes: Scorpion probes are other types of probes or we say, a type of molecular beacon in which instead of two different probes and primer, the hairpin loop is incorporated directly at the 5′ end of the primer. The 3′ end contains the complementary sequence to our target DNA. The scorpion probe is even more specific than the molecular beacons.

18. How it works: 1.Reaction plate 2.Filters 3.Excitation light source 4.Emission light from fluorophore 5.Camera (takes measurement at every elongation step) Bio Rad CFX96 real time PCR instrument

19. Instrumentation of REAL TIME PCR(Bio Rad CFX96) Real-time PCR instrument consist of: 1)Reaction module  — samples are heated and cooled to precise temperatures to promote nucleotide denaturation, annealing, and then polymerase-mediated extension for each round of DNA amplification Temperature Control Peltier  — this method is employed by most thermal cyclers in use today. It uses a solid-state active heat pump that transfers heat from one side to the other against a temperature gradient with the consumption of electrical energy. One very useful feature of Peltier blocks is that a thermal gradient (Figure 1) can be established, permitting optimization of an assay's annealing step in a single run (fig -1)Thermal gradient of the CFX96 Touch reaction block

20. Heated and cooled air  : This type of instrument uses a chamber in which tubes are suspended and air of a defined temperature is circulated for specified periods of time as required for PCR Formats of reaction blocks : Reaction blocks come in multiple formats with the most common being a 96-well block (Figure 2) with reaction volumes ranging from 1 to 125 µl. Blocks with more than 384 wells typically use microfluidics with volumes in the pico to nanoliter range. (fig -2) CFX96 Touch reaction block

21. 2.Light Sources: Light-emitting diodes (LEDs)  — there can be individual or multiple LEDs present in a shuttle mechanism, which is positioned above each well so that each one is independently illuminated (Figure 3), or these can be arranged in a stationary array that excites multiple wells at a time (Figure 4) Halogen lamp  — this light source emits broad-spectrum white light, which is then filtered to excite specific fluorophores (Figure 5) (fig -3) (fig -4)

22. 3.Detectors: A photodiode (Figures 3 and 5) is a type of photodetector that, when exposed to light, causes a current to flow. These have a wide spectral range, are rugged, with low failure rates, and can be quite compact in size A CCD (Figure 4) converts the light that it captures into digital data. The quality of the image captured is determined by the resolution (measured in megapixels). CCDs are typically used to capture an image of the reaction plate, whose content is then interpreted by instrument software A photomultiplier tube (PMT; Figure 6) multiplies the current that is produced by incident light (fig -5) (fig -6)

23. 4.Instrument Software : CFX Manager Software Real-time instrument software consists of the following major components: Protocol setup , Plate setup , Data collection , Data analysis. Protocol Editor window  Touch Plate Editor screen 

24.   Run Details window  Quantification window

25. STUDYS FOR GENE REGULATION The process of turning on a gene to produce RNA and protein is called gene expression Due to lack of nuclear membrane in prokaryotes the processes of transcription and translation occur almost simultaneously. So that , the control of gene expression is mostly at the transcriptional level. While in Eukaryotic organisms transcription occurs in the nucleus and the Translation of RNA to protein occurs in the cytoplasm so due to this complexity nature . Regulation of genes in eukaryotes may occur at 1.Epigenetic level ( when the DNA is uncoiled and loosened from nucleosomes to bind transcription factors) 2.Transcriptional level (when the RNA is transcribed) 3.Post-transcriptional level (when the RNA is processed and exported to the cytoplasm after it is transcribed) 4.Translational level (when the RNA is translated into protein) 5.Post-translational level (after the protein has been made)

26. Prokaryotic Gene Regulation Operons: Proteins that are needed for a specific function are encoded together in blocks called operons. In prokaryotic cells, there are three types of regulatory molecules that can affect the expression of operons 1.Repressors (prevent transcription of a gene by binding to operator regions.) E.g. trp (Tryptophan) Operon 2.Activators (increase the transcription of a gene by binding to promoter site) E.g. Catabolite Activator Protein (CAP) 3.Inducers. (either activate or repress transcription depending on the needs of the cell and the availability of substrate) E.g. lac (lactose) Operon

27. 1.The trp Operon: A Repressible Operon Bacteria such as Escherichia coli need amino acids to survive, and are able to synthesize many of them. Tryptophan is one such amino acid that E. coli can either ingest from the environment or synthesize using enzymes that are encoded by five genes. These five genes are next to each other in what is called the tryptophan (trp) operon When tryptophan is plentiful, two tryptophan molecules bind the repressor protein at the operator sequence. This physically blocks the RNA polymerase from transcribing the tryptophan genes. When tryptophan is absent, the repressor protein does not bind to the operator and the genes are transcribed. Fig: The tryptophan operon.

28. 2.Catabolite Activator Protein (CAP): A Transcriptional Activator When glucose levels fall, E. coli may use other sugars for fuel but must transcribe new genes to do so. As glucose supplies become limited IN E. coli, cAMP levels increase. This cAMP binds to the CAP protein which is a positive regulator that binds to a promoter region upstream of the genes required to use other sugar sources. This CAP binding stabilizes the binding of RNA polymerase to the promoter region and increases transcription of the associated protein-coding genes Fig : Transcriptional activation by the CAP protein .

29. 3.The lac Operon: An Inducible Operon In lac operon, lactose acts as an inducer

30. Eukaryotic Gene Regulation 1. Epigenetic Gene Regulation Histone proteins can move along the string (DNA) to expose different sections of the molecule. If DNA encoding a specific gene is to be transcribed into RNA, the nucleosomes surrounding that region of DNA can slide down the DNA to open that specific chromosomal region and allow for the transcriptional machinery (RNA polymerase) to initiate transcription

31. 2.Transcription Gene Regulation PROMOTER Unlike prokaryotic cells, the eukaryotic RNA polymerase requires other proteins, or transcription factors, to facilitate transcription initiation. RNA polymerase by itself cannot initiate transcription in eukaryotic cells. promoter region is immediately upstream of the coding sequence. The purpose of the promoter is to bind transcription factors that control the initiation of transcription. Within the core promoter region resides the TATA box. The TATA box has the consensus sequence of 5’-TATAAA-3’. The TATA box is the binding site for a protein complex called TFIID, Binding of TFIID recruits other transcription factors, including TFIIB, TFIIE, TFIIF, and TFIIH. Some of these transcription factors help to bind the RNA polymerase to the promoter, and others help to activate the transcription initiation complex.

32. Transcriptional Repressors Transcriptional Repressors in prokaryotic cells, eukaryotic cells also have mechanisms to prevent transcription. Transcriptional repressors can bind to promoter or enhancer regions and block transcription. Like the transcriptional activators, repressors respond to external stimuli to prevent the binding of activating transcription factors. 3.Post-transcriptional Gene Regulation Alternative RNA Splicing The RNA contains both introns(unwanted sequences) and exons (wanted sequences) the introns are removed by splicing. Splicing is done by spliceosomes, ribonucleoprotein complexes that can recognize the two ends of the intron, cut the transcript at those two points, and bring the exons together for ligation.

33. Alternative RNA splicing is a mechanism that allows different protein products to be produced from one gene when different combinations of exons are combined to form the mRNA This alternative splicing can be haphazard, but more often it is controlled and acts as a mechanism of gene regulation,

34. 4.Translational and Post-translational Gene Regulation By Initiation Complex In translation, the complex that assembles to start the process is referred to as the translation initiation complex. a protein initiation factor called eukaryotic initiation factor-2 (eIF-2), binds to the high-energy molecule guanosine triphosphate (GTP). The tRNA-eIF2-GTP complex then binds to the 40S ribosome. The binding of eIF-2 to the RNA is controlled by phosphorylation. If eIF-2 is phosphorylated, it undergoes a conformational change and cannot bind to GTP. Therefore, the initiation complex cannot form properly and translation is impeded When eIF-2 remains unphosphorylated, the initiation complex can form normally and translation can proceed.

35. Chemical Modifications Proteins can be chemically modified with the addition of groups including methyl, phosphate, acetyl, and ubiquitin groups. The addition or removal of these groups from proteins regulates their activity or the length of time they exist in the cell.

36. REFERENCES: Real-time PCR: Principle, Procedure, Advantages, Limitations and Applications, Genetic Education,   PCR technology  / By  Dr Tushar Chauhan https://www.bio-rad.com/en-in/applications-technologies/introduction-qpcr-instrumentation?ID=LUSO5YMNI Chapter 16 gene regulation (https://www.theexpertta.com/book-files/OpenStaxBio2e/Chapter%2016%20-%20Gene%20Expression.pdf)