POLYMERASECHAINREACTION.pptx important topic

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1. PCR, RT PCR & SANGER SEQUENCING Presenter: Dr. Pankhuri Gupta Moderated by: Dr. Sangeeta Maam

2. Polymerase chain reaction (PCR) is one of the most important techniques in molecular pathology . With the help of PCR, the single DNA or the pieces of target DNA can be amplified many folds. This technique is an integral part in every modern laboratory for both the diagnos tic & research use. A cts like a “molecular photocopying” machine and amplifies the specific target DNA .

3. MATERIALS

4. PRIMERS: S mall pieces of artificially made DNA strands that are actually the complementary strands of the 3′ end of each strand of target DNA. U sually consist of 20–30 nucleotides. DNA POLYMERASE (TAQ POLYMERASE): A type of DNA polymerase enzyme that extends the new DNA strand . It combines at the end of the primer and then sequentially adds new nucleotides to the DNA strand at 3′ end complementary to the target DNA . Taq polymerase has the unique characteristic to work efficiently in higher temperature. This Taq DNA polymerase is extracted from the bacteria Thermus aquaticus. These bacteria live in hot spring and can survive there. DEOXYNUCLEOTIDE TRIPHOSPHATES ( d NTPS ): These are the raw material or the basic building blocks of the new DNA strands. The Taq polymerase captures these dNTPs from the working solution and attach them to the terminal part of the primers to extend the DNA chain.

5. STEPS

6. Denaturation : Heat breaks double-stranded DNA to single-stranded DNA. Annealing : Forward and reverse DNA primer bind with the complementary DNA strand in 3′ region of each separated DNA strand. Extension : Heat-resistant DNA polymerase (Taq polymerase) enzyme grabs the nucleotides and extends the DNA strand from 3′ to 5′ direction. Repetition of this thermal cycle for 25–30 times increases the DNA product.

7. DENATURATION STEP, 94 °C : DNA is heated to 94 °C to make it single stranded . Only 1–2 min are given to this heating process in each cycle. Initial denaturation : At 94 °C for 1 minute then Denaturation : At 94 °C for 30 seconds A NNEALING , 54 °C : The temperature is rapidly cooled . In this lowered temperature, the primer quickly anneals with the respective site of DNA . With the help of Taq polymerase , the reaction starts at the primer- DNA template site . 50–60 °C for 30 seconds: The temperature may vary depending on the primer used. EXTENSION, 72 °C. The complementary nucleotides are attached from the 3′ to 5′ end of DNA . There is an exponential increment in the number of genes in each cycle. Extension : 72 °C for 1 minute per kilobase of the PCR product. Final extension : 72 °C for 10 minute At least 30 cycles is done in each PCR. 4. Termination : The reaction is terminated by chilling the mixture to4 °C.

8. THE TARGET DNA FROM THE SAMPLE : E xtracted from the sample . BUFFER SOLUTION : Provides the optimum chemical environment for the reaction to occur. MAGNESIUM CHLORIDE (MgCl2): Magnesium chloride works as a cofactor of the Taq polymerase enzyme .

9. TYPES : Conventional PCR RT PCR Asymmetric PCR Multiplex PCR Nested PCR Quantitative PCR Digital PCR

10. REVERSE TRANSCRIPTASE PCR (RT- PCR): A mplify ribo -nucleic acid (RNA) targets. In this process, cDNA is first produced from RNA targets by reverse transcription cDNA is amplified by PCR. Limitations : RNA Instability Expensive Technical expertise needed Risk of contamination- False positive

11. ASY M METRIC PCR: In this technique unequal concentration of primers are used. The great excess of primers is used for the targeted DNA strand that we need to amplify. As the reaction proceeds, only the adequate amount of primer in the reaction mixture produces the particular DNA strand in excess.

12. U ltimately single-stranded DNA ( ssDNA ) is formed as PCR product. As the reaction is slow and goes on arithmetically, so many more cycles are needed in this technique. Asymmetric PCR is used for DNA sequencing and hybridization as only one strand is needed in such conditions.

13. IN SITU PCR: T he reaction takes place within a cell on the glass slide . The PCR product is accumulated within the cell, so it is possible to locate the origin of the amplified DNA . The specially designed PCR machine is used to put the slide within it. This technique is used to amplify the nucleic acid in the fixed tissue and cell instead in solution . Use : Detection and location of virus within the tissue Detection and also localization of the Malignant cells G enetic mutation in case of inherited genetic disease L ocation of gene expression within the tissue

14. SINGLE STRAND CONFORMATION POLYMORPHISM (SSCP): The basic principle of SSCP is that the single-stranded DNA has a specific conformation . Any alteration of the single base change due to mutation may lead to different migration pattern of the single-stranded DNA, and therefore in electrophoresis one can distinguish wild-type DNA from mutant DNA. Application : The detection of single base change mutation and polymorphism in essential hypertension, carcinoma, diabetes, etc.

15. 6. Real- time PCR : Real- time PCR is also known as quantitative PCR (qPCR) as it constantly monitors the quantity of the amplified DNA in the reaction process. In case of qPCR, the amplified DNA is fluorescently labelled , and the emitted fluorescent is directly proportional to the amount of the amplified fluorescent dye . Therefore in each cycle, the amount of the product can be directly monitored , and it is also possible to quantitate the initial amount of the target DNA in the sample

16. 7. Nested PCR : M ore than two pairs of primers are used for DNA amplification . The first PCR is a conventional PCR, and the primer is used for the DNA template of the sample . In secondary PCR , the product of the first PCR is used as the target of the second set of primers . The DNA sequence of secondary PCR is different , and therefore there is no chance of undesired PCR product formation . This prevents the contamination or wrong amplification of DNA because if any wrong locus is amplified by error then the second time it will not be amplified again by the second PCR primer.

17. 8. INVERSE PCR: Inverse PCR (IPCR) amplifies anonymous DNA sequence . It helps to identify the flanking DNA sequence of the genome outside the boundary of the known target sequence . This is particularly employed to find out the clonality of lymphoma and the insertion site of viral DNA. Uses : Identification of flanking sequence Identification of viral gene insertion within the genome Chromosomal rearrangement of oncogene

18. 5′ → 3′ Exonuclease -Based Real-Time RT-PCR Method One-step RT-PCR with Taq Man probes. Fluorescent reporter (5′) and quencher ( 3 ′) dyes used. During PCR extension, Taq polymerase cleaves the probe → fluorescence increases. Fluorescence detected in real time (ABI PRISM 7700). Data expressed as Δ Rn (reporter signal – baseline).

19. Primers :​ bcl-2 mbr -F (forward primer for major breakpoint region) ​bcl-2 mcr -F (forward primer for minor breakpoint region) ​JH-R (reverse primer, NED fluorescent dye) ​cy-F (forward primer for cyclophilin gene)​ cy-R (reverse primer for cyclophilin gene)​ Probes : ​bcl-2 mbr -P (probe for major breakpoint region, FAM fluorescent dye) ​bcl-2 mcr -P (probe for minor breakpoint region, FAM fluorescent dye) ​cy-P (probe for cyclophilin gene, VIC fluorescent dye)​ The cyclophilin gene is a housekeeping gene used as an internal control to confirm the quality of the extracted DNA

20. 40 PCR cycles performed UNG activation (50°C, 2 min) → Taq Gold activation (95 °C, 12 min) Cycling: 95 °C denaturation (30 s)58°C annealing (20 s)72 °C extension (45 s) Final elongation: 72°C for 10 min Cyclophilin gene used as internal control for DNA quality & normalization

21. Key Results: JH-NED primer tested for sensitivity of real-time PCR assay Ct values remained consistent across 5-log range of DNA dilutions No interference from NED in detection Reliable detection of bcl-2mbr/JH and bcl-2mcr/JH fusion sequences

22. Data Analysis q PCR Analysis: The fluorescence emission data is analyzed to determine the threshold cycle (Ct) value for each sample. The Ct value is the cycle number where the fluorescence signal rises above an arbitrary threshold, indicating an increase in the PCR product. Gene Scan Analysis: PCR products are also analyzed using a 310 Genetic Analyzer with a POP4 polymer. This uses capillary electrophoresis (CE) and fluorescent dyes to analyze the size of the amplified DNA fragments. Standard Curves: Generated by plotting the Ct values versus the amount of target DNA from serially diluted cell line DNA known to have the translocation. This allows for the quantification of the fusion sequences in a test sample.

23. Key Findings and Significance The assay can detect and quantify the t(14;18) translocation. The use of cyclophilin as an internal control ensures the quality of the DNA and normalizes the data. Ct values are used as a measure of the initial amount of target DNA in the sample. The Gene Scan analysis and internal size standards ensure correct base calling and alignment of the peak patterns, confirming the size of the amplified products.