PCR and RT-PCR

In this slide contains contents, steps, different and application of PCR and RT-PCR
Presented by: RAMYA NAGARAJU GARI (Department of pharmacology).
RIPER, anantapur

1. 1 PCR and RT-PCR A Seminar as a part of curricular requirement for M. Pharmacy, I Year - I semester Presented by N. Ramya (20L81S0110) PHARMACOLOGY Under the guidance of A. Sudheer, M. Pharm Associate Professor Dept. of pharmacology

2. 2 RT-PCR PCR Contents of PCR Steps in PCR PCR VS RT-PCR Applications References contents

3. 3 principle In RT- PCR, the RNA template is first converted into a Complementary DNA (cDNA) using a reverse t ranscriptase (RT). The cDNA is then used as a template for exponential amplification using PCR. The two technique use the same process except that RT-PCR has an added step of reverse transcription of RNA to DNA to allow for amplification. RT-PCR:

4. 4

5. 5 optimized one- step RT- PCR condition, supports the reverse t ranscription of the RNA from unpurified or crude samples, such as whole blood and serum However, the starting RNA However, the starting RNA templates are prone to degradation in the one- step approach, and the use of this approach is not recommended when repeated assays from the same sample is required.

6. 6 Polymerase Chain Reaction Polymerase chain reaction (PCR) is a technique used in molecular biology. To amplify a single copy or a few copies of a segment of DNA generating thousands to millions of copies of a particular DNA sequence.

7. 7 Components Of PCR constitutes the following: DNA Template – The DNA of interest from the sample. DNA Polymerase – Taq Polymerase is used. It is thermostable and does not denature at very high temperatures. Oligonucleotide Primer s- These are the short stretches of single-stranded DNA complementary to the 3’ ends of sense and anti-sense strands. C omponents Of PCR

8. 8 4.Deoxyribonucleotide triphosphate – These provide energy for polymerization and are the building blocks for the synthesis of DNA. These are single units of bases. 5. Buffer System– Magnesium and Potassium provide optimum conditions for DNA denaturation and renaturation. It is also important for fidelity, polymerase activity, and stability.

9. 9 There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time. Denaturation. Annealing. Extension. Procedure:

10. 10 Denaturation at 94°C : During the denaturation, the reaction mixture is heated to 94°C for 1 min, which causes separation of DNA double stranded. Now, each strand acts as template for synthesis of complimentary strand.

11. 11 Annealing at 54°C : The reaction temperature is lowered to 54-60℃ for around 20-40 seconds. Here, the primers bind to their complementary sequences on the template DNA. Primers are single-strand sequences of DNA or RNA around 20 to 30 bases in length. They serve as the starting point for the synthesis of DNA.

12. 12 The two separated strands run in the opposite direction and consequently there are two primers- a forward primer and a reverse primer.

13. 13 At this step, the temperature is raised to 72-80℃. T he bases are added to the 3’ end of the primer by the Taq polymerase enzyme. This elongates the DNA in the 5’ to 3’ direction. The DNA polymerase adds about 1000bp/minute under optimum conditions. Taq Polymerase can tolerate very high temperatures. It attaches to the primer and adds DNA bases to the single strand. As a result, a double-stranded DNA molecule is obtained. Extension:

14. 14

15. 15 PCR RT-PCR PCR is a technique to amplify a segment of DNA, generating millions of copies of a DNA Sequence. RT-PCR is a variant of PCR used in detection of gene expression in molecular biology. Denaturation, annealing, and extension are the three steps. RT-PCR is followed by PCR. A double-standard DNA molecule serve as the template. A single standard RNA molecule is the template for the reverse transcription. DNA Polymerase is used as the enzyme. Reverse transcriptase and DNA polymerase are used as enzyme. Used in functional analysis of genes, diagnosis, and monitoring of heredity diseases, DNA Cloning, DNA Sequencing. Used in detection of gene expression.

16. 16 Gene Insertion: RT-PCR can also be very useful in the insertion of eukaryotic genes into prokaryotes. Genetic Insertion Diagnosis: RT-PCR can be used to diagnose genetic diseases such as Lesch -Nyhan symdrome. Cancer Detection: scientists are working on ways to use RT-PCR in cancer detection to help improve prognosis and monitor response to therapy . Applications

17. 17 Julia Bachman. Reverse Transcription PCR. Elsevier. 2013, 530. 67-74. Freeman WM, Walker SJ. Quantitative RT-PCR. Elsevier. 2012, 26(1): 124-125. Alexander A. Morley. Digital PCR. Elsevier. 2014, 272. 12-13. References