Polymerase Chain Reaction (PCR) - Cihan University Sulaymaniyah PDF

Cihan University- Sulaymaniyah Medical Microbiology Department Molecular Biology Lecturer: Dr. Alla Polymerase Chain Reaction prepared by: Sonia Kamal & Sakar Mohammed Tabl...

Cihan University- Sulaymaniyah Medical Microbiology Department Molecular Biology Lecturer: Dr. Alla Polymerase Chain Reaction prepared by: Sonia Kamal & Sakar Mohammed Table of contents 01 02 03 What's PCR? What are its What's PCR’s types? purpose? 04 05 06 The uses of PCR What's the Steps of DNA requirements replication WHAT’S PCR? The Polymerase Chain Reaction (PCR) machine, also known as a thermal cycler, is a laboratory instrument used to amplify and replicate a specific segment of DNA, through a process called PCR. This technique allows for the production of millions of copies of a particular DNA sequence, enabling researchers to study and analyze genetic material in various applications such as medical diagnostics, forensic analysis, and molecular biology research. Types of PCR Real Time PCR Nested PCR Multiplex PCR Quantitative PCR Arbitrary primed PCR Now that we are familiar with the different types of PCR i wanna talk about, Real Time PCR  Uses of PCR Research Applications Evolutionary Biology PCR is used for gene PCR aids in the study of expression analysis, muta- evolutionary biology by genesis studies, sequencing allowing scientists to analyze genomes, and phylogenetic ancient DNA sequences from studies among others. archaeological samples. Personalized Medicine It helps in identifying genetic mutations that may affect an individual’s response to certain drugs, hence guiding personalized treatment plans. Purpose of PCR The purpose of PCR, is to amplify a specific segment of DNA, that is, to create millions to billions of copies of a particular DNA sequence. This process is fundamental in molecular biology and has a myriad of applications across different scientific fields. To understand the purpose of PCR, lrt’s talk about it in logical components:  Purpose of PCR cont. Molecular Cloning & Genetic Diagnostic Applications Engineering in medical diagnostics, PCR generate sufficient quantities of a DNA can detect the presence of fragment for cloning into a vector for infectious agents by genetic manipulation. This is an targeting sequences that ar important step in creating genetically unique to pathogens; modified organisms or in gene bacteria & viruses. therapy research. 1 2 3 4 DNA Amplification Enabling Detection PCR works by making enough PCR allows for the detection of copies of DNA to be analyzed or very small amounts of DNA. This manipulated for various is useful when the sample size is purposes. PCR mimics the natural limited, such as detecting process of cell division in a pathogens in a clinical sample controlled laboratory setting. Requirements of PCR To understand the requirements for PCR and why they are necessary, we need to delve into the fundamentals of this molecular biology technique. hence it amplifies a specific DNA sequence, it allows researchers to obtain sufficient quantities of DNA for further analysis or experimentation. the requirements are as follows… 1) DNA template. 2) Buffer Solution. 3) Primer. 4) Polymerase. 5) Neuclotides. 6) PCR machine. Steps of DNA replication Denaturation: During this step, the reaction mixture is heated to 94–98°C for 20–30 seconds. This causes the melting of the DNA template by breaking the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules. Steps of DNA replication Annealing: The reaction temperature is lowered to 50–65°C for 20–40 seconds allowing primers to bind to their complementary sequences on the single-stranded DNA template. The temperature at this step depends on the melting temperature (Tm) of the primers used. Steps of DNA replication Extension/Elongation: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80°C, and commonly a 1 minute per kilobase of DNA to be amplified is considered adequate. During this time, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in the direction of 5’ to 3’, starting from the primer Steps of DNA replication Steps repitation: The three steps of the PCR cycle are repeated for bout 20-40 times. As more and more reaction cycles are carried out, the double-stranded DNA are synthesized more in number. After 20 cycles, the original DNA has been amplified a million-fold (million strands of DNA) and this rises to a billion fold (1000) million after 30 cycles. Polymerase chain reaction  PCR reagents PCR as a cyclic PCR reaction process 94c 10 DNA Primers template Denaturation Inal buffer Nucleotides taq 52-68°C 2 polymer ase Annealing PCR reaction mixture 72°C Extension Exponential DNA amplification thermocycler THANK YOU Any Questions?