Molecular Biology Techniques Lecture Notes PDF

Biochemistry II lab 8 Molecular Biology Techniques  We’ll learn about… Techniques Gel 01 classification 03 electrophoresis TaqMan assay, PCR components, 04 FRET 02 principle, phases,...

Biochemistry II lab 8 Molecular Biology Techniques  We’ll learn about… Techniques Gel 01 classification 03 electrophoresis TaqMan assay, PCR components, 04 FRET 02 principle, phases, types SYBR green dye 05 assay  We can classify molecular biology techniques into two main categories: Techniques that deal with Techniques that deal with nucleic acid, they include: proteins, they include: PCR Western blotting Cloning ELISA Southern blotting and Flow cytometry Northern blotting Polymerase Chain Reaction (PCR)  Polymerase Chain Reaction (PCR) It is in vitro technique used to amplify (creating many copies) a specific sequence of genetic material. Why called “Polymerase”? Because the only enzyme used in the reaction is DNA polymerase (called Taq polymerase). Why called “Chain”? Because the products of the first reaction become substrates of the following one, and so on.  The “Reaction” Components 1) Target DNA - contains the sequence to be amplified. 2) Primers - short single sequence of nucleotides, antiparallel and complementary to the target gene. 3) dNTPs – deoxynucleotide triphosphates, building block for new DNA strand. 4) Taq DNA Polymerase - Thermostable enzyme, isolated from “Thermus aquaticus” a thermophilic bacteria, makes new strands of DNA. 5) Mg⁺⁺ ions - cofactor of the enzyme. 6) Buffer solution - maintains pH of the reaction suitable for enzyme activity.  Principle of PCR - The cycling reactions: There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated thermal cycler, which can heat and cool the tubes with the reaction mixture in a very short time.  Steps of PCR I. Denaturation at (95°C): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step. II. Annealing at (50-65°C): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA. III. Extension at (72°C): Raise the reaction temperature so Taq polymerase extends the primers, synthesizing new strands of DNA. Steps of PCR 95°C 72°C 50°C  There are three phases of PCR amplification: The exponential The linear phase The plateau phase phase ▪ The first phase of PCR ▪ The second phase of PCR ▪ The final phase of PCR amplification. amplification. amplification. ▪ Complete reaction and ▪ Excess reaction components. ▪ The reaction components no more products are being generated. ▪ Real-Time PCR takes its are being consumed. measurements during this ▪ Amplification slows. ▪ Traditional PCR takes its measurements during phase of PCR.. this phase of PCR.  Types of PCR Conventional PCR Droplet digital PCR Reverse- Real Time Transcriptase PCR PCR  1- Conventional PCR ✓ The traditional PCR is a qualitative technique, which is performed in a tube and when the reaction is complete, the products (amplified DNA fragments) are analysed and visualised by gel electrophoresis. ✓ It requires only a thermal cycler and horizontal gel electrophoresis unit. ✓ Also known as endpoint PCR, where the detection of the amplified sequence are performed at the end of the reaction after the last PCR cycle. Gel Electrophoresis  Gel Electrophoresis Electrophoresis is a separation technique based on the mobility of charged molecules in an electric field according to molecular weight or charge. It is used mainly for the analysis and purification of large molecules such as proteins or nucleic acids. Agarose gel is the most commonly used matrices in research laboratories for separation of nucleic acids. As molecules are forced through the gel by an applied voltage, larger molecules are retarded in their migration more than smaller molecules Gel Electrophoresis  2- Real-Time PCR (qRT-PCR) ❖ Also known as quantitative PCR (qPCR) and is used to measure the quantity of a PCR product. ❖ Permits the analysis of the products while the reaction is actually in progress. ❖ This is achieved by using various fluorescent dyes that react with the amplified product and can be measured.  2- Real-Time PCR (qRT-PCR) ❖ Fluorescent dyes used in real-time PCR include: - Double-stranded DNA (dsDNA)–binding dyes. - Dye molecules attached to PCR primers. - Probes that hybridize with PCR products during amplification.  Real-time PCR fluorescence detection systems Labeling PCR products to be quantified: - The most widely used are: TaqMan probe SYBR green based assay dye based assay  A- TaqMan Assays TaqMan probes are oligonucleotides that contain a fluorescent dye at the 5′ base and a quenching dye typically at the 3′ base. During the amplification process, the Taq polymerase enzyme (hence the name "TaqMan") cleaves the probe between the two dyes. This separation causes the fluorescent dyes to no longer be in proximity, resulting in the release of fluorescence that can be detected. Which is a phenomenon called fluorescence resonance energy transfer (FRET). Fluorescence Resonance Energy Transfer (FRET) Polymerization Cleavage Polymerization Completed  B- SYBR Green dye ▪ It is a fluorescent DNA-binding dye that binds to double- stranded DNA. ▪ Fluorescent signal of DNA-bound SYBR Green dye is much stronger compared to unbound dye. TaqMan probe is more specific, however SYBR Green assay is less expensive.  SYBR Green I Chemistry Polymerization Polymerization completed Forward Primer 5' 5' 3' 5' 3' 5' 5' 3' 5' 3' 5' 5' Reverse Primer  2- Real-Time PCR (qRT-PCR) The advantages of real-time PCR include: Quantitative. Monitor the progress of the PCR reaction as it occurs in real time. Amplification and detection occur in a single tube, eliminating post-PCR manipulations.  3- Droplet Digital PCR The Droplet Digital PCR System is advanced and highly sensitive technique for nucleic acid amplification. It could be used in case of low concentrations of nucleic acids while Real-time PCR requires specific concentration for amplification (50-250 ng/μl). 4- Reverse-Transcriptase (RT–PCR) - It uses reverse transcriptase enzyme to synthesize cDNA “complementary DNA“ from target RNA, then amplify cDNA.  Thanks! CREDITS: This presentation template was created by Slidesgo, and includes icons by Flaticon, and infographics & Slidesgo Flaticon images by Freepik Freepik Please keep this slide for attribution