PCR Theory and Primer Design - Lecture 12 - DNA Extraction and PCR Theory PDF

RDNA202: Biotechnology and Genomics Cassie [email protected] Biotechnology and Genomics - Topics DNA extraction PCR Theory Primer Design DNA Sequencing Introduction to Bioinformatics Lecture 12: DNA extraction & PCR Theory RDNA202 Cassie [email protected] Biotech...

RDNA202: Biotechnology and Genomics Cassie [email protected] Biotechnology and Genomics - Topics DNA extraction PCR Theory Primer Design DNA Sequencing Introduction to Bioinformatics Lecture 12: DNA extraction & PCR Theory RDNA202 Cassie [email protected] Biotechnology and Genomics Lecture 12: DNA extraction & PCR Theory Lecture 13: PCR Theory Lecture 14: Primer Design Lecture 15: Tutorial / Lecture continuation PCR Theory and Primer Design Weier 1988 Biotechnology “Applied biology” The use of molecular methods with biological systems – living organisms or parts of them: To modify the genetic materials of living cells So that they produce new substances or perform new functions  Biotechnology Primarily used in: Medicine Agriculture industries Species identification studies Rats species – Araujo et al. (2014) Antibiotic production Biotechnology Facilitates the manipulation of genetic material: DNA RNA Procedures: 1. DNA extraction 2. DNA amplification – Polymerase Chain Reaction (PCR) 3. Analysis of PCR products – Gel electrophoresis DNA Extraction A method that purifies DNA using physical and/or chemical methods Goal is to separate DNA from cell components including: Cell membrane Proteins Other cell components Isolation technique needs to produce DNA that is: Good quality Good quantity Pure Devoid of contaminants – RNA and proteins DNA Extraction DNA extraction involves: Lysing the cells Solubilising DNA Chemical or enzymatic methods to remove macromolecules, lipids, RNA or proteins Process often includes the use of kits Techniques could include: Organic extraction –Phenol-chloroform method Non-organic method – Salting out and proteinase K treatment Adsorption method – Silica-gel membrane  DNA Extraction A lysis buffer – Solution used to break open cells Enzymes break apart lipid molecules in cell membranes and nuclear membranes: Enzymes like proteases Break down proteins & inactivate macromolecules Ribonucleases (RNAses) Break down RNA Alcohol (ethanol/isopropyl alcohol) Precipitate DNA DNA Extraction – Quality and Quantity Gel electrophoresis Nanodrop Note: DNA absorbs at A260 Spectrophotometer Proteins absorb at A280 Ratio of A260/A280 = DNA Qubit quality  DNA Extraction – Quality and Quantity Gel electrophoresis Separates DNA by size For visualization and detection of purity Nanodrop Used to measure the concentration and purity of DNA, RNA, and proteins Concentration measured by placing 1-2 µl on a pedestal Measures using absorbance  DNA Extraction – Quality and Quantity Spectrophotometer DNA concentration – determined by measuring absorbance at 260 nm (A260) Done via spectrophotometry using a quartz cuvette Qubit Useful to check DNA quality by measuring intact dsDNA Measures based on fluorometry  Polymerase Chain Reaction (PCR) Technique used to amplify specific DNA regions for further analysis In about 2 hours – billions of copies of sequence made Polymerase Chain Reaction (PCR) Can work with very small amounts of DNA – Advantage Uses in Palaeontology - Criminology Used for several applications: Sequencing of genes – diagnosis of hereditary and genetic diseases Identification of genetic fingerprints Detection and diagnosis of infectious diseases Determining paternity Identifying DNA contamination in a sample PCR components Several components are required for the PCR process: 1. DNA template – contains DNA fragment to be amplified 2. Dual primers – Regulate the start and end of region to be amplified 3. Taq polymerase – Copies amplified region 4. Nucleotides – DNA polymerase uses these to create new DNA amplicons 5. Buffer – Offers appropriate chemical environment for DNA polymerase PCR components 1. DNA template Serves as the starting DNA Obtained via DNA extraction 1pg – 10ng DNA usually used as starting concentration (2 - 5µl) PCR components 2. Dual primers Forward and reverse primer Identify particular DNA product to be amplified Small DNA fragments = complement to target region Around 18-30 nucleotides long Complementary to sequence at 3’end of each DNA strand to be copied PCR components 3. Taq polymerase DNA polymerase Isolated from thermostable bacterium Thermus aquaticus Can withstand high temperatures used during PCR PCR components 4. Nucleotides dNTPs – Deoxynucleotide triphosphates dATP, dGTP, dTTP, DCTP Building blocks of DNA PCR components 5. Buffer Provides optimum ionic environment – e.g. pH for PCR MgCl2 – cofactor for thermostability of Taq High concentrations may prevent DNA denaturation Low concentration impairs polymerization  PCR components - Mastermix PCR components combined into a master mixture = Mastermix This is added to solution of DNA Contains: Taq dNTPs Buffer Add this solution to primers to make final mastermix PCR components – Mastermix e.g. Number of reactions Stock solutions 1 10 20 28 µl µl µl µl water 0.8 16 22.4 Buffer (10x) 2.5 50 70 MgCl2 (25mM) 4 40 80 112 Forward primer (6µM) 4 40 80 112 Reverse primer (6µM) 4 40 80 112 dNTPs (100mM) 0.5 5.0 10 114 Taq (5units / µl) 0.2 2.0 4 5.6 TOTAL 16 160 320  PCR components - Mastermix What are the final concentrations of each component? If each PCR tube gets 16 µl of mastermix a. Buffer (10X) – 10 x(2.5/16) = 1.56X b. MgCl2 (25mM) – 25 x (4/16) = 6.25mM c. Forward primer (6µM) – 6 x (4/16) = 1.5µM d. Reverse primer (6µM) – 6 x (4/16) = 1.5µM e. Each dNTP (100mM) – 100 x (0.5/16) = 3.125µM  PCR conditions PCRs are run on a Thermal Cycler – “cycles heat” Run in 5 stages: Initial Denaturation Denaturation Annealing Repeated as necessary e.g. X 35 Extension Final Extension  PCR conditions Denaturation 94 – 960C Results in separation of the DNA strand – becomes single stranded Annealing 45 – 650C Sample cooled to allow primer attachment (annealing) to DNA sequence Extension 720C Ensures binding between primers and DNA sequence Taq polymerase synthesizes new DNA strands – catalyzes primer elongation Polymerase can withstand high temperatures used in PCR  PCR Reaction Theoretical amplification = 2n times 20 cycles – 1 048 576 times 30 cycles – 1 073 741 824 times PCR Summary https://app.jove.com/embed/player?id=10819&access=db1e8  Tomorrow: Lecture 13 & 14: PCR Theory & Primer Design RDNA202 Cassie [email protected]