DNA Extraction, PCR, & Electrophoresis PDF

DNA EXTRACTION, PCR, & DNA ELECTROPHORESIS Mahmoud Alfaqih BDS PhD Department of Medical Biochemistry DNA extraction ■ DNA isolation is a process of purification of DNA from sample using a combination of physical and chemical methods. ■ This is used in science and medicine – For genotyping – In fo...

DNA EXTRACTION, PCR, & DNA ELECTROPHORESIS Mahmoud Alfaqih BDS PhD Department of Medical Biochemistry DNA extraction ■ DNA isolation is a process of purification of DNA from sample using a combination of physical and chemical methods. ■ This is used in science and medicine – For genotyping – In forensics – Paternity testing – In medical genetics to identify mutations or diseases DNA extraction ■ It involves the following steps: – Digestion: (mechanical, chemical) – Precipitation: Ice cold ethanol – DNA storage: Tris-EDTA (TE) buffer (to prevent DNA from degradation) PCR ■ 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 across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence ■ The purpose of PCR is to make a large number of copies of a gene or gene segments PCR Cycle - Step 1 – Denaturation by Heat Target Sequence Target Sequence PCR Cycle - Step 2 – Biotinylated Primers Anneal to Ends of Target PCR Cycle - Step 3 – Taq DNA Polymerase Catalyses Primer Extension as Nucleotides are Added End of the 1st PCR Cycle – Two Copies of Target Sequence Target Amplification No. of Cycles No. Amplicon Copies of 1 cycle = 2 Amplicon 2 cycle = 4 Amplicon 3 cycle = 8 Amplicon 4 cycle = 16 Amplicon 1 2 2 4 3 8 4 16 5 32 6 64 5 cycle = 32 Amplicon 6 cycle = 64 Amplicon 20 1,048,576 7 cycle = 128 Amplicon 30 1,073741824 DNA electrophoresis ■ Electrophoresis is a procedure that separates molecules (protein or DNA), through a stationary material (gel), in an electrical field. ■ Separation based on: – Molecule size – Molecule charge – Molecule conformation ■ Migration of DNA towards positive charged electrode (DNA is negatively charged) How does electrophoresis work? • The gel is made from agar • Derived from seaweed • No net electric charge • DNA is a negatively charged • Molecules sort based on • Charge • Size • shape DNA electrophoresis ■ Agarose gel: a sugar → heat (to dissolve) → cool (to polymerize/solidify) ■ Loading dye: ■ Prevent sample floating ■ Sample monitoring ■ Ethidium bromide stains DNA and emit (release) fluorescence if exposed to UV light. ■ Ladder standard → a known base pair (bp.) size used to compare the test product (DNA). Terms ■ Agarose is a polysaccharide (carbohydrate) polymer material, generally extracted from seaweed. ■ Gel electrophoresis is a method that uses an electrical current and a gel matrix to separate molecules like DNA and proteins. ■ Buffer a solution containing either a weak acid and its salt or a weak base and its salt, which is resistant to changes in pH. ■ Ethidium Bromide a fluorescent chemical that intercalates between base pairs in a double stranded DNA molecule. Commonly used to detect DNA following gel electrophoresis. ■ DNA Ladder consists of known DNA sizes used to determine the size of an unknown DNA sample. The DNA ladder usually contains regularly spaced sized samples which when run on an agarose gel looks like a "ladder”. ■ Power Supply a source of electric current Pipetting exercise ■ Many tasks in molecular biology labs require highly accurate transfer of small volumes of reagents. ■ This high degree of precision is essential and can be carried out by specific tools known as the micropipettes ■ You can aspirate: – Small volumes (e.g. 1-5 uL) – Medium volumes (e.g. 20-50 uL) – Large volumes ( e.g 100-500 uL)