Molecular Biology Techniques PDF

220 BRANCHED CHAIN DNA (bDNA) 1. Uses a series of hybrid probes to elicit a signal amplification- chemiluminescence REMEMBER! 2. Detect specific RNA sequences Tech PENS...

220 BRANCHED CHAIN DNA (bDNA) 1. Uses a series of hybrid probes to elicit a signal amplification- chemiluminescence REMEMBER! 2. Detect specific RNA sequences Tech PENS Load Gels! Electrophoresis Technology P = Pore size of gel ELECTROPHORESIS OF NUCLEIC ACIDS - E = electric field N = negative DNA charge 1. Separation based on size and charge through a sieve-like matrix (agarose 01· polyacryla1nide) S = size of DNA CJ CJ CJ CJ - 2. Migration in electrical field at a rate inversely proportional to loglO of molecular size (number ofbase pairs) 3. DNA (negatively charget{) migrates toward anode (positively charged) FACTORS AFFECTING MIGRATION RATE Tortoises are big and slow Bunnies are small and fast 1. Matrix type and porosity (%) of the gel (gel castinB) Movement through a gel depends on the size of the DNA particle, its charge, and the pore size of the 2. Net charge of nucleic acid molecule gel. All negatively charged DNA particles move toward the anode, but the larger pieces have a 3. DNA conformation harder time squeezing through the small pores in the gel and cannot move as fast, i.e., as far, as the 4. Electric field strength smaller pieces. 5. Temperature of gel Blotting Techniques 6. ucleic acid base composition SOUTHERN BLOT 1. Detects specific DNA sequences 7. Presence of intercalating dyes 2. DNA denatured in the gel by an 8. Type and strength of buffer increase in pH PULSED FIELD Ga ELECTROPHORESIS OF DNA (PFGE) 3. DNA transferred to a membrane by 1. Analysis of DNA fragments up to 100 capillary action with a high salt kb in size solution 2. Separation accomplished using a pulsed electrical field 4. Labeled complementary probe used for 3. PFGE commonly used for genotyping detection prokaryotes Paper towels Nitrocellulose I filter membta ne Gel Methods, instruments, reagents & controls Sponge Routine and special procedures to verify test results High Salt Solution  221 5. Procedure: WESTERN BLOT a. Genomic DNA cut with restriction 1. Protein run on SDS- polyacrylamide enzymes gel electrophoresis (PAGE) b. DNA electrophoresed c. Gel submerged in an alkaline 2. Protein electrically transferred to solution to denature the DNA membrane ( el ectro-transfer) d. DNA transferred onto a nitrocellulose membrane by 3. Membrane incubated with a primary capillary action antibody and blocking solution e. Membrane mixed with a solution containing labeled probe 4. Membrane washed and incubated with ❖ Prohe will hybridize to secondary antibody and blocking complementary piece of DNA on solution gel 5. Membrane washed and rinsed with f. Membrane washed to remove substrate buffer excess, unbound probe g. Membrane developed and visualized 6. Substrate added and developed using either radioactive isotopes, SOUTHWESTERN BLOT chemiluminescent dyes , or 1. DNA-binding proteins colorimetric techniques REMEMBE Western Blot - Protein Southern Belle named "Dee" for DNA Western Cowboy Eats his Protein NORTHERN BLOT 1. Detect specific sequences of RNA DOTI SLOT BLOTS 2. RNA transferred to membrane by 1. Quick analysis of DNA and RNA capillary action using a high salt solution 2. Does not determine the size of target 3. Labeled complementary probe used for 3. Applied to: detection a. Expression analysis b. Mutation anaysis c. Amplification analysis «FD\ REMEMBER! REVERSE DOT BLOTS ~ Northern Blot - RNA 1. Reverse allele sp ecific oligonucleotide 2. Hybridization Northern Eskimo with an RNA Virus because it's Cold ~ 3. Important method for genotyping common human mutations l(~ STRINGENCY 1. Describes the conditions under which hybridization takes place 2. Salt, heat and formamide increase stringency