University:
Boston University
Course:
CAS BI 107 | Biology 1
Academic year:

2024
Views:

167

Pages:

19
Author:

Drobtinciq3u5

3 Steps of PCR • Step #1 Denaturation – Heat (94oC) to denature DNA template strands • Step #2 Annealing – Cool (52oC) to anneal primers to template • Step #3 Extension – Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA • Repeat 35 cycles Step #1 Denaturation 5’ 3’ 3’ 5’ 94oC 5’ 3’ 3’ 5’ Heat causes DNA strands to separate Primers Anneal • Primers bind to the template sequence Primers anneal at 52oC 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ • Taq polymerase recognizes 3’ end of primer + template strand Taq extends at 72oC 5’ 3’ 5’ 3’ 3’ 3’ 5’ 5’ What is happening in the PCR tube while in the thermocycler? • PCR ANIMATION Cycle 1 Cycle 2 Cycle 3 Generation of Precise-length fragments Food Fight !!! • Crime Scene Evidence (CS) – DNA isolated from a half eaten sandwich • Suspects – A: Dylan – B: Sabrina – C: Aristides – D: Tarialas How to use a micropipette • Press the plunger down to the first soft stop. You will feel it stop naturally • Lower the tip into the loading dye and slowly let the plunger come back up and fill the pipet. • Press down to the first soft stop to deliver the loading dye. If it all doesn't come out you can push down to the hard stop to give it one last chance to come out. • Eject tip Set up PCR reactions 1. Find the PCR tubes at your station. 2. Label them ‘CS’ for Crime Scene DNA, ‘A’ for Suspect A DNA, ‘B’ for Suspect B DNA, ‘C’ for Suspect C DNA, and ‘D’ for Suspect D DNA. 3. Keeping the tubes on ice, add 20 μl of Master Mix + blue primers to each tube. 4. Keeping the tubes on ice, add 20 µl of each DNA to the appropriately labeled tube. 5. USE A FRESH TIP EACH TIME! 6. Mix and put in thermal cycler 7. Cycle ~3 hours (OVERNIGHT) To visualize PCR products Crime Scene investigators use gel electrophoresis TH01 alleles (14) (13) (12) (11) (10) (9) (8) (7) (6) (5) (4) (3) Allele ladder Mother Father Child C Child D Child E Load Gel • Obtain 5 PCR Reactions – CS –A –B –C –D • Add 10μl of Orange Loading Dye (LD) to each tube Load Gel Lane Description 1 20 μl of CSI allele ladder 2 20 μl of Crime Scene PCR reaction 3 20 μl of Suspect A PCR reaction 4 20 μl of Suspect B PCR reaction 5 20 μl of Suspect C PCR reaction 6 20 μl of Suspect D PCR reaction Agarose Gel Electrophoresis Individual 1’s DNA Individual 2’s DNA A A B B C C D D E -- Wells where DNA samples are loaded D B C A B C A E + D Agarose Gel Smaller fragments will move farther through the dense gel than larger fragments, so bands near the top are bigger! Agarose gel • Agarose – gelatanous substance derived from algae – polymer of galactose • Forms a matrix – Separate fragments of DNA – Smaller fragments travel farther than larger • Higher percentage of agarose separates small molecules • To make a gel – Grams of agarose/100ml of buffer Gel Electrophoresis • Place gel in gel box • Pour buffer in box until gel wells are covered. Gel Electrophoresis Place 20ul of samples into appropriate wells Set up electrophoresis chamber by putting top in place and connecting it to the power supply Gel Electrophoresis • Gel runs from negative to positive • DNA slightly negative Gel running Analysis of Results BXP007 alleles AL CS A B C D 15 10 7 5 AL: Allele ladder CS: Crime Scene A: Suspect A B: Suspect B C: Suspect C D: Suspect D 4 3 2 1 5-2 7-4 5-2 genotype • Who can’t be excluded? 7-2 10-3 COmbined DNA Index System (CODIS) • A federally maintained database used by law enforcement officials Real STR analysis •Four different fluorescent tags have been used to identify 7 amplified loci •Allele ladders are indicated by arrows