RESTRICTION ENZYMES & GEL ELECTROPHORESIS ANALYSIS OF PRECUT LAMBDA DNA

VIDEO INTRODUCTION AP Bio Lab 6 - Molecular Biology — bozemanscience AP Bio Lab 6 - Molecular Biology — bozemanscience Restriction Enzymes 2.19 “Molecular Scissors” Restriction Enzymes 2.19 Finds 5 specific sequences…..# cuts? # fragments?

OBJECTIVES Understand the use of restriction enzymes as biotechnology tools Become familiar with principles and techniques of agarose gel electrophoresis Generate a standard curve from a series of DNA size standards Estimate DNA fragment sizes from agarose gel data

RESTRICTION ENZYMES “ENDONUCLEASES” Biomolecules that cut DNA at specific sites. Recognize specific sequences of DNA base pairs and cut (chemically separate) DNA at that specific site First isolated in bacteria that use them as a natural defense mechanism to cut up the invading DNA of bacteriophages. “1 st biological immune system” Thousands of restriction enzymes. Named for the bacterium where they were isolated (and #) EcoRI (Escherichia coli-1st) A-A-T-T HindIII (Haemophilus Infulenzae- 3rd)A-G-C-T PstI (Providencia stuartii)T-G-C-A

STICKY ENDS (UNPAIRED BASES) VS BLUNT PALINDROMIC PsiI Action of enzyme: produces restriction fragments cut DNA at specific sequences restriction site symmetrical “palindrome” produces two double stranded DNA sticky ends will bind to any foreign (complementary) by hydrogen bonding “racecar” “radar”

MANY USES OF RESTRICTION ENZYMES… Now that we can cut DNA with restriction enzymes… we can cut up DNA from different people… or different organisms… and compare it why? forensics medical diagnostics paternity and more…

LAMBDA PHAGE GENOME Genome = 48,000 base pairs Cut into several DNA fragments; dye 4 samples: 3 precut; 1 undigested From (-) to (+) Fragment size : rate ? Effects of 3 restriction enzymes on lambda genomic DNA *Smaller DNA fragments will travel farther than larger ones

GEL ELECTROPHORESIS A method of separating DNA in a gelatin-like material using an electrical field when it’s in an electrical field it moves toward the positive side + – DNA         “swimming through Jello”

DNA moves in an electrical field… so how does that help you compare DNA fragments? size of DNA fragment affects how far it travels small pieces travel farther large pieces travel slower & lag behind GEL ELECTROPHORESIS + – DNA        “swimming through Jello”

GEL ELECTROPHORESIS longer fragments shorter fragments power source completed gel gel DNA & restriction enzyme wells - +

RUNNING A GEL 12 cut DNA with restriction enzymes fragments of DNA separate based on size 3 Stain DNA ethidium bromide binds to DNA fluoresces under UV light

DNA TECHNOLOGY AND GENOMICS abbench/lab6/praprob2.html abbench/lab6/praprob2.html Recombinant DNA technology 1.13 Isolates and amplifies DNA sequences; cloned for use with restriction enzyme and transformation technology polymerase chain reaction Denaturing > Anneal primer > DNA polymerase > repeat