ANALYSIS OF PRECUT LAMBDA DNA

R E S T R I C T I O N E N Z Y M E S & G E L E L E C T R O P H O R E S I S

VIDEO INTRODUCTION

• • AP Bio Lab 6 - Molecular Biology — bozemanscience 11.57

Restriction Enzymes 2.19

“Molecular Scissors”

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) HindIII (Haemophilus Infulenzae- 3rd) PstI (Providencia stuartii) A-A-T-T A-G-C-T T-G-C-A

STICKY ENDS (UNPAIRED BASES) VS BLUNT PALINDROMIC PsiI “racecar” “radar” • 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

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…

Effects of 3 restriction enzymes on lambda genomic DNA

LAMBDA PHAGE GENOME

*Smaller DNA fragments will travel farther than larger ones

• • • • Genome = 48,000 base pairs Cut into several DNA fragments; dye 4 samples: 3 precut; 1 undigested From (-) to (+) Fragment size : rate ?

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” +

GEL ELECTROPHORESIS

• 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

– DNA

      

“swimming through Jello” +

GEL ELECTROPHORESIS

DNA & restriction enzyme wells gel power source

-

+ longer fragments completed gel shorter fragments

RUNNING A GEL

fragments of DNA separate based on size cut DNA with restriction enzymes 1 2

Stain DNA • • ethidium bromide binds to DNA fluoresces under UV light

3

DNA TECHNOLOGY AND GENOMICS

• http://www.phschool.com/science/biology_place/l 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 -1.28

• Denaturing > Anneal primer > DNA polymerase > repeat