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

ANALYSIS OF PRECUT LAMBDA DNA

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. “1st 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 BLUNTPALINDROMIC

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

powersource

completed gel

gel

DNA &restriction enzyme

wells

-

+

RUNNING A GEL

1 2

cut DNA with restriction enzymes

fragments of DNAseparate based on size

3

Stain DNA• ethidium

bromide binds to DNA

• fluoresces under UV light

DNA TECHNOLOGY AND GENOMICS

• http://www.phschool.com/science/biology_place/labbench/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