Prentice Hall c2002 Chapter 19 1

Recombinant DNA Technology

• Endonucleases• RFLPs• DNA fingerprinting• Polymerase Chain

reaction• DNA sequencing

Prentice Hall c2002 Chapter 19 2

Restriction Endonucleases

• Enzymes that recognize specific DNA sequences

• Cut both strands of DNA at the binding site, producing fragments that can be degraded by exonucleases

• Host cells protect their own DNA by covalent modification of bases at the restriction site (e.g. methylation)

Prentice Hall c2002 Chapter 19 3

Restriction endonuclease properties

• Type I - catalyze both the methylation of host DNA and cleavage of unmethylatedDNA at a specific recognition sequence

• Type II - cleave double-stranded DNA only, at or near an unmethylatedrecognition sequence

• More than 200 type I and type II are known

• Most recognize “palindromicsequences” (read the same in either direction)

Prentice Hall c2002 Chapter 19 4

Palindromic sequences

Prentice Hall c2002 Chapter 19 5

Uses of Restriction Endonucleases

• Developing restriction maps (indicates specific cleavage sites in a DNA fragment)

• Map of bacteriophage λ showing cleavage sites of some restriction enzymes

Prentice Hall c2002 Chapter 19 6

• Restriction digest of bacteriophage λ

• Four restriction enzymes used

• Sizing gel separates fragments (smallest move fastest)

Prentice Hall c2002 Chapter 19 7

DNA Fingerprinting

• DNA sequence can be used to identify individuals in a large population

• Highly variable regions give restriction fragments that are as unique as fingerprints

Prentice Hall c2002 Chapter 19 8

Fig 19.34

• DNA Fingerprinting

Prentice Hall c2002 Chapter 19 9

PCR analysis & technique

Prentice Hall c2002 Chapter 19 10

DNA sequencing

Prentice Hall c2002 Chapter 19 11

DNA sequencing

Prentice Hall c2002 Chapter 19 12

DNA sequence—Fluorescent dyes