Restriction enzymes (endonucleases)

• Cleave a specific DNA sequence• Protect bacteria from phage infection by

digesting phage DNA after infection.• Cellular DNA is protected by methylases -

block restriction enzyme activity

Each organism has a specific set

of restriction enzymes:

• EcoRI from Escherichia coli • BamHI from Bacillus

amyloliqueraciens• PvuI and PvuII are different

enzymes from same strain.

Ch. 3-1

Restriction enzymes are used for cloning and analyzing DNA

fragmentsRI

PstI

HindIII

A

EcoRi/HindIII Digest

Purify Frgs.

AA

BB

RI

PstI

HindIII

A

BPstI

HindIII

RI

RI

HindIII

BA

HindIII

RI

PstI PstI

Ligate

BPstI

HindIII

RI

AB

Sequence Recognition and cleavage:

a) 5' overhang EcoRI GAATTC G AATTCCTTAAG CTTAA G

b) 3' overhang KpnI GGTACC GGTAC CCCATGG C CATGG

c) Blunt end SmaI CCCGGG CCC GGGGGGCCC GGG CCC

Ch. 3-2

Sticky ends

• The overhangs on cleaved DNA can serve as “sticky ends” or unpaired bases that can be used to link pieces of DNA.

• Use the same enzyme, or one that leaves the same overhang to cut two DNA sources.– Complementary bases will pair. – Ligase will seal.

Vectors

In order to study a DNA fragment (e.g., a gene), it must be amplified and eventually purified.

• Do this by cloning the DNA into a vector, generally a small, circular DNA molecule that replicates inside a bacterium such as Escherichia coli.

Ch. 1-1

Cloning Scheme

Digest Ligate Amplify and Prep

1-1

Vector Types

There are three commonly used types of vectors:1) plasmid vectors (e.g., pUC plasmids)

• These are the most common in biotechnology

2) bacteriophage vectors (e.g., phage )

3) phagemid (hybrid) vectors

Each has a different use, and there are many derivatives of these basic building blocks.

Ch. 1-1

Plasmids• Circular DNA molecules found in bacteria

• Replicated by the host’s machinery independently of the genome. This is accomplished by a sequence on the plasmid called ori, for origin of replication.

• Some plasmids are present in E. coli at 200-500 copies/cell

• Plasmids also contain selectable markers:

• These are genes encoding proteins which provide a way to rapidly and easily find bacteria containing the plasmid.

• Commonly- provide resistance to an antibiotic like ampicillin.

• Thus, bacteria will grow on medium containing these antibiotics only if the bacteria contain a plasmid with the appropriate selectable marker.

Plasmid Engineering

Ch. 1-2

Safety Features

• Modern cloning plasmids have been engineered to be incapable of transfer between bacterial cells

• Provide a level of biological containment.

• Naturally occurring plasmids with their drug resistance genes have produced antibiotic-resistant bacteria.

Ch. 1-2

Transforming plasmids Into bacteria

Ch. 1-2

Screening for Inserts

• Transform bacteria with plasmids containing gene for ampicillin resistance; small number will transform.

• Spread bacteria on plates containing nutrient agar and ampicillin.

• Only transformed cells will survive and form colonies.

1-3