Cloning VectorsMerebeth Ann V. Pedroso

Definition

Cloning vector is a strand of foreign DNA

molecule introduced into host cells. It then

replicates itself to have more copies of

itself and the foreign DNA.

Three features of all cloning vectors

sequences that permit the propagation of

itself in bacteria (or in yeast for YACs)

Three features of all cloning vectors

a cloning site to insert foreign DNA; the most

versatile vectors contain a site that can be

cut by many restriction enzymes

Three features of all cloning vectors

a method of selecting for bacteria (or yeast

for YACs) containing a vector with foreign

DNA; usually accomplished by selectable

markers for drug resistance

General Steps of Cloning with Any Vector

1. Prepare the vector and DNA to be cloned by digestion

with restriction enzymes to generate complementary

ends.

General Steps of Cloning with Any Vector

2. Ligation

General Steps of Cloning with Any Vector

3. introduce the DNA into bacterial cells (or

yeast cells for YACs) by transformation

General Steps of Cloning with Any Vector

4. select cells containing foreign DNA by

screening for selectable markers (usually drug

resistance)

Types of Vectors

Plasmids

Phage

Cosmids

Yeast Artificial Chromosomes (YAC)

Bacterial Artificial Chromosome (BAC)

Plasmid

 an extrachromosomal circular DNA molecule that

autonomously replicates inside the bacterial cell;

cloning limit: 100 to 10,000 base pairs or 0.1-10

kilobases (kb)

A plasmid vector is made from natural plasmids by

removing unnecessary segments and adding

essential sequences.

Plasmids

Plasmids can contain:

• Polylinker

• Drug-resistance gene

• Replication Origin

Plasmids

Types of Plasmids (According to their ability to

transfer to bacteria):

• Conjugative

• Non-conjugative

• Intermediate classes

Plasmids

Classes of Plasmids according to Function:

• Fertility F-plasmids

• Resistance (R) Plasmids

• Col Plasmids

• Degredative Plasmids

• Virulence Plasmids

Phage

• Lambda genome exists as a linear, double-stranded complimentary ends

• λ phages are viruses that can infect bacteria. 

• High transformation efficiency• They could complete a life cycle even if

there are foreign DNA was inserted in its genome.

Phage

Phage

Phage

In Vitro Assembly System

1. If the two genes for expression of Nu1 and A

are mutated in the λ DNA, it cannot be

packaged into the pre-assembled head. ^

2. When the extract is mixed with recombinant  λ

DNA and proteins Nu1 and A, the complete λ

 virion carrying recombinant l DNA will be

assembled.

Cosmid

a combination of the plasmid

vector and the COS site

allows the target DNA to be

inserted into the λ head. 

Cosmid

It has the following advantages:

• High transformation efficiency.

• The cosmid vector can carry up to

45 kb whereas plasmid and  λ

phage vectors are limited to 25 kb.

Cosmid

YAC

• Yeast Artificial Chromosome

•  an artificially constructed system

that can undergo replication

• capable of carrying a large DNA

fragment (up to 2 Mb)

• transformation efficiency is very low

YAC

Techniques for cloning genomic DNA into yeast artificial chromosomes (YAC) make it possible to analyze very long DNA sequences like human genes.

YAC

Key Components:

1. Centromere

2. Two Telomeres

3. Origins of replication

4. Antimicrobial-resistant gene

5. Recognition sites

YAC

BAC

• Bacterial Artificial Chromosomes

• developed to hold much larger

pieces of DNA than a plasmid can

• originally created from part of an

unusual plasmid present in some

bacteria called the F’ plasmid

BAC

•  F’ plasmid allows bacteria to reproduce

sexually^

• could hold up to a million basepairs of DNA

from another bacteria

•  F’ has origins of replication and bacteria have

a way to control how F’ is copied

• Hiroaki Shizuya took the parts of F’ that were

important, cleaned it up, and turned it into a

vector

BAC

BAC

• 350 kbp of DNA

•  replication origins, antibiotic

resistance genes, and convenient

places where clone DNA can insert

itself

BAC

• The Institute for Genomic

Research (TIGR) in the technique

of shotgun cloning that was

employed in the sequence

determination of the human

genome