Plant Biotechnology and Genetics: Principles, Techniques and Applications

7.2 DNA Vectors

In molecular biology, a cloning vector is a DNA molecule that carries foreign DNA
fragments into a host cell and allows them to replicate. Cloning vectors are frequently
derived from plasmids, a generic term first coined by Joshua Lederberg in 1952, to
describe any extrachromosomal hereditary determinant. Plasmids, found in bacteria but
not in plants and other “higher” organisms, are convenient vectors used to manipulate
DNA for genetic engineering. Plasmids were discovered in bacteria as double-stranded,
covalently closed circular, extrachromosomal DNA molecules. They have evolved
mechanisms to maintain a stable copy number in their host, to ensure that copies are
shared between daughter cells and to encodegenesthat provide a selective advantage
to their host.
DNA replication determines the plasmid copy number and this is rigorously controlled
and closely coordinated with thecell cycle. The process of DNA replication is initiated at
distinct sites known asorigins of replication(ori) and proceeds in both directions along the
DNA. In simple organisms, such asE. coli, there is only one origin (oriC); however, more
complex organisms, with larger genomes, require many origins to ensure complete DNA
synthesis prior to cell division. Origins are usually defined by a segment of DNA, compris-
ing several hundred base pairs, which binds DNA polymerase and other proteins required to
initiate DNA synthesis. The plasmid DNA must also replicate in its host organism to ensure
that each daughter cell receives a copy of the plasmid. The regulation of this replication
determines the number of plasmid copies contained within each cell. Control of plasmid

TABLE 7.1. Restriction Endonucleases

Enzyme Source

Recognition

sequence Cut Ends

EcoRI Escherichia coliRY13 GAATTC
CTTAAC

G

CTTAA

AATTC

G

50 overhangs

BamHI Bacillus amyloliquefaciensH GGATCC
CCTAGG

G

CCTAG

GATCC

G

50 overhangs

HindIII Haemophilus inflenzaeRd AAGCTT
TTCGAA

A

TTCGA

AGCTT

A

50 overhangs

KpnI Klebsiella pneumoniae GGTACC
CCATGG

GGTAC

C

C

CATGG

30 overhangs

NotI Nocardia otitidis GCGGCCGC
CGCCGGCG

GC

GGCCGC

CGCCGG

CG

50 overhangs

PstI Providencia stuartii CTGCAG
GACGTC

CTGCA

G

G

ACGTC

30 overhangs

SmaI Serratia marcescens CCCGGG
GGGCCC

CCC

GGG

GGG

CCC

Blunt ends

SacI Streptomyces achromogenes GAGCTC
CTCGAG

GAGCT

C

C

TCGAG

30 overhangs

SstI Streptomyces stanford GAGCTC
CTCGAG

GAGCT

C

C

TCGAG

30 overhangs

TaqI Thermophilus aquaticus TCGA
AGCT

T

AGC

CGA

T

50 overhangs

XbaI Xanthomonas campestris pv. badrii TCTAGA
AGATCT

T

AGATC

CTAGA

T

50 overhangs

7.2. DNA VECTORS 163