Gene manipulation and recombinant DNA technology - Pharmaceutical Biotechnology: Concepts and Aapplications - page 51

DNA recognition sequence and cleavage site a Restriction enzyme Source

5 ' -T↓GATCA-3 '
3 ' -ACTAG↑T-5 '

BclI Bacillus caldolyticus

5 ' -A↓GATCT-3 '
3 ' -T C TAG↑A-5 '

BglII Recombinant E. coli carrying BglII gene

from Bacillus globigii

5 ' -PyAC↓GTPu-3 '
3 ' -PuTG↑CAPy-5 '

BsaAI Recombinant E. coli carrying BsaAI gene

from Bacillus stearothermophilus A

5 ' -C↓CNNGG-3 '
3 ' -GGNNC↑C-5 '

BsaJI B. stearothermophilus J

5 ' -CGPuPy↓CG-3 '
3 ' -GC↑PyPuGC-5 '

BsiEI B. stearothermophilus

5 ' -GAT↓ATC-3 '
3 ' -CTA↑TAG-5 '

EcoRV Recombinant E. coli carrying EcoRV

gene from the plasmid J62 pIg 74

5 ' -GCNNNNN NNGC-3 '

MwoI Recombinant E. coli carrying cloned MwoI

gene from Methanobacterium wolfeii

↓

3 ' -CGNN NNNNNCG-5 ' ↑

Tsp509I Thermus sp. 5 ' - AATT-3 '

3 ' -T TA A

↓

↑ -5 '

XbaI Recombinant E. coli carrying XbaI gene

from Xanthomonas badvii

5 ' -T CTAGA-3 '

3 ' -AGATC

↓

↑ T-5 '

XhoI Recombinant E. coli carrying XhoI gene

from X. holcicola

5 ' -C TCGAG-3 '

3 ' -GAGCT

↓

↑ C-5 '

a G: guanine; C: cytosine; A: adenine; T: thymine; Pu: any purine; Py: any pyrimidine; N: either a purine or pyrimidine.
Arrow indicates site of cleavage.

lacZ

Polylinker
region

amp R

pUC 18

lacI

ori

Figure 3.13 The plasmid pUC18 is often used for cloning purposes. It contains three genes: the ampicillin
resistance gene ( amp R ), the lacZ gene, which codes for the enzyme β -galactosidase, and the lacI gene, which
codes for a factor that controls the transcription of lacZ . Also present is an origin of replication (ori), essential
for plasmid replication within the cell. Note the presence of a short stretch of DNA called the polylinker region
located within the lacZ gene. The polylinker (also called a multiple cloning site) contains cleavage sites for
13 different REs. This allows genetic engineers great fl exibility to insert a DNA fragment for cloning into this
area. The polylinker has been designed and positioned within the lacZ gene so as not to prevent the expression
of functional β -galactosidase. However, if a piece of DNA for cloning is introduced into the polylinker region,
then the increased length does block β -galactosidase expression. The full sequence of the 2.69 kb plasmid
is known and sequence analysis confi rms the presence of multiple additional RE sites outside the polylinker
region. There are at least six target sites for commonly used restriction enzymes within the amp R gene