Restriction mapping
Site-specific restriction endonucleases are used to identify DNA molecules
What are restriction endonucleases (REs)?
How can REs be used to identify DNA molecules?
How can I find RE recognition sites in the MET plasmids?
Restriction endonucleases are part of a bacterium’s defense against invaders
Restriction-modification systems allow the
bacterium to distinguish self from non-self DNA
Restriction: bacterial endonucleases
cleave both strands of foreign DNA at
specific recognition sites
Modification: bacteria protect their
own DNA by adding a methyl group to
the recognition sites in their own DNA
electron micrograph by Graham Colm
of bacteriophage infecting a bacterium
Type II restriction enzymes are widely used in molecular biology:
enzymes cleave, but do not modify, their specific recognition sites
REs with 6-nucleotide recognition sites (6-cutters) are widely
used in molecular biology
Sites would randomly be
expected every 1/4096
nucleotides (1/46)
Actual sizes vary widely with
average of ~4000 bp
Recognition sites are often palindromes
Crystal structure 2CKQ
EcoRI bound to DNA
RE
Strain of origin
Recognition site
EcoRI
E. coli (strain RY13)
GAATTC
Hind III
H. influenza
AAGCTT
BamHI
B. amyloliquefaciens
GGATCC
5’
3’
GAATTC
CTTAAG
3’
5’
EcoRI recognition site is a
palindrome with an axis of
symmetry
5’
3’
GAATTC
CTTAAG
3’
5’
EcoRI dimer binds sequence
and catalyzes doublestrand cleavage
5’
3’
G
CTTAA
AATTC
G
Products have “sticky
3’ ends”: unpaired
5’ hydrogen bonds on
nitrogen bases
The sticky ends generated by REs are useful in generating
recombinant DNA molecules (more later........)
REs are the scissors—ligases are the paste
5’
3’
AATTC
G
G
CTTAA
3’
5’
Sticky ends from two
molecules form hydrogen
bonds
DNA ligase
5’
3’
GAATTC
CTTAAG
3’
5’
Recombinant
molecule
What are restriction endonucleases (REs)?
How can REs be used to identify DNA molecules?
How can I find RE recognition sites in the MET plasmids?
Preparing a restriction map
pBG1805-MSRA is digested with:
Acc I
MSRA (555 bp)
inserted here
BsaA I
Hinc II
Restriction fragments are
separated on 1% agarose gels
pBG1805 (6573 bp)
Each restriction enzyme produces a distinct set of fragments
21,228
5148, 4973
4268
3530
2027
1904
1584
1375
947
831
564
Hinc II
BsaA I
Acc I
Size (bp)
RE Digests
Uncut
Standards
EcoRI and HindIII
digest of lambda DNA
Markers
RE digests of pBG1805 containing YER042W ORF
Your task:
Design a strategy to distinguish your three plasmids with restriction
endonucleases
S. cerevisiae
ORF
pBG1805 (6573 bp)
S. pombe
ORF or LacZ
pYES2.1 (5886 bp)
What are restriction endonucleases (REs)?
How can REs be used to identify DNA molecules?
How can I find RE recognition sites in the MET plasmids?
Program for finding restriction sites in DNA sequences
Overview
Find the vector (plasmid) sequence
Access the pBG1805 sequence in NCBI’s Nucleotide database
The pYES2.1 sequence is available on Blackboard*
Paste the sequence into NEB cutter and give the file a name
Find the MET gene sequence in SGD (yeastgenome.org)
Paste the MET coding sequence at the end of the vector sequence
Indicate that the sequence is circular and click submit
Use NEB cutter to find restriction sites for four restriction
endonucleases:
AccI
HincII
ScaI
XbaI
*Control pYES2.1-LacZ sequence can be pasted directly into NEB cutter