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 bacteriums defense against invaders ID: 917162
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Slide1
Restriction mapping
Site-specific restriction endonucleases are used to identify DNA molecules
Slide2What are restriction endonucleases (REs)?
How can REs be used to identify DNA molecules?
How can I find RE recognition sites in the
MET
plasmids?
Slide3Restriction endonucleases are part of a bacterium’s defense against invaders
electron micrograph by Graham
Colm
of bacteriophage infecting a bacterium
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 sitesModification: bacteria protect their own DNA by adding a methyl group to the recognition sites in their own DNA
Type II restriction enzymes are widely used in molecular biology:
enzymes cleave, but do not modify, their specific recognition sites
Slide4REs with 6-nucleotide recognition sites
(6-cutters) are
widely used in molecular
biology
S
ites would randomly be expected every 1/4096 nucleotides (1/4
6
)
Actual sizes vary widely with average of ~4000
bp
RE
Strain of origin
Recognition site
EcoR
I E. coli (strain RY13) G A A T T CHind III H. influenza A A G C T TBamHI B. amyloliquefaciens G G A T C C
Recognition sites are often palindromes
Crystal structure 2CKQ
EcoRI
bound to DNA
Slide5GAATTC
CTTAAG
3’
5’
5
’
3
’
GAATTC
CTTAAG
3’
5’
5
’
3
’
G AATTC
CTTAA G
3’
5’
5
’
3
’
EcoRI
recognition site is a palindrome with an axis of symmetry
EcoRI
dimer binds sequence and catalyzes double-strand cleavage
Products have “sticky ends”: unpaired hydrogen bonds on nitrogen bases
Slide6The sticky ends generated by REs are useful in generating recombinant DNA molecules (more later........)
REs are the scissors—ligases are the paste
G
CTTAA
5
’
3
’
AATTC
G
3’
5’
GAATTC
CTTAAG
3’
5’
5
’
3
’
DNA ligase
Sticky ends from two molecules form hydrogen bonds
Recombinant molecule
Slide7What are restriction endonucleases (REs)?
How can REs be used to identify DNA molecules?
How can I find RE recognition sites in the
MET
plasmids?
Slide8Preparing a restriction map
pBG1805-
MSRA
is digested with:
Acc
I
BsaA
I
Hinc
II
MSRA (555
bp
) inserted here
pBG1805 (6573
bp
)Restriction fragments are separated on 1% agarose gels
Slide9Markers
Uncut
Acc
I
BsaA
I
Hinc II
RE Digests
21,228
Size (bp)
5148, 4973
4268
3530
2027
1904
1584
1375
947
831
564
Standards
EcoRI and HindIII digest of lambda DNA
RE digests of pBG1805 containing YER042W ORF
Each restriction enzyme produces a distinct set of fragments
Slide10pBG1805 (6573
bp
)
S. cerevisiae
ORF
pYES2.1 (5886
bp
)
S. pombe ORF or
LacZ
Your task:
Design a strategy to distinguish your three plasmids with restriction endonucleases
Slide11What are restriction endonucleases (REs)?
How can REs be used to identify DNA molecules?
How can I find RE recognition sites in the
MET
plasmids?
Slide12Program for finding restriction sites in DNA sequences
Slide13Overview
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 submitUse 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