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

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 sites

Modification: 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

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

RE Strain of originRecognition site

EcoRI E. coli (strain RY13) G A A T T C

Hind III H. influenzaA A G C T T

BamHI B. amyloliquefaciens G G A T C C

Recognition sites are often palindromes

Crystal structure 2CKQEcoRI bound to DNA

GAATTCCTTAAG

3’

5’

5’

3’

GAATTCCTTAAG

3’

5’

5’

3’

G AATTCCTTAA 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

The 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’

GAATTCCTTAAG

3’

5’

5’

3’

DNA ligase

Sticky ends from two molecules form hydrogen bonds

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

BsaA I

Hinc II

MSRA (555 bp) inserted here

pBG1805 (6573 bp)

Restriction fragments are separated on 1% agarose gels

Mar

kers

Unc

ut

Acc

I

BsaA

I

Hin

c II

RE Digests

21,228

Size (bp)

5148, 497342683530

2027190415841375

947831

564

StandardsEcoRI and HindIII digest of lambda DNA

RE digests of pBG1805 containing YER042W ORF

Each restriction enzyme produces a distinct set of fragments

pBG1805 (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

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) sequenceAccess the pBG1805 sequence in NCBI’s Nucleotide databaseThe 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

sequenceIndicate that the sequence is circular and click submit

Use NEB cutter to find restriction sites for four restriction endonucleases:

AccIHincIIScaIXbaI

*Control pYES2.1-LacZ sequence can be pasted directly into NEB cutter