Restriction Enzyme Digestion
Introduction
For molecular biologists, the 1970’s, were marked by discovery of site-specific bacterial DNases used as defence against foreign (usually viral) DNA. The DNases, referred to as restriction enzymes, hydrolyze ATP in a reaction that breaks the phosphodiester bonds between nucleotides in a strand of DNA. Endonucleases cleave both strands of dsDNA strands within the sequence, while exonucleases cut at either the 3’ or the 5’ ends of dsDNA. Isoschizomers are restriction enzymes from different organisms that recognize the same sequence and cut in the same location, and neoschisomers are isoschizomers that cut within different regions of the same recognition sequence.
Sequence Recognition and Digestion
The majority of restriction enzymes recognize unique 4-8 bp DNA sequences, many of which are palindromic (read the same backwards and forwards). Within a given DNA sequence, the frequency of restriction site occurrence (and therefore cuts) for a particular restriction enzyme is predicted by the exponential formula below which assumes 4 nucleotides occur in equal proportions within DNA sequence:
Predicted bases between cuts for a RE = 4n (where n = # of bases in recognition sequence)
Restriction enzymes can cleave directly through both dsDNA strands to produce “blunt ends”. Alternatively, digestion may produce complementary 5’ or 3’ overhangs referred to as “sticky ends”.
Conditions for Restriction Digestion
The optimal ionic strength for enzyme activity typically ranges from pH 7.4-8.0. Ionic strength is established by salts (ex. NaCl) and divalent cations, usually Mg2+. Sub-optimal ionic strength compromises both enzymatic activity and sequence specificity which may result in non-specific aberrant cutting is referred to as “star activity”. Optimal temperature for enzyme activity is usually 37°C, but enzymes isolated from thermophilic bacteria function optimally at 50-65°C while others with short half life at 37°C are incubated at 25°C. Restriction enzyme inactivation is accomplished by altering optimal conditions. Incubation at 70°C heat-inactivates most restriction enzymes. Chelating agents (ex. EDTA) can chelate di-valent cations (ex. Mg2+) required as co-factors for enzymatic activity to render the restriction enzyme functionally inactive. The optimal conditions required by specific restriction enzymes are listed in the following link:
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp
Note: Refer to composition of “NE buffers” for details regarding ionic strength
Distinguishing Endogenous Genome from Foreign DNA
Bacterial DNA methyl transferases methylate the bacterial genome at or near restriction sites. This methylation distinguishes the endogenous bacterial genome from unmethylated foreign (viral) DNA which is left susceptible to restriction digestion.
Designing a Restriction Digest Reaction
1) Determine Composition of Restriction Digest Mix
a. Amount of DNA to be digested (minimum 1 microgram)
b. Ionic conditions (see above)
c. Amount of restriction enzyme to add
d. Incubation temperature and time
Refer to aforementioned section on reaction conditions, and consult manufacturer online catalogue for specific conditions
2) Verify Successful Restriction Digest
a. Resolve an aliquot of digested DNA via agarose gel electrophoresis
b. Compare observed DNA fragment lengths to expected lengths from restriction map
For molecular biologists, the 1970’s, were marked by discovery of site-specific bacterial DNases used as defence against foreign (usually viral) DNA. The DNases, referred to as restriction enzymes, hydrolyze ATP in a reaction that breaks the phosphodiester bonds between nucleotides in a strand of DNA. Endonucleases cleave both strands of dsDNA strands within the sequence, while exonucleases cut at either the 3’ or the 5’ ends of dsDNA. Isoschizomers are restriction enzymes from different organisms that recognize the same sequence and cut in the same location, and neoschisomers are isoschizomers that cut within different regions of the same recognition sequence.
Sequence Recognition and Digestion
The majority of restriction enzymes recognize unique 4-8 bp DNA sequences, many of which are palindromic (read the same backwards and forwards). Within a given DNA sequence, the frequency of restriction site occurrence (and therefore cuts) for a particular restriction enzyme is predicted by the exponential formula below which assumes 4 nucleotides occur in equal proportions within DNA sequence:
Predicted bases between cuts for a RE = 4n (where n = # of bases in recognition sequence)
Restriction enzymes can cleave directly through both dsDNA strands to produce “blunt ends”. Alternatively, digestion may produce complementary 5’ or 3’ overhangs referred to as “sticky ends”.
Conditions for Restriction Digestion
The optimal ionic strength for enzyme activity typically ranges from pH 7.4-8.0. Ionic strength is established by salts (ex. NaCl) and divalent cations, usually Mg2+. Sub-optimal ionic strength compromises both enzymatic activity and sequence specificity which may result in non-specific aberrant cutting is referred to as “star activity”. Optimal temperature for enzyme activity is usually 37°C, but enzymes isolated from thermophilic bacteria function optimally at 50-65°C while others with short half life at 37°C are incubated at 25°C. Restriction enzyme inactivation is accomplished by altering optimal conditions. Incubation at 70°C heat-inactivates most restriction enzymes. Chelating agents (ex. EDTA) can chelate di-valent cations (ex. Mg2+) required as co-factors for enzymatic activity to render the restriction enzyme functionally inactive. The optimal conditions required by specific restriction enzymes are listed in the following link:
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp
Note: Refer to composition of “NE buffers” for details regarding ionic strength
Distinguishing Endogenous Genome from Foreign DNA
Bacterial DNA methyl transferases methylate the bacterial genome at or near restriction sites. This methylation distinguishes the endogenous bacterial genome from unmethylated foreign (viral) DNA which is left susceptible to restriction digestion.
Designing a Restriction Digest Reaction
1) Determine Composition of Restriction Digest Mix
a. Amount of DNA to be digested (minimum 1 microgram)
b. Ionic conditions (see above)
c. Amount of restriction enzyme to add
d. Incubation temperature and time
Refer to aforementioned section on reaction conditions, and consult manufacturer online catalogue for specific conditions
2) Verify Successful Restriction Digest
a. Resolve an aliquot of digested DNA via agarose gel electrophoresis
b. Compare observed DNA fragment lengths to expected lengths from restriction map