Restriction Enzymes
What do restriction enzymes do?
Recognise specific sequences within a DNA molecule (often 4-6 base pairs long) and cut them in a specific manner.
How is the problem of inter/intramolecular association with T4 DNA ligase avoided?
Treat the cloning vector with alkaline phosphatase which will remove the 5' terminal phosphates from the plasmid - thereby preventing self-ligation. The DNA of interest can still be ligated in as it has 5' phosphates and the nicks will be repaired when the construct is reintroduced into E. coli.
What is agarose gel electrophoresis?
When agarose (a highly pure form of agar) is mixed with a buffer, heated and cooled, it forms a transparent gel matric that can be used to separate DNA molecules with electrophoresis.
What is the method for agarose gel electrophoresis?
- Agarose and buffer are heated until the agarose dissolves. - Cool to approximately 50oC, add ethidium bromide. - Pour onto glass plate gel former and add a comb to produce the wells. - Remove the comb, place the gel in the electrophoresis tank and cover it with electrophoresis buffer. - The digested DNA is mixed with loading buffer which contains a dye so the DNA can be seen. It also contains sucrose/glycerol to make the sample heavier than the buffer to make the sample sink to the bottom of the well. - DNA moves from the cathode towards the anode. The gel matrix separates the DNA according to their size (small fragments move further). - The agarose gel matrix acts as a filter to slow down the larger fragments. This makes you end up with a size fractionation of the DNA fragments.
What is the method for restriction mapping?
- Digest the DNA with each enzyme on its own. - Perform double digests in which all combinations of enzymes are used in pairs. - Separate the single and double digests on an agarose gel together with appropriate molecular weight markers. - Accurately size all of the fragments so then you can work out the relative positions on the DNA molecule.
What are some examples of restriction endonucleases?
- EcoRI from E. coli (GAATTC, sticky) - BamHI from Bacillus amyloliquefaciens (GGATCC, sticky) - AluI from Arthrobacter luteus (AGCT, blunt) - Sau3A from Staphylococcus aureus (GATC, sticky)
What do type II restriction endonucleases do?
- Recognise a specific DNA sequence (often 4 to 6 nucleotides in length) - Cut precisely or near this site - Sequence often reads the same in both directions (palindromic)
How can DNA fragments be visualized after agarose gel electrophoresis?
DNA fragments need to be stained with EtBr either during or after electrophoresis (EtBr has the property of making the DNA fluoresce when exposed to UV light). You can detect more than 10 ng of DNA.
What is the function of DNA ligase?
DNA ligase will join DNA molecules together - any blunt ends can be joined (however this is tricky as there is no base pairing to rely on) and compatible sticky ends are joined easily as there is base pairing.
What is the problem with inter and intra molecular association with T4 DNA ligase?
During a ligation reaction, there is no control as to which molecules will join together - as long as they have compatible sticky ends, they can equally join to themselves as they can to others. This means that only a few ligation products are the desired recombinant molecules.
How can the size of DNA fragments be measured after electrophoresis?
Fragments of DNA with known molecular weight are run on the gel in order to determine the size of unknown fragments (size markers - often HindIII digest of lambda bacteriophage DNA). Sizing can either be done roughly by visually comparing relative positions on the gel, more accurately by plotting a graph of the known marker sizes against the distance travelled from the wells.
What is restriction mapping used for?
Restriction mapping can be useful for cloning particular genes or for comparing molecules from different sources.
What is interesting about BamHI, BglIII and Sau3a?
They recognise different sites but produce the same sticky ends. This allows DNA fragments produced by these different enzymes to be joined together again via base-pairing.
How can the average frequency of occurrence of a recognition site be calculated?
A tetranucleotide (a recognition site of 4 base pairs) is found once every 4^4 times. A hexanucleotide is found once every 4^6 times. DNA isn't always random so these figures aren't exact.
What enzyme catalyses the insertion of a gene into a cloning vector?
T4 DNA ligase (originating from the bacteriophage T4 where its function is to repair DNA). This reaction requires ATP.
What is the natural function of a restriction enzyme?
Their natural function is to destroy foreign DNA entering the cell by cleaving the bacteriophage DNA to prevent infection. The cell's own DNA is modified by methylation to protect it from its own enzyme. Each restriction enzyme has a specific methylase.
What are sticky ends?
When restriction enzymes cut in a staggered fashion across its recognition site. They're staggered because the ends can base pair together via hydrogen bonds, which is helpful if you want to rejoin the ends.
What are blunt ends?
When restriction enzymes cut straight across the double stranded DNA. Blunt ends can be re-joined regardless of the enzyme that produced them, though with reduced efficiency compared to sticky ends as they don't have hydrogen bonding.
What is restriction mapping?
When you can work out the relative proportions of the restriction sites for particular enzymes in the larger molecule from which the fragments are derived (from an agarose gel)
How are restriction enzymes used in the lab?
• The reaction is carried out in a microfuge tube in a small volume (usually 20 µl) using sterile components. • Requires an appropriate buffer for the enzyme (optimum). • Typically a few µg of DNA is digested. • Usually incubated at 37oC for 1 hour for digestion to go to completion. When the digestion is complete, the reaction is stopped and the resultant DNA fragments are ready for analysis. • Enzyme can be inactivated by EDTA, head or phenol.