Restriction enzymes Flashcards
What is the difference between endonucleases and restriction endonucleases?
Endonucleases cut randomly rather than at specific points.
What is the natural function of restriction endonucleases?
They occur naturally in bacteria to protect the bacteria from bacteriophage.
Why doesn’t the bacterial genome get cleaved by the restriction enzymes?
Bacterial DNA is methylated at the restriction sites and is therefore protected.
What are Type II restriction endonucleases?
They recognise a specific DNA sequence that is often 4 or 6 nucleotides in length. They cut precisely at or near this site.
What does palindromic mean and what relevance does this have to type II restriction endonucleases?
It means the sequence will be read the same in each direction. Type II restriction endonuclease restriction sites are often palindromic.
What is the recognition site of EcoRI and does it create blunt or sticky ends?
GAATTC, sticky.
What is the recognition site of BamHI and does it cut blunt or sticky?
GGATCC, sticky.
What is the recognition site of AluI and does it cut blunt or sticky?
AGCT, blunt.
What is the restriction site of Sau3A and does it cut blunt or sticky?
GATC, sticky.
What is the benefit of sticky ends?
The ends can base pair together via hydrogen bonds which is helpful if you want to rejoin ends.
What is the benefit of the same sticky ends being produced by different restriction enzymes?
Different fragments can be joined together even if they have been cut by different restriction enzymes.
What is the frequency of restriction sites in a tetranucleotide?
Once every 245 nucleotides.
What is the frequence of a hexanucleotide restriction site?
Once every 4096 nucleotides.
What is the process for using restriction enzymes?
Reaction is carried out in a microfuge tube using sterile components. It requires an appropriate buffer for the enzyme and is usually incubated at 37 degrees for an hour for the digestion to complete.
How can the restriction enzyme be inactivated?
EDTA, heat or phenol.
How can agarose gel electrophoresis be used together and provide use?
Used for analysing, mapping and purifying DNA molecules.
What is agarose?
A highly pure form of agar, extracted from seaweed.
How can an agarose gel be prepared?
Various plastic shit, buffer, agarose, ethidium bromide, pour into a casting tray and wait for it to set.
How can you prepare DNA for electrophoresis?
Mix the digested DNA with a loading buffer that contains a dye so you can see what you’re doing. The buffer will also contain sucrose/glycerol to make the sample heavier than the elctrophoresis buffer. The sample will sink to the bottom of the well.
Which direction does DNA move in electrophoresis?
From the cathode towards the anode.
Do smaller or larger fragments move further throughout the electrophorese?
Smaller fragments move further.
How can the DNA fragments be seen after the electrophorese?
Stain with ethidium bromide during or after the electrophorese and place the gel on a UV transilluminator.
How can the size of the unknown fragments be determined?
Using size markers, usually a HindIII digest of lambda bacteriophage DNA run next to the unknown sample.
What is restriction mapping?
Working out the relative positions of the restriction sites for particular enzymes.
