a. Restriction enzymes type 2 and 4 and type 2 reach over. These looks for a site and cut at the site. They can
generate 3’ or 5’ overhangs. The type 2 reach over, will bind to a sequence and reach over 20 bps before cutting. Be careful
for start activity with the wrong buffer.
b. Uracil-DNA Glycosylase – cuts out uracil from a strand.
c. Homing endonuclease – One can think of these like restriction enzymes with a very, very long recognition sequence,
although there is some degeneracy allowed. These are naturally there to induce recombination. There are “reach over and cut”
homing endonuclease. We are typically interested in inducing recombination in order to take a sick gene i.e. sickle cell anemia
and taking a good normal gene and recombining them in vivo to cure diseases.
a. Dna ligase – needs a 3’ hydroxyl and 5’ phosphate. Requires energy. Note pcr products will not have a 5’ phosphate,
you need to digest it or have something to do stuff to it. Works the worse on single base pair overhangs (a/t).
b. Rna ligase – acts on dna and rna, but no double stranded. Also it needs the 5’ phosphate and 3’ OH.
c. Vaccinia Topoisomerase – will take an “a” and “t” one bp end and ligate them together. I’m not sure how the
orientation is conserved.
d. Alkaline Phosphotase- will remove phosphate off of anything. Used to prevent the reclosure of ends.
a. Recombinase. These will recognize 2 binding sites which must have homology between them. The recombinase will
invert the sequence between the two recognition sites.
b. Reach over Recombinase Doesn't exist, but I wish it did.
IV. Block/ Protect
a. CpG Methylation – eukaryotic methylation.
b. Dam and Dcm methylation – the capitalized ones are methylated. dam gAtc; dcm cCwgg
c. DPN1- kills methylated DNA that’s Dam/Dcm methylated.
a. Blunting – t4 dna polymerase will cut off 3’ overhangs and fill in 5’ overhangs. For the 3’ excision, there needs
to be almost no dntps (2um) to drive the reaction to excision. There needs to be a trace amount of dntps to keep the polymerase
from cutting past the 3’ end.
b. Mung bean nuclease- Will blunt all overhangs through excision.
a. taq polymerase – will add an A or T at the end. Works in a good combo with Vaccinia Topoisomerase.
b. terminal transferase – randomly adds bases to the end of the chain. Usually used for labeling.
a. dna polymerase – will repair nicks.
b. strand displacement – certain dna polymerases will strand displace some will not. Strand displacement is removing
the existing strand and continue to add bps.
c. mutS – cuts out mismatches for better fidelity of dna. The parent strand is methylated.
a. Tat – there are tons of tat pathways but in general Tat will fold a protein in the cytosol which is a reducing
b. Sec – there are also a ton of sec pathways but in general sec will fold in the periplasm which is oxidative.
a. heat sensitive origins/ varying origins- Used in datsenko/wanner to remove a unneeded strand. Varying heat or
machinery to read different origins can be used as a control system for replication.
Yet to Organize
a. Gateway – transfer between gateway sites.
b. Phage – a phage will take a tag sequence from a parent then go through recombination with a target and input that
gene. This isn’t always so clean though. An alternative may be transposons http://www.ncbi.nlm.nih.gov/pubmed/17164785