Manipulating DNA. Recombinant DNA: a combination of DNA in ways that “nature never intended” Usually, DNA from different organisms combined Terminology Clones: organisms, cells, or molecules that are identical copies (in this case, usually DNA) Tools: restriction enzymes, vectors
How do you know which of the few bacteria actually took up the plasmid?
How can you know your gene of interest is actually in the plasmid?
Selection: bacteria that don’t have the plasmid are unwanted (and numerous). Ampicillin in culture medium kills those.
Screening: Not all plasmids contain inserted DNA. Beta-galactosidase (lac Z) turns “X-gal” blue; insert in lac Z gene = white colonies.
YAC starts circular.
Contains a centromere, an origin to allow its replication, and two BamH1 sites that open up the circle leaving telomeres at the end.
DNA can be cloned into a restriction site (e.g. EcoR1).
DNA synthesis, whether PCR or DNA replication in a cell, is carried out by DNA polymerase.
DNA polymerases have three requirements:
Only work in a 5’ to 3’ direction
Require a template: DNA to copy.
Require a primer: a free 3’ OH end to add to.
PCR uses Taq polymerase, a heat stable polymerase from the thermophilic bacterium Thermus aquaticus.
Poly-T synthetic oligonucleotide acts as primer, mRNA is the template.
DNA-RNA hybrid is denatured, RNA is digested away, and DNA polymerase makes a ds DNA of the ss DNA.
This piece of DNA, representing an active gene, can now be cloned.
ddNTPs (Dideoxy NTPs) are missing an -OH group at the 3’ position. Remember that this is where the next nucleotide must attach during DNA synthesis.
This picture shows a few of the different sized molecules that can result.
Notice that each ends with a particular ddNTP; in modern practice, this will have a color associated with it and thus be identified.
derived from opbs.okstate.edu/.../ img015.jpg
Inexpensive, older method:
Four reaction tubes, each containing a different ddNTP:
ddATP, ddCTP, etc.
Contents of each tube run in a different lane, stained to see the bands.Gel read from bottom up.
ddCTP = blue
ddATP = green
ddTTP = red
ddGTP = grey