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
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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