selection methods used to screen for host cells that have taken up the recombinant DNA molecules?

3 Comments

1. archaeadoc says:

   November 6, 2009 at 6:23 pm

   Your first two answers here as of this writing are correct. I would just add a little detail…

   Usually if you use e. coli, then you select with antibiotic resistance markers. When you use mammalian cells you also use antibiotic resistance. For yeast it is more common to use nutritional markers like amino acids.

   Also, a words about selection and screening. With selection using drugs or nutritional markers there is a selective pressure, and as the other guy said, only the ones that have taken up the plasmid or other DNA construct will be able to grow. After that though you still need to screen those cells that survive the selection process to verify that they have exactly the kind of molecule you want. For example, it is very possible that only a portion of a plasmid is taken up, and that portion of the plasmid is the part where the selectable marker is. That is why you screen E. coli plasmid preps by restriction analysis, to make sure they look exactly as they should.

   Depending on the purpose of the cloning I often then screen again by sequence analysis across the cloning junction to make sure that everything is as it should be to the last base pair. A loss of a single nucleotide can change the frame of the coding region.
2. Peter S says:

   November 6, 2009 at 6:23 pm

   Use recombinant plasmids containing a gene for antibiotic resistance. Only those bacteria which have taken up the plasmid will be able to grow in the presence of the antibiotic.
3. jcrew says:

   November 6, 2009 at 6:23 pm

   Put it in a solution that would normally kill the host cell but would not if the host cell has taken up recombinant DNA. Then see if it’s dead or not.