Flashcards in Recombinant DNA Deck (11):
the isolation and manipulation of DNA sequences, usually involving combining nonconsecutive sequences
what are the 2 types of enzymes that make DNA recombination possible
restriction endonuclease- cuts specific palandromic sequences, usually leaving sticky ends. usually palandrome sequences are 4/6/8 sequences long. they are named after the bacteria they are derived from
DNA ligase- reseals nicks in the backbone after insertion of DNA sequence
What are the key characteristics of vectors?
1. separable from chromosomal DNA
2. restriction enzyme sites
3. OR and autonomous replication
4. able to tolerate sequence insertion
5. ability to be reinserted back into bacteria
why are plasmids good vectors?
1. easily separable b/c of size
2. autonomous replication
3. tolerate insertion of ~10kb
4. can be engineered w/ restriction enzyme sites
5. encode selection factors
what are alternatives to plasmids for vectors?
cosmids and bacterial artificial chromosomes- can encode bigger sequences but there are fewer per cell and they replicate much more slowly
what is a genomic clone?
represents any part of the genome (including non coding material). therefore, can be taken from any cell. usually need cosmid or BAC b/c of larger protein sizes
what is a library?
a genomic clone of the entire genome
what is a cDNA clone and how is it different than genomic DNA
it represents a clone of only mRNA (has no introns). It matters where you get it from b/c tissues selectively express proteins. can be done with plasmids b/c they are generally smaller. you make a poly T tail as a primer, then RNase to degrade the original template
describe how to run a southern blot
use restriction endonucleases to cut DNA. denature it and run it through a gel to separate by size. use capillary action to transfer the DNA onto a nitrocellulose sheet and soak it in solution with radioactive probe. visualize probe with x-rays- signal is autoradiography
what is autoradiography
the signal at the end of a southern blot