Week 8 Flashcards
DNA Cloning
when are recombinant proteins formed
when DNA from one species is transplanted into another species
where are restriction enzymes present
in prokaryotes
what is the purpose of restriction enzymes in their natural hosts
to recognize foreign DNA sequences and cut it into pieces for further degradation by host exonucleases
what was the first therapeutic product to be synthesized using recombinant DNA
insulin
what type of DNA sequences do restriction enzymes recognize
inverted palindromic sequences
what are the type of cleavage ends
- blunt ends
- sticky 5’ ends
- sticky 3’ ends
in which direction is the recognition site read
5’ to 3’
define: electrophoresis
the movement of charged molecules in an electric field
how does gel electrophoresis distinguish DNA fragments
size
what do restriction maps show
the order of different size fragments within a clone
define: vector
specialized chromosome-like carrier used for cloning
define: DNA clone
vectors with inserts that are replicated in cells
what is a common type of vector
plasmids
what specialized regions do vectors contain
- origin of replication
- multiple cloning site
- selectable marker
define: multiple cloning site
region with several unique restriction enzyme recognition sites
how large are plasmids
2-4 kb
how large can inserts in plasmids be
up to 15 kb
what connects vector and DNA fragment
ligase
what are the products of ligation
- linear vector
- linear inserts
- circular vector
- circular insert
- vector plus insert
- vector plus multiple inserts
how do you determine which plasmids contain inserts for E. coli
put them in a media that has ampicillin and X-gal
what des ampicillin resistance indicate for cloning
there is a plasmid
what do white colonies in a media with X-gal indicate
recombinant plasmid, they disrupt transcription of lacZ
plasmids with no insert are blue
what does YAC stand for
yeast artificial chromosomes; linear vectors built to mimic a yeast chromosome
how large of inserts can YACs carry
up to 1 million base pairs
how do you retrieve the clone from the plasmid
digest with restriction enzyme and separate by gel electrophoresis
define: prions
proteinaceous infectious particles
what is mad cow disease and scrapie caused by
prions
define: genomic library
a collection of DNA clones that theoretically contains copies of every DNA fragment in the whole genome
define: complimentary DNA library
DNA sequences that have been copied from mRNA transcripts; only contain the exon sequences
what converts mRNA to cDNA
reverse transcriptase
what are the differences between genomic libraries and cDNA libraries
genomic:
- contain all sequences
- same info for every cell type of that organism
- used for prokaryotes or sequencing projects
cDNA:
- only contains exon sequences
- conditional upon cell type
- used for eukaryotes
