DNA technology Flashcards
colne made possible by the discovery of ?
restraction enzymes
plasmid DNA vectors
DNA cutting enzymes
restraction enzyme
circular form of self-replicating DNA
plasmid DNA vectors
restraction enzyme is primalry found in ?
bacteria
what is the function of rastraction enzyme ?
cut DNA by cleaving the phosphodiester bond that joins adjacent nucleotide
bind to ;recognize and cut DNA within specific sequences of bases in restraction site
why there are 4 or 6 cutters ?
becuse they recognize restraction site with a sequences of 4 or 6 nucleotides
how the fragments with cohesive ends are produced?
by digastion of DNA by EcoRI
what blocks the DNA clevage ?
methylation of the EcoRI restraction
restraction of single strand ends ///
double strands//
sticky-cohasive
blunt ends
which end is prefered for cloing DNA ?
cohasive end because it easily joind togather
by forming weak H bound
what is the vector in cloing DNA ?
plasmid DNA
RECOMBINANT
transformation
by adding forgin dna
electroportion making tiny holes that allow the DNA to enter
selection
1 antibiotics
2 blue-white selection
if funcional lacZ is present
blue colony
if funcional lacZ is not ?
white colony
library screening with DNA probe to identify a cloned gene interest
colony hybridaztion
library screening rarely result in
the coloing of the full length
hybridization filter
nylon then it washed
filter is incubeted with probe
advantage of cDNA libraries ?
mRNA is isolated
introns are not cloned
making double strnad of DNA
1 complementary single strand DNA is is catalyzed by reverse transcriptase
secnod one is synthesized by DNA pol
mRNA is degraded //?
either with an enzyme or alklaine sloution
disadvantage of genomic libraries
introns are cloned in addtion to exons
time consuming
searching for gene of interst is difficult with genomec libraries?
many organisms have very large genome
