Lec 16 Genetic Engineering Flashcards
Molecular cloning
Restrictive enzyme what is it
AKA restriction endonuclease (RE)
found in ONLY bacteria and archaea providing a defense mechanism against invading viruses
Molecular cloning
Restrictive enzyme
what is restriction site
cut double stranded DNA at specific recognition sequences
Molecular cloning
restrictive enzyme
how many base pair cutter and what is an example
6-bp cutter
ex: EcoRI (1st enzyme isolated from E. coli Strain R)
Molecular cloning
restrictive enzyme
what is palindromic sequences
palindromic sequence
ex: GAATTC for EcoRI
Molecular cloning
restriction enzyme
what generates overhang
A zig-zag cut generates overhang (protruding) ends- by most Res
What joins DNA fragments in the presence of ATP
DNA Ligase
what are the three vectors
plasmid
viruses
artificial chromosomes
first vector plasmid
what is it
a plasmid is an extra chromosomal DNA molecule which is capable independently from the chromosomal DNA
Plasmid has Ori which is what
replication origin
Vector plasmid what is selectable marker example
ampicillin resistance gene
vectors plasmid what is LacZ
multiple restriction sites usually within a gene
what is vector artificial chromosomes
for larger insert and large-scale analysis of genomes
Molecular cloning (genetic engineering) what are the four steps
1) DNA cleavage
2) Ligation of gene into vector by DNA ligase in the presence of ATP. Now the recombinant plasmid is made
3) transformation
4) Screening
Molecular cloning (genetic engineering)
what is DNA cleavage
Both gene fragment & vector are digested by restriction enzyme
Molecular cloning (genetic engineering)
what is transformation in the steps
transformation- introducing the recombinant DNA into bacteria
Molecular cloning (genetic engineering)
what is screening
using selective medium containing antibiotic and substrate for lacZ enzyme called X-Gal
Molecular cloning (genetic engineering)
Screening antibiotic ex
screening X-Gal what is it
antibiotic- ampicillin-clones that contain the plasmid will grow in the medium with ampicillin
X-Gal-clones that have the gene fragment inserted in the LacZ gene will turn white
PCR stands for what and what is it
polymerase chain reaction and its using the concept of DNA replication
PCR protocol
1) DNA template, gene specific primer pairs, free nucleotides & Taq DNA polymerase
2) Thermocycle program
Thermocycle
94C
94C to denature the DNA template
Thermocycle
55C
55C for primers to anneal to the template
thermocycle
70C
for DNA synthesis (or primer extension)
PCR does what
exponential production of specific DNA fragment
Memorize Hae(3) and EcoR1 and Hind(3) and Not1
