DNA cloning Flashcards
what is recombinant DNA?
any DNA molecule derived from two different sources
what does plasmid cloning vector include?
origin of replication, polylinker, antibiotic resistance
what is the function of antibiotic resistance?
it codes for an enzyme that allows the cell to destroy penicillin
why is antibiotic resistance a selectable marker?
because cell received the plasmid can grow in the presence of penicillin but cell not received cannot survive.
how does DNA fragments cut and join?
cut by restriction enzymes and join b DNA ligase
how do restriction enzyme recognize restriction site?
they recognize the palindromic sequence which reads the same from both strands
what is the enzyme that cut blunt ends?
Sma I
CCC | GGG
how much energy is consumed by T4 DNA ligase to join DNA fragments and vector DNA?
2 ATPs are hydrolyzed to 2 AMP + 2PPi
procedure for DNA cloning?
- insertion into vector
- transformation and selection.
plasmid entered the cell and transformed the cell. transformed cell survived; cell that do not take up plasmid die on ampicillin plates.
what are the two types of DNA library?
- cDNA library
2. genomic DNA library
what is the difference between cDNA library and genomic DNA library?
- genomic DNA includes introns, intergenic regions, mobile DNA elements
steps of cDNA library?
- reverse transcription of mRNA to get cDNA (complementary to an mRNA sequence)
- make double-stranded cDNA
- cut cDNA with restriction enzyme
- insert cDNA into a vector also cut by restriction enzyme
why would you want to express foreign proteins in bacteria or in eukaryotic cells?
for biotechnology:
- produce large amounts of a protein that may be very rare in natural materials
- difficult to purify, expensive
- expressed at high levels in bacteria
example: leukocyte growth factor G-CSF used to fight immunosuppressive side-effects of cancer chemotherapy. human insulin, human growth hormone
for research:
can express wild type, or designed mutant or fragments of the protein to study protein function in a living cell
what is needed for expression of recombinant proteins?
a promoter to drive transcription of the inserted protein-coding DNA
which promoter is used in production of recombinant protein in bacteria?
lacZ promoter
if lactose is around or we add IPTG, lacZ promoter can be very active and start transcription
what is IPTG?
it is lactose analogue which induces transcription form lac promoter
how does lacZ gene be expressed? and the function of protein lacZ expressed?
lacZ gene –> lacZ mRNA –> beta-galactosidase
beta-galactosidase encodes an enzyme that can be detected because it produces an insoluble blue product from the colorless substrate X-Gal.
what are the two types of expression of recombinant protein in eukaryotic cells?
transient transfection stable transfection (transformation)
why transient transfection is transient?
we tend to lose the plasmid because plasmid replication cannot keep up with the cell division
can lacZ promoter be used in transient transfection?
NO. transcription in eukaryotic cells requires a eukaryotic promoter
what are used to transfect cultured cells in transient transfection and stable transfection?
lipid treatment or electroporation
which resistance is added to the vector for stable transfection?
neomycin resistance which help animal cells resistance to a drug that would normally kill them.
why is stable transfection stable?
the whole vector can break and inserted into host chromosome. so it becomes the descendent of that cell.
procedures for transgenic mice?
- inject foreign DNA into one of the pronuclei
- transfer injected eggs into foster mother
- about 10%-30% of offspring will contain foreign DNA in chromosomes of all their tissues and germ line.
- breed mice expressing foreign DNA to propagate DNA in germ line.
* most random insertions do not disrupt other genes
