DNA cloning

Question 1

what is recombinant DNA?

Answer 1

any DNA molecule derived from two different sources

Question 2

what does plasmid cloning vector include?

Answer 2

origin of replication, polylinker, antibiotic resistance

Question 3

what is the function of antibiotic resistance?

Answer 3

it codes for an enzyme that allows the cell to destroy penicillin

Question 4

why is antibiotic resistance a selectable marker?

Answer 4

because cell received the plasmid can grow in the presence of penicillin but cell not received cannot survive.

Question 5

how does DNA fragments cut and join?

Answer 5

cut by restriction enzymes and join b DNA ligase

Question 6

how do restriction enzyme recognize restriction site?

Answer 6

they recognize the palindromic sequence which reads the same from both strands

Question 7

what is the enzyme that cut blunt ends?

Answer 7

Sma I

CCC | GGG

Question 8

how much energy is consumed by T4 DNA ligase to join DNA fragments and vector DNA?

Answer 8

2 ATPs are hydrolyzed to 2 AMP + 2PPi

Question 9

procedure for DNA cloning?

Answer 9

1. insertion into vector
2. 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.

Question 10

what are the two types of DNA library?

Answer 10

1. cDNA library

2. genomic DNA library

Question 11

what is the difference between cDNA library and genomic DNA library?

Answer 11

1. genomic DNA includes introns, intergenic regions, mobile DNA elements

Question 12

steps of cDNA library?

Answer 12

1. reverse transcription of mRNA to get cDNA (complementary to an mRNA sequence)
2. make double-stranded cDNA
3. cut cDNA with restriction enzyme
4. insert cDNA into a vector also cut by restriction enzyme

Question 13

why would you want to express foreign proteins in bacteria or in eukaryotic cells?

Answer 13

for biotechnology:

1. produce large amounts of a protein that may be very rare in natural materials
2. difficult to purify, expensive
3. 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

Question 14

what is needed for expression of recombinant proteins?

Answer 14

a promoter to drive transcription of the inserted protein-coding DNA

Question 15

which promoter is used in production of recombinant protein in bacteria?

Answer 15

lacZ promoter

if lactose is around or we add IPTG, lacZ promoter can be very active and start transcription

Question 16

what is IPTG?

Answer 16

it is lactose analogue which induces transcription form lac promoter

Question 17

how does lacZ gene be expressed? and the function of protein lacZ expressed?

Answer 17

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.

Question 18

what are the two types of expression of recombinant protein in eukaryotic cells?

Answer 18

transient transfection 
stable transfection (transformation)

Question 19

why transient transfection is transient?

Answer 19

we tend to lose the plasmid because plasmid replication cannot keep up with the cell division

Question 20

can lacZ promoter be used in transient transfection?

Answer 20

NO. transcription in eukaryotic cells requires a eukaryotic promoter

Question 21

what are used to transfect cultured cells in transient transfection and stable transfection?

Answer 21

lipid treatment or electroporation

Question 22

which resistance is added to the vector for stable transfection?

Answer 22

neomycin resistance which help animal cells resistance to a drug that would normally kill them.

Question 23

why is stable transfection stable?

Answer 23

the whole vector can break and inserted into host chromosome. so it becomes the descendent of that cell.

Question 24

procedures for transgenic mice?

Answer 24

1. inject foreign DNA into one of the pronuclei
2. transfer injected eggs into foster mother
3. about 10%-30% of offspring will contain foreign DNA in chromosomes of all their tissues and germ line.
4. breed mice expressing foreign DNA to propagate DNA in germ line.
   * most random insertions do not disrupt other genes