Recombinant DNA Technology

Question 1

Explain 3 pharmaceutical uses of recombinant dna tech

Answer 1

Human insulin for diabetes

Factor VIII for haemophilia

Hepatitis B vaccine produced antigens

Question 2

Name the 4 cells used potentially in dna recombination

Answer 2

Bacteria cells - prokaryotes host cells

Yeast cells - eukaryotic

Insect cells - eukaryotic

Mammalian cells - eukaryotic

Question 3

Name the 4 enzymes involved in dna technology

Answer 3

Restriction endonucleases - cleaves dna fragments

Reverse transcriptase - turns mrna for gene into cDNA

Taq polymerase - used in PCR

Dna ligase - synthesise the 2 strands together in vector and injected dna

Question 4

Why do restriction endonucleases originate from bacteria

Answer 4

Bacteria use it to cleave phage dna which infects bacteria

Question 5

Why are restriction endonucleases called specific

Answer 5

They cleave dna at specific sites in DNA called palindromic sequences (same base sequence on 2 strands)

Question 6

Why do restrictionenzymes recognise palindromic sequences

Answer 6

They are 2 homodimers (2 proteins) which are identical so on each strand identify same sequence

Question 7

What are the 2 ways DNA sequences are cut my restriction enzymes

Answer 7

Symmetrical cleavage - blunt ends

Asymmetrical cleavage - sticky ends used by dna ligase

Question 8

What is the process called where dna ligase will join the dna fragments and the plasmid dna where it has been cut by SAME enzyme

Answer 8

Annealing

Question 9

What are 3 things vectors must have to be used in recombinant dna tech

Answer 9

Unique restriction sites where they can be cleaved
Oric- where dna is cloned
Gene to allow selection (identifying the recombinant hosts) - this can be eg the Lac Z gene for enzyme B galactosidase

Question 10

Plasmids are the most common vector, explain why they are used

Answer 10

They replicate independantly and quick in bacterial

They have genes such as Ampicillin resistance to allow selection

They have restriction sites

Some have expression factors (able to express a gene into a protein)

Question 11

Bacteriophages are also vectors. How are they used

Answer 11

The dna fragments are inserted into them so when they inject bacteria, they inject the dna fragment

This then joins the plasmid (PROPHAGE)

Question 12

What are phagemids and why are they good vectors

Answer 12

Hybrids where plasmids are contained in phages - larger fragment can be cloned

Question 13

To isolate random DNA sequences there are 2 techniques - reverse transcriptase or using restriction endonucleases, explain both

Answer 13

Restriction enzymes are used to cleave random dna into fragments when extracted from cells

Reverse transcriptase uses mrna strands eg isolated from genes for eg insulin

Then transcribes it into cDNA (double stranded)

Question 14

How are specific fragments isolated from dna using PCR

Answer 14

Dna strands are hybridised with primers specific to that base sequence (strands are seperated)

These are then cloned many times to produce many fragments of the same DNA sequence

Question 15

When dna fragments are inserted then into plasmids via dna ligase and restriction endonucleases (same ones), explain how the bacteria is then transformed to accept the recombinant plasmid

Answer 15

The bacteria goes through HEAT SHOCK AND CACL is added (calcium chloride)
This forms pores in the bacteria membrane

The pores allow diffusion of the plasmid into the bacteria where it is kept on ice

When warmed to 37C the cell fixed the cell wall and bacteria are grown on agar plates

Question 16

Why is the plasmid kept on ice to transform the bacteria

Answer 16

Prevent cell growth - because cell wall is damaged/pores the bacteria would undergo apoptosis and die

Question 17

How is antibiotic Eg ampicillin resistance used to select the hosts with insert plasmids

Answer 17

The insert hosts will have ampicillin resistance in the plasmid so when added to the medium they survive and wild type don’t

Question 18

What is an issue with using the antibiotic resistance principle to select inserts

Answer 18

Some bacteria will have turned the plasmid back into normal plasmid when joined by dna ligase - they would still have the amp resistance gene

Question 19

How is INSERTIONAL INACTIVATION used to select for host inserts

Answer 19

The dna fragment can be inserted into genes such as the Lac Z gene which produces B galactosidase

If the fragment is inserted it inactivated the b galactosidase

Can’t digest X gal into blue pigment

Blue pigment won’t show up in inserts

Question 20

What checks can be made to see if the dna wanted is in the host

Answer 20

Using dna probes which only recognise the base sequence

Question 21

What are the 3 ways recombinant dna is used after gene tech

Answer 21

1- to express the gene into proteins
2- recover plasmids and manipulate them - by isolating from bacteria cells
3- transfer into useful mamalian cells

Question 22

What do plasmids need to be able to induce expression of the protein

Answer 22

An expressive factor - they need promoter sequences next to the gene to allow transcription and translation

Question 23

Why are 2 restriction endonucleases needed to cut the plasmids

Answer 23

To allow for the right orientation of the DNA sequence - allowing expression of the gene

Question 24

Advantages of using bacterial cells as hosts

Answer 24

Simple
Short generation time
Large yield of product

Question 25

What is the biggest disadvantage of bacterial cells

Answer 25

No post translational modification such as splicing or glycolysation

Proteins can be toxic to the bacteria and they die

Not eukaryotes- the protein can fail to fold properly and lose activity

Question 26

Advantages and disadvantages of yeast cells

Answer 26

+ they are eukaryotic so proteins can Fold properly
They are simple and fast replication
Have POST TRANSLATIONAL MODIFICATION

• Have proteases which can degrade the recombinant proteins

Question 27

Ads and dis ads of insect cells

Answer 27

+ correctly folded (eukaryotes) , post translational modification, cheaper than mammal

• modification differs from mammalian cells still

Question 28

Advantages and disads of mammalian cells

Answer 28

Accurate product
Correctly folded protein
Post translational modification

-
Complex
Expensive
Grow to lower densities (few cells cloned)

Question 29

Insulin is a protein consisting of A and B chain joined by disulfide bonds. How many introns does it have

Answer 29

2 introns in the insulin gene

Question 30

Why didn’t insulin from pigs or cows work

Answer 30

Not an identical protein than humans

Question 31

Explain how reverse transcriptase is used to make pro insulin in bacterial plasmids

Answer 31

The mRNA strand for pro insulin is transcribed into cDNA

It’s inserted into a plasmid and the transformed bacteria then produces pro insulin

Question 32

How was producing the CD4 receptor from dna technology useful in research for hiv

Answer 32

Found that when they added the receptor to a normal non CD4 cell it became infected with hiv - FOUND CD4 cells are cellular receptors for hiv infection

Question 33

How can ingesting ecoli with expression vector for Sirna help gene knockdown in c elegans

Answer 33

Because they will then be able to transcribe for ds sirna