Gene technologies allow the study and alteration of gene function

Question 1

what is recombinant DNA?

Answer 1

transferred fragments of DNA from one organism, or species, into another

Question 2

why can the transferred DNA be translated in the host cell?

Answer 2

1. DNA is universal

2. transcription and translation are universal mechanisms

Question 3

what are the methods for forming DNA fragments?

Answer 3

1. conversion of mRNA to complementary DNA (cDNA) using reverse transcriptase
2. using restriction endonucleases to cut a fragment containing the desired gene from the DNA
3. creating the gene in a gene machine

Question 4

describe how reverse transcriptase is used to form DNA fragments

Answer 4

1. mRNA is isolated from a cell that readily synthesizes the protein coded for by the desired gene
2. mix mRNA with DNA nucleotides and reverse transcriptase; RT uses the mRNA as a template to form a single strand of complementary DNA
3. Add more DNA nucleotides and DNA polymerase forms the 2nd strand of DNA using cDNA as the template; forms double stranded DNA= desired gene

Question 5

describe how restriction endonucleases are used to form DNA fragments

Answer 5

different restriction endonucleases cut DNA at specific sequence of bases- ‘recognition sites’
the shape of the recognition site is complementary to the active site
some restriction enzymes cut in a staggered fashion
this forms sticky ends

Question 6

what are the 2 outcomes of using restriction endonucleases to form DNA fragments?

Answer 6

blunt ends and sticky ends

Question 7

describe how the gene machine is used to form DNA fragments

Answer 7

1. Examine the desired protein and identify the amino acid sequence
2. mRNA codons are looked up and the complementary DNA triplets are worked out
3. The DNA sequence of nucleotides bases is fed into a computer
4. Checked for biosafety and biosecurity (the protein being produced is safe and ethical to produce)
5. The computer designs small overlapping single strands of nucleotides called oligonucleotides which can be assembled into a gene
6. The oligonucleotides are joined to make a gene🡪 it does not contain introns or non coding DNA
7. The gene is amplified using PCR and then made into a double stranded gene
8. The gene can be inserted in to a bacterial plasmid using sticky ends. It is now a vector which can be stored, cloned or transferred

Question 8

what are the advantages of using reverse transcriptase to form DNA fragments?

Answer 8

1. mRNA present in cell from actively transcribed genes

2. lots of mRNA to make cDNA

Question 9

what are the advantages of using restriction endonucleases to form DNA fragments?

Answer 9

sticky ends on DNA fragments make it easier to insert into a vector to form recombinant DNA

Question 10

what are the advantages of using the gene machine to form DNA fragments?

Answer 10

exact DNA fragment synthesized

Question 11

what are the disadvantages of using reverse transcriptase to form DNA fragments?

Answer 11

lots of steps so very time consuming; more difficult

Question 12

what are the disadvantages of using restriction endonucleases to form DNA fragments?

Answer 12

DNA fragment has introns

Question 13

what are the disadvantages of the gene machine to form DNA fragments?

Answer 13

need to know the amino acid sequence

Question 14

what are the 2 methods to amplify DNA fragments?

Answer 14

1. in vivo; culture of transformed host cells

2. in vitro: PCR

Question 15

describe the how enzymes can be used to insert a gene/DNA fragment into a plasmid (3 marks)

Answer 15

1. Restriction endonuclease is used to cut the plasmid, the same restriction endonuclease is used to cut the DNA fragment; forming sticky ends
2. so they have complementory sticky ends
3. DNA ligase is used to join the 2 DNA strands to gether; by forming phosphodiester bonds

Question 16

what must be done to the DNA fragments in a culture of transformed host cells to allow the DNA fragments to be amplified

Answer 16

1. add promotor and terminator regions

2. allows gene to be transcribed and translated into a protein

Question 17

what are methods of identifying if a host cell has taken up the recombinant DNA?

Answer 17

1. antibiotic resistance markers
2. fluorscent markers
3. enzyme action markers

Question 18

how does antibiotic resistance show host cells have taken up the recombinant DNA?

Answer 18

1. antibiotic resistance acts as a marker
2. bacteria cell contains 2(or more) antibiotic resistant genes
3. one of these genes is disrupted when DNA fragment is inserted
4. recombinant plasmid is then inserted into the bacteria cell
5. bacteria cell is grown on agar; treated with one of the antibiotics
6. cells that have taken up that plasmid will be resistant
7. Replica plate grown; treated with the other antibiotic
8. only those that have taken up the recombinant DNA will die out (as there is no longer any resistance)

Question 19

how do fluorescent markers show host cells have taken up recombinant DNA?

Answer 19

1. gene for fluorescence is inserted into a plasmid; acts as a marker (GFP)
2. DNA fragment is inserted into the fluorescent gene (gene is disrupted, cannot be expressed)
3. only those bacteria cells that DID take up the recombinant DNA will NOT glow

Question 20

how does enzyme action show host cells have taken up recombinant DNA?

Answer 20

1. enzyme action acts as a marker
2. DNA fragment inserted into the gene for lactase
   - lactase turns certain colorless solutions blue
3. insertion disrupts the lactase gene; not expressed
4. colorless spots on agar show host cells have taken up recombinant DNA

Question 21

why do not all bacteria cells take up the recombinant DNA?

Answer 21

1. recombinant plasmid doesnt get inside cell; cell surface membrane not permiable enough
2. plasmid rejoins
3. DNA fragment rejoins; forms a plasmid

Question 22

how are bacteria host cells transformed in order to be able to take up the recombinant DNA?

Answer 22

1. placed in ice-cold calcium ion solution
2. heat chocked
3. allows cell surface membrane to become more perimiable

Question 23

what are the 3 overall steps of PCR as a method to amplify specific DNA fragments?

Answer 23

1. strand separation (95)
2. annealing (55)
3. DNA strand synthesis (72)

Question 24

what is needed to carry out PCR as a methods of amplifying specific DNA fragments?

Answer 24

1. thermocycler
2. DNA fragment
3. DNA polymerase (taq polymerase)
4. DNA primers
5. Nucleotides

Question 25

describe the process of PCR as a method of amplifying specific DNA fragments

Answer 25

1. at 95, seperate DNA strands by breaking weak hydrogen bonds between the bases 2. At 55, allow primers to anneal to DNA fragment strand, forming hydrogen bonds 3. primer is complemtary to template DNA 4. DNA polymerase binds primer to start synthesis; adds nucleotides from the 3' end to 5' 5. 2 different primers required as DNA is aniparallel 6. At 72, DNA nucelotides align next to complmentary exposed bases 7. DNA polymerase joins adjacent nucleotides, forming phosphodiester bonds

Question 26

why is taq polymerase used?

Answer 26

enzyme can withstand higher temperatures for PCR that human DNA polymerase cannot

Question 27

what is the function of the primers in PCR?

Answer 27

1. marks the region of DNA that needs to be replicated | 2. enzymes needs a starting strand in order to attach nucleotides

Question 28

why do 2 different primers have to be used in PCR

Answer 28

1. DNA strands are anti-parallel | 2. nucleotides will only bind from the 3' end to the 5' end

Question 29

explain why each temperature is used at each stage of PCR?

Answer 29

1. 95- breaks the h-bonds holding complementary DNA bases 2. 55- allows H-bonds to re-form and allows the primers to attach 3. 72- optimal temperature for taq polymerase to function

Question 30

what are some advantages to using PCR as a method of amplifying DNA fragments?

Answer 30

1. rapid process 2. doesn't require living cells 3. useful when we want to introduce a gene into another organism

Question 31

what are some disadvantages to using PCR as a method of amplifying DNA fragments?

Answer 31

1. risk of contamination by unwanted DNA 2. cannot cut out specific gene 3. doesn't produce transformed bacteria