3.8.4.1- Recombinant DNA technology

Question 1

Advantages of In vitro cloning (3)

Answer 1

1) automated (more efficient)
2) rapid
3) doesnt require living cells

Question 2

In vitro cloning- process (3)

Answer 2

1) Temp increased to 95 degrees, to break H bonds & split DNA> 2
2) Temp decreased to 55 degrees, so primers can ANNEAL
3) DNA polymerase (taq) then attaches complementary free nucleotides & synthesises new strand (done at 72 degrees)

Question 3

In vivo cloning- 5) identifying transformed cells- c) enzyme marker (4)

Answer 3

1) Lactase gene inserted into plasmid
2) DNA fragment inserted into middle of gene, disrupting it
3) Bacteria grown on agar, with colourless substance
4) Ones that turn from colourless> blue contain original plasmid; others are transformed cells

Question 4

In vivo cloning- 5) Identify transformed cells- b)Fluorescent markers (3)

Answer 4

1) GFP gene (that codes for fluorescence) inserted into plasmid
2) DNA fragment inserted in middle of GFP gene, disrupting it
3) Grow bacteria on agar; non-fluorescent colonies contain recombinant plasmid

Question 5

In vivo cloning- 5) Identify transformed cells- a) antibiotic resistance (6)

Answer 5

1) DNA fragment inserted into middle of TETRACYCLINE resistance gene; disrupting it (on a plasmid with genes r to both T & A)
2) Grow bacteria on AGAR
3) Transfer colonies using Velvet Block
4) Place on new agar plate, with AMPICILLIN antibitoic; non resistant die
5) Transfer remaining to plate with TETRACYCLINE antibiotic
6) Those on 2nd plate but not 3rd, are transformed cells (as resistant to A but not T)

Question 6

In vivo cloning- issues that can occur in host cell transformation (3)

Answer 6

1) plasmid RE-JOINS before DNA fragment entered
2) DNA fragment sticks to itself
3) Recombinant plasmid doesn’t get inside cell

Question 7

In vivo cloning- 5) Identify transformed cells- Types of marker gene to identify (3)

Answer 7

Marker genes=

1) antibiotic resistance genes
2) Genes coding for fluorescent proteins
3) Genes coding for enzymes

Question 8

In vivo cloning- 4) Transform host cells with vector (2)

Answer 8

1) Cell membrane needs to be MORE permeable, to take up vector
2) Host cells are mixed with Ca2+ & heat-shocked to increase permeability

Question 9

In vivo- 3) Insert DNA fragment into vector- process= (2)

Answer 9

1) Plasmid cut open using SAME RE; this creates SAME SE (DNA F’s SE complementary to P’s SE)
2) DNA fragment & P combined & enzyme ligase ANNEALS them together (by catalysing C reaction to form phosphodiester bonds between nucleotides)

Question 10

In vivo- 2) modification (3)

Answer 10

1) Restriction Endonucleases left sticky ends; DNA F need modification to ensure gene T can occur
2) Promoter region added at START of DNA fragment
This provides a binding site for RNA polymerase to enable transcription)
3) Terminator region added at END of gene
This causes RNA polymerase to detach & stop transcription, so only 1
gene at a time copied > mRNA

Question 11

In Vivo cloning- stages (6)

Answer 11

1) create DNA fragment (via restriction endonucleases)
2) modification of DNA
3) Insertion of DNA fragment> vector
4) Host cell transformation
5) Identify transformed cells
6) clone & make copies

Question 12

which method of amplification is used following restriction endonucleases method of creating DNA fragments?

Answer 12

In Vivo cloning

Question 13

Advantage of gene machine method of creating DNA fragments (3+1)

Answer 13

-process is quick, accurate & makes intron-free DNA so can be transcribed in prokaryotic cells

Question 14

Creating DNA fragments- 3)Gene machine- process (5)

Answer 14

1) Protein examined to identify AA sequence & thus mRNA & DNA sequence
2) DNA sequence entered into computer which checks for biosafety & biosecurity
3) computer creates small sections of overlapping single nucleotide strands (OLIGONUCLEOTIDES)
4) Oligonucleotides joined to create DNA for entire gene
5) Then enters PCR for amplification

Question 15

Creating DNA fragments- 2) Restriction Endonucleases- process (4)

Answer 15

1) RE have an active site complementary to a specific DNA base sequence (recognition sequence)
2) RE cut DNA at specific location
3) Some cut at SAME location in double strand to create BLUNT end, others cut to create STAGGERED ends & exposed DNA bases
4) Exposed staggered ends are PALINDROMIC & called ‘sticky ends’ as they can join to DNA with complementary base pairs

Question 16

Advantage of reverse transcription process of creating DNA fragments- (1)

Answer 16

cDNA is intron-free as based on mRNA which has been spliced

Question 17

Creating DNA fragments- 1) Reverse transcription- process (4)

Answer 17

1. Cells that naturally produce protein of interest is selected (as have large amounts of mRNA for the protein)
2. Reverse transcriptase joins DNA nucleotides with complementary bases (to the mRNA sequence)
3. cDNA made
4. To make double-stranded, DNA polymerase is used

Question 18

3 methods of creating DNA fragments (3)=

Answer 18

1. Reverse transcription
2. Restriction endonucleases
3. Gene machine

Question 19

Recombinant DNA technologies= (3)

Answer 19

1. Creating DNA fragments
2. Genetic fingerprinting
3. Genetic screening, counselling & locating genes