3.8.4.1: Recombinant DNA technology

Question 1

What is recombinant DNA?

Answer 1

Refers to a cell having two or more sources of DNA

Question 2

What are the five steps of recombinant DNA technology?

Answer 2

1) Isolation of genes
2) Insertion
3) Transformation
4) Identification
5) Cloning

Question 3

What are there three steps of isolation?

Answer 3

1) Reverse transcriptase
2) Restriction endonuclease
3) Gene machine

Question 4

Process of isolation: Reverse transcriptase

Answer 4

1) Reverse transcriptase joins DNA nucleotides with complementary bases to the mRNA sequence to make single stranded DNA (cDNA)

2) DNA polymerase and nucleotides makes the DNA double stranded

Question 5

Two advantages of reverse transcriptase isolation

Answer 5

1) cDNA is intro free because it is based on mRNA sequence

2) mRNA is easier obtained and purified

Question 6

What are restriction endonuclease?

Answer 6

Enzymes that hydrolyse DNA at specific recognition sequences.

Question 7

Process of isolation: Restriction endonuclease

Answer 7

1) Different restriction endonuclease hydrolyse DNA at different recognition sequences because the shape of the sequence is complementary to the active site

2) Recognition sequences are palindromic and the cut creates sticky ends which have the ability to join to DNA complementary base pairs

Question 8

Why might the gene not code for functional proteins using restriction endonucleases?

Answer 8

Is the recognition sequence for the selected restriction endonuclease occurs within the DNA fragment for isolation.

Question 9

How might the gene be expressed using restriction endonuclease process?

Answer 9

Promoter region which allows transcription factors and RNA polymeraseto bind allows gene to be expressed.

Question 10

Process of isolation: Gene machine

Answer 10

1) Examine protein to identify amino acid sequence to work out mRNA and DNA sequence

2) DNA sequence is entered into the computer

3) Computer creates small sections of overlapping single strands of nucleotides (oligonucleotides)

4) Oligonucleotides joined to create DNA for gene

5) PCR amplifies the quantity to make a double strand

Question 11

What are two advantages of gene machine isolation

Answer 11

1) DNA is made from proteins so has no introns

2) Artificial genes are easily transcribes and translated by prokaryotes as they have no introns

Question 12

Definition of a vector

Answer 12

DNA carrier which transfers foreign DNA into cells e.g. plasmid or virus

Question 13

Process of insertion:

Answer 13

1) Plasmid is cut open using the same restriction endonuclease

2) DNA fragment sticky ends are complementary to sticky ends of plasmids

3) DNA fragment anneals to vector DNA by DNA ligase which catalyses the condensation reaction to form phosphodiester bonds between nucleotides to create sugar phosphate backbone

Question 14

What is transformation?

Answer 14

Process which recombinant DNA vector is transferred into a host cell e.g. bacteria

Question 15

Describe the process of transformation?

Answer 15

Host cells are heat shocked and mixed with calcium phosphate to increase the permeability of the host cell membrane. Vector contained recombinant DNA enters bacteria.

Question 16

Why must be identify transformed cell? (2)

Answer 16

1) Not all vectors take up target DNA to become recombinant

2) Not all host cells take up recombinant vectors

Question 17

Define marker gene.

Answer 17

Allows easy identification of cells that have taken up genetically transformed plasmi d

Question 18

Describe the process of antibiotic resistant marker genes in identification

Answer 18

1) Gene has resistance to tetracycline and ampicillin

2) DNA fragment inserted causing disruption to tetracycline resistance so the gene.

3) Grow bacteria on agar

4) transfer colonies on replica ampicillin plate in agar. If bacteria grows it contains the plasmid

5) Transfer colonies to a plate with tetracycline in agar. The colonies that didn’t grow from the last replica plate have the plasmid with desired DNA

Question 19

How do fluorescent markers work?

Answer 19

1) Fluorescent gene inserted into plasmid

2) DNA fragment inserted in fluorescent gene which disrupts it

3) Bacteria grown on agar and exposed to UV light. If they don’t glow they contain recombinant plasmid

Question 20

How do enzyme markers work?

Answer 20

1) Gene for lactase inserted into plasmid

2) DNA fragment disrupts lactase gene to prevent lactase production

3) Grow bacteria on agar plate with colourless substance. If it doesn’t turn blue it contains recombinant plasmid

Question 21

What is in vivo cloning?

Answer 21

Cloning by binary fission

Question 22

Describe the process of PCR (6)

Answer 22

1) Heat DNA to 95 degrees to break weak hydrogen bonds and separate strands

2) Add primers and nucleotides

3) Cool to 50 degrees to allow binding of nucleotides

4) Add DNA taq polymerase

5) Heat to 75 degrees

6) DNA polymerase joins nucleotides

7) repeat cycle many time

Question 23

What is a primer?

Answer 23

Short piece of single stranded DNA with complementary base sequences to DNA fragment for isolation. They prevent DNA strands stocking back together.