Recombinant DNA technology

Question 1

How is recombinant DNA tech possible

Answer 1

-Only works because DNA code is universal
-Transcription + translations are also universal

Question 2

Stages

Answer 2

1. Isolation of DNA fragment with the desired gene
2. Insertion of DNA fragment into a vector (molecule that carries gene/DNA into host cell)
   3.Transformation-DNA into host cell
3. Identification of host cell with gene in
4. Cloning of pop. of host cells

Question 3

Reverse transcriptase (Isolation)

Answer 3

-Reverse transcriptase converts the mRNA strand into a strand of cDNA
-DNA polymerase creates second strand=double stranded cDNA

Question 4

Restriction endonucleases (Isolation)

Answer 4

-Bacteria are infected by viruses, they cut the viral DNA using restriction endonuclease
-Each type of RENDase cuts DNA at a specific sequence, called recognition sequence

Question 5

Gene machine (Isolation)

Answer 5

1. Amino acid sequence + mRNA are determined from protein
2. cDNA triplets, are fed into a computer
3. Small overlapping strands of nucleotides designed
4. Assembled by adding one nucleotide at a time, then joined
5. Cloned, and inserted into vector using sticky ends

Question 6

Insertion (2)

Answer 6

Once DNA is identified it’s inserted into a vector, to be cloned
-The first DNA has extra lengths added to (promoter + terminator

Question 7

In vivo

Answer 7

experiments or processes carried out within a living organism
-Transferring into a host cell using vector

Question 8

In vitro

Answer 8

Outside of a living organism
-PCR

Question 9

Transformation (3)

Answer 9

-Plasmids & bacteria cells mixed
-Calcium ions are added, and change in temperature, to make cell membranes permeable, so plasmids enter
-Not all bacterial cells take up plasmids: some plasmids close again, and some DNA fragments form their own plasmids

Question 10

Identification (4)

Answer 10

Identifying the gene we want
-Marker genes
-use a second easily identifiable marker gene to identify whether the desired gene has been taken up

Question 11

Antibiotic-resistant marker gene (Identification)

Answer 11

-Cells within the plasmid can be identified via replica plating
-The gene for tetracycline resistance is cut, so the enzyme that breaks down tetracycline cant be made, so no resistance to TC
-If grown on plated with TC, bacteria dies
-Replica plating is used so that the cells containing the desired plasmids (gene) aren’t destroyed

Question 12

Fluorescent marker genes (Identification)

Answer 12

-Green fluorescent protein (GFP) from jellyfish is inserted into plasmid
-The desired gene is inserted into the GFP gene
-Any fluorescing bacteria that don’t have the plasmid with the desired gene

Question 13

Enzyme marker genes (Identification)

Answer 13

-Lactase turns a colorless substrate blue
-The required gene is transplanted into the center of the gene that makes lactase
-If plasmid with the required gene is present the colonies won’t reproduce lactase
-So won’t be able to turn colorless substrate blue

Question 14

5. Cloning

Answer 14

Takes place in vivo e.g. bacteria