Genetic engineering

Question 1

Genetic engineering:

Answer 1

deliberate manipulation of genetic material to modify an organism’s characteristics, often involving gene transfer.

Question 2

Recombinant DNA:

Answer 2

• DNA that is altered to contain nucleotides from two different organisms.
• It allows DNA fragments to be transferred between organisms

Question 3

Genetically modified or transgenic organisms:

Answer 3

Organisms that receive transferred DNA fragments

Question 4

Key stages in gene transfer:

Answer 4

1. Desired gene is identified and isolated
2. Multiple copies of the gene are made using the polymerase chain reaction (PCR)
3. The gene is inserted into a vector
4. The vector delivers the gene into cells in a different organism
5. Cells with the new gene are identified, such as by using marker genes
6. Cells with the new gene are cloned

Question 5

Why can transgenic organisms express the transferred gene:

Answer 5

• Produce encoded protein and express new genetic traits
• Due to universal nature of the genetic code and the similarity of transcription and translation across different organisms

Question 6

How are DNA fragments produced?

Answer 6

1. Making complementary DNA (cDNA) using reverse transcriptase and mRNA
2. Cleaving DNA from a donor organism with restriction enzymes

Question 7

Reverse transcriptase method:

Answer 7

1. extract mRNA
2. mRNA is reverse transcribed using enzyme reverse transcriptase and DNA nucleotides
3. cDNA identical strand to original DNA strand formed (single stranded). cDNA isolated from mRNA strand
4. Double-stranded DNA is reformed: cDNA, free nucleotides, DNA polymerase
5. Reform desired gene

Question 8

Using restriction enzymes to cut DNA:

Answer 8

1. DNA incubated with chosen restriction endonucleases
2. Restriction enzymes identify palindromic sequences in the DNA double helix and cut double-stranded DNA if their recognition sequence is present
3. Recognition sequences at either end of a desired DNA fragment allow restriction enzymes to separate the fragment from the rest of DNA to obtain desired gene
4. Enzymes cut target gene fragment out via hydrolysis reaction
5. Different restriction enzymes cut at different sequences based on their active site shape.

Question 9

Sticky ends:

Answer 9

• Short overhanging sequences of unpaired bases that can bind to other DNA fragments when they are inserted into vectors

Question 10

‘in vivo cloning’ or ‘in vivo DNA amplification’

Answer 10

• Process of producing large quantities of target DNA fragment in living cells
• Begins with inserting target DNA fragment into a vector
• Vector transfers to the host

Question 11

Steps in inserting DNA fragments into vectors:

Answer 11

1. A vector is cut open at a specific site using a restriction enzyme, creating sticky ends
2. The same restriction enzyme is used to cut the target DNA fragment, creating complementary sticky ends
3. DNA ligase forms phosphodiester bonds between the sugar and phosphate groups on the two strands of DNA, joining the sticky ends of the vector and DNA fragmented group
4. Newly formed combined DNA molecule known as recombinant DNA

Question 12

Step 2 - transferring recombinant DNA into host cells: 2 methods

Answer 12

• Vectors are either plasmids or bacteriophages

Question 13

Plasmid vectors:

Answer 13

• Small, circular DNA molecules found in bacteria
• Host cells are treated to enhance uptake of plasmids that have recombinant DNA
• Apply calcium ions and temperature shifts can make bacterial membranes more permeable to plasmids
• Electroporation uses an electrical current to make bacterial membranes more porous, helping plasmids enter bacterial cells (and can also transform DNA fragments into eukaryotic cells)

Question 14

Bacteriophage vectors:

Answer 14

• Viruses that infect bacteria
• Bacteriophages inject DNA into host bacterial cells during infection
• The phage DNA now carrying the recombinant DNA, inserts into hosts DNA

Question 15

How to identify uptake of DNA if 5% of host cells uptake?

Answer 15

• Marker genes are inserted into vectors alongside target genes.
• Transformed cells are cultivated on selective agar plates.
• Only transformed cells display the characteristics encoded by marker genes.
• These transformed cells can then be cultured to mass-produce the target DNA fragment through cellular replication.

Question 16

Types of marker genes:

Answer 16

• A marker gene for a specific trait, like antibiotic resistance, ensures that only transformed cells form colonies.
• A marker gene that is visible under UV light like green fluorescent protein (GFP).
• Inserting a marker gene within the GFP gene inhibits fluorescence if it is successfully incorporated.
• A marker gene coding for an enzyme that alters the colour of a specific substrate.