Control of Gene Expression: Recombinant DNA Technology - Amplifying DNA Fragments

Question 1

Which techniques are used to amplify DNA fragments?

Answer 1

• In vivo cloning

- In vitro cloning

Question 2

What is in vivo cloning?

Answer 2

• Where gene copies are made within a living organism

- As the organism grows and divides, it replicates the DNA, creating multiple copies of the gene

Question 3

What is in vitro cloning?

Answer 3

• Where gene copies are made outside a living organism using polymerase chain reaction (PCR)

Question 4

What are the main stages of in vivo cloning?

Answer 4

• Making recombinant DNA
• Transforming cells
• Identifying transformed cells

Question 5

Explain what happens during the making recombinant DNA stage of in vivo cloning

Answer 5

• Isolating and cutting open vector DNA

• Vector DNA is isolated
• Vector DNA cut open with same restriction endonuclease used to isolate DNA fragment containing target gene
• Sticky ends of vector DNA complementary to sticky ends of DNA fragment

• Ligation

• Vector DNA and DNA fragment mixed with DNA ligase
• DNA ligase joins sticky ends of DNA fragment to sticky ends of vector DNA in process called ligation
• New combination of bases in DNA called recombinant DNA

Question 6

What is a vector? Give some examples.

Answer 6

• Something used to transfer DNA into a cell
• Can be plasmids (small, circular molecules of DNA in bacteria)
• Can be bacteriophages (viruses that infect bacteria)

Question 7

Which enzyme joins the sticky ends of DNA fragments to sticky ends of vector DNA during in vivo cloning?

Answer 7

• DNA ligase

Question 8

What is recombinant DNA?

Answer 8

• A combination of DNA from two different organisms

Question 9

Explain what happens during the transformation of cells stage of in vivo cloning

Answer 9

• Vector with recombinant DNA used to transfer genes into host cells
• Host cells taking up target gene from vector are said to be transformed
• Bacterial host cells are mixed with vectors in ice-cold calcium chloride solution, then heat shocked to encourage cells to take in plasmids

Question 10

What are transformed cells?

Answer 10

• Host cells that take up vectors containing target gene

Question 11

Explain what happens during the identification of transformed cells stage of in vivo cloning

Answer 11

• Insertion of marker genes

• Marker genes can be used to identify transformed cells
• Marker genes can be inserted into vectors at same time as target gene is inserted or are naturally found in vector DNA
• Transformed cells contain marker gene and target gene

• Growth and identification of transformed cells

• Host cells grown on agar plates
• Marker genes can code for fluorescence under UV light or antibiotic resistance etc. to be identified
• Identified transformed cells grow to produce many copies of target gene

Question 12

What are marker genes?

Answer 12

• Additional gene inserted into a plasmid used to aid in the identification of transformed cells
• Marker genes are easily recognisable e.g. fluoresce or provide antibiotic resistance

Question 13

What must vectors used in in vivo contain to allow transformed cells to produce proteins coded for by target gene?

Answer 13

• Vectors must contain specific promoter and terminator regions
• Promoter regions are DNA sequences telling RNA polymerase to start producing mRNA
• Terminator regions are DNA sequences telling RNA polymerase where to stop producing mRNA
• If regions not present, DNA fragment won’t be transcribed and protein won’t be made
• These regions may be present in vector DNA or may need to be added

Question 14

Explain what happens during in vitro cloning or the polymerase chain reaction (PCR)

Answer 14

• Setting up reaction mixture
- Reaction mixture contains DNA sample, free nucleotides, primers and DNA polymerase

• Addition of primers

• Mixture heated to 95°C to break hydrogen bonds between 2 strands of DNA
• Mixture cooled to 50°C-65°C depending on primer used, so primers can anneal to strands

• Action of DNA polymerase

• Mixture heated to 72°C so DNA polymerase can work
• DNA polymerase lines free DNA nucleotides along template strand
• Specific base pairing allows complementary bonds to be formed

• Result

• 2 new copies of DNA fragment formed
• Cycle starts again
• Each PCR cycle doubles the amount of DNA

Question 15

Which temperatures is the reaction mixture in PCR heated and cooled down to and why?

Answer 15

• Break hydrogen bonds between 2 DNA strands
- 95°C

• To allow primers to anneal to strands
- 50°C-65°C (depends on primer)

• Allow DNA polymerase to work
- 72°C

Question 16

What is a primer?

Answer 16

• Short pieces of DNA that are complementary to bases at start of desired DNA fragment

Question 17

What does DNA polymerase do?

Answer 17

• Turns single template strand into a double-stranded DNA molecule by using free complementary nucleotides

Question 18

How much does the amount of DNA increase by in each PCR cycle?

Answer 18

• Each PCR cycle doubles the amount of DNA