8.2 Genome Projects And Gene Technologies

Question 1

What is a genome?

Answer 1

All genes in an organism/cell

Question 2

What is the proteome?

Answer 2

All of the proteins that can be made by the genes in an organism/cell

Question 3

Why is it easier to determine the proteome of simple organisms rather than complex organisms from their genome?

Answer 3

Simple organisms have less non-coding DNA

Question 4

What must be done to DNA in order to sequence the entire genome?

Answer 4

Must be broke into fragments

Question 5

What is recombinant DNA technology used for?

Answer 5

To combine genetic material from different sources

Question 6

What are organisms that contain transferred DNA known as?

Answer 6

Transgenic organisms

Question 7

What are the three ways that DNA fragments can be produced/isolating the gene?

Answer 7

1 - reverse transcriptase
2 - restriction endonuclease enzymes
3 - gene machine

Question 8

What are the 5 stages of gene cloning?

Answer 8

1- Isolation of the DNA
2 - Insertion
3 - Transformation
4 - Identification
5 - Cloning/growth

Question 9

Explain the process of using reverse transcriptase to isolate a gene

Answer 9

• Use a cell that expresses desired protein
• Since cells only contain two copies of each gene, obtaining a DNA fragment is hard so mRNA is obtained instead since many mRNA molecules can be synthesised from a gene.
• Isolate mRNA from the cell and use reverse transcriptase to convert it to cDNA (complementary DNA)
• Use DNA polymerase to produce double-stranded molecule

Question 10

Explain the process of using restriction endonucleases to isolate a gene

Answer 10

• Restriction enzymes directly cut the gene out
• They cut DNA at specific sites which are called recognition sequences or restriction sites and are palindromic
• Straight ends - cuts occur between two opposite base pairs leaving blunt ends
• Staggered ends - Cuts occur that are not opposite base pairs leaving sticky ends

Question 11

Explain the process of using a gene machine to isolate a gene

Answer 11

• The sequence for the target gene is obtained from a database.
• Nucleotides are added in the correct order to synthesise the correct base sequence.
• Protecting groups are added throughout the synthesis to make sure the correct nucleotides are added and no side branches are produced.

Question 12

What are the two different types of gene cloning

Answer 12

• In vivo - within a living organism
• In vitro - outside of a living organism

Question 13

Explain the process of inserting a gene

Answer 13

• The vector DNA (usually a plasmid) is isolated
• The vector DNA is cut open using the same restriction endonuclease as was used to isolate the gene. This means the sticky ends (if sticky) of the vector DNA are complementary to the sticky ends of the DNA fragment containing the gene
• Ligase is the enzyme that joins the gene fragment and the vector together. This process is known as ligation.
• The new combination of bases in the DNA is called recombinant DNA (rDNA)

Question 14

Explain the process of transformation

Answer 14

• The vector with the rDNA is used to transfer the genes into cells (host cells)
• The host cell taking up the vector containing the desired gene is said to be transformed

Question 15

Explain the process of identifying transformed cells

Answer 15

• Only some of the host cells will take up the vector and the desired gene.
• Marker genes can be used to identify cells which have been transformed
• The marker gene can code for antibiotic resistance so host cells are grown on agar plates containing a specific antibiotic. Only transformed cells that have the marker gene will survive and grow.
• Or the marker gene can code for fluorescence so the agar plate is places under a UV light, cells that fluoresce contain religated vectors and cells that contain desired gene will not fluoresce as the gene interrupts the second marker.
• A final way of identification is to add a substrate, if the colour does not change, then the enzyme doesn’t function meaning that the desired gene has been inserted.

Question 16

What reaction does in vitro cloning use?

Answer 16

The polymerase chain reaction (PCR)

Question 17

Explain the different stages of PCR

Answer 17

• DNA is heated to 95 degrees to break the hydrogen bonds between the DNA strands
• The mixture is then cooled to 55 degrees and primers and DNA nucleotides are added.
• Primers bind (anneal) to the ends of the strands to stop them re-joining together
• The reaction mixture is heated to 72 degrees so DNA polymerase can work and create new strands of DNA alongside the template strands.

Question 19

How many new copies of the DNA does one PCR cycle create?

Answer 19

2