Genetechnology

Question 1

Define Gene technology

Answer 1

The manipulation of an organism’s DNA to produce an organism or product that we want/need

Question 2

Define genome

Answer 2

All the genes possessed by an individual; the complete DNA of an organism

Question 3

Why do we need to sequence genomes

Answer 3

• To treat genetic illnesses/disease by looking to see if any mutations
• To look at evolutionary relatonships
• Individual DNA can be analysed to identify genes e.g. ones that may increase risk of heart disease
• Identifying genes coding from proteins to see how important they are
• Compare genomes of pathogens to see which genes cause disease

Question 4

What is DNA profiling used for

Answer 4

Used in forensic crime scene analysis and paternity testing

Question 5

What is genetic sequencing

Answer 5

• Used in research into the function of genes and regulatory DNA sequences
• Involves breaking DNA into short fragments and determining their base sequences

Question 6

What are the stages of sequencing a genome

Answer 6

1) DNA is denatured and separated into 2 strands
2) Primers are added to the DNA fragments and bind to the start of the gene with the complementary code
3) Free nucleotides are added and so are enzymes like DNA polymerase which replicates strand of DNA
4) More free nucleotides are added which either have a dye that will fluoresce when illuminated when join; different colour for 4 types of bases or a nucleotide is double deoxidised which stops the reaction when, by chance it randomly joins to the DNA
5) Results in many copies of DNA with different lengths and coloured tags
6) Mixture of lengths then are pulled along by electrophoresis; shortest move through fastest
7) Computer records sequence of colours to look at DNA base sequence

Question 7

Define primer

Answer 7

A short length of single-stranded DNA which is around 20 bases long which starts synthesis of DNA

Question 8

What does DNA polymerase do

Answer 8

An enzyme that makes complementary copies of DNA

Question 9

Define genetic engineering

Answer 9

• Using technology to change the genetic material of an organism
• Used to produce pharmaceutical chemicals

Question 10

Define recombinant DNA

Answer 10

DNA that has had DNA from a different source inserted into it (often from another species)

Question 11

What is a transformed organism

Answer 11

Transformed organism An organism that has had foreign DNA inserted into it

Question 12

What is a transgenic organism

Answer 12

An organism that has had foreign DNA inserted into it

Question 13

What is a GM organism

Answer 13

An organisms whose DNA has been modified using gene technology e.g. by the removal of some of its genes or by addition of DNA from another organism

Question 14

Describe the steps of gene transfer

Answer 14

1) Gene that is required is identified and is either cut out of chromosome or made my reverse transcription of mRNA
2) Multiple copies of gene are made using PCR technique
3) Gene is inserted into vector
4) Vector inserts gene into the cells
5) The cells that have been successfully transformed are identified and cloned

Question 15

How do you extract a gene

Answer 15

• Length of DNA containing the gene is treated with restriction enzymes
• Enzymes are made by bacteria and are used to attack and destroy DNA that has been inserted into them by viruses
• Restriction enzyme cuts the DNA staggered (not in a straight line) leaving unpaired bases on both pieces called sticky ends
• DNA probes and electrophoresis can help identify bits of DNA you need
• Multiple copies can be made by PCR

Question 16

How do you insert a gene into a vector

Answer 16

• Gene is inserted into the vector e.g. a plasmid
• To get the plasmids, the bacteria containing them can be treated with enzymes to dissolve their cell walls
• Then separated
• Circular DNA plasmid making up plasmid is cut open using restriction enzyme
• This leaves sticky ends that are complementary to those on the required gene
• Plasmid and gene are mixed together and so some plasmid’s sticky ends will pair up with the genes and some won’t
• (DNA) ligase is then used to link deoxyribose-phosphate backbone of DNA to produce closed circle of double-stranded DNA

Question 17

How do you insert the plasmid into bacteria

Answer 17

• Calcium ions are added to the mixture of plasmids and make it easier for them to take up plasmids
• Small proportion of bacteria will take up the plasmids containing the gene wanted
• To sort out which have been transformed, they use the antibiotic resistance genes in the plasmids
• Restriction enzymes may cut through some antibiotic resistant genes inactivating them
• This shows that any that have survived have taken up the plasmid
• Still don’t know whether transformed or unaltered plasmid and so sample of each colony placed on agar jelly containing the antibiotic resistant gene
• Only the ones with the working resistant gene will survive and so shows they HAVEN’T taken up the transformed plasmid and so the other colony which haven’t grown will have the transformed gene and so they are used

Question 18

What does DNA ligase do

Answer 18

(DNA) ligase An enzyme that links nucleotides together by catalysing formation of covalent bonds between deoxyribose and phosphate groups

Question 19

What is a vector

Answer 19

Something that is used to transfer DNA into the organisms to be genetically transformed e.g. plasmids and liposomes

Question 20

Define plasmid

Answer 20

• Small, circular DNA found in bacteria which act as vectors

- Often contain antibiotic resistant genes which can be transferred between bacteria which is bad for us

Question 21

Define liposome

Answer 21

A tiny ball of lipid which contains substances like DNA or protein and act as vectors and have been attempted to be used for human gene therapy

Question 22

Define restriction enzyme

Answer 22

• Enzymes produced by bacteria to destroy viral DNA entering the cell
• Used widely in gene technology to cut DNA into smaller lengths and create sticky ends

Question 23

Define sticky ends

Answer 23

• Short stretched of unpaired nucleotides at the end of a DNA molecule
• Are classed as ‘sticky’ because they can easily form hydrogen bonds with similar ends to other pieces of DNA

Question 24

What is replica plating

Answer 24

Technique used to determine which of the several colonies of bacteria have successfully taken up the plasmid with the desired gene, by testing for antibiotic resistance

Question 25

What are genetic markers

Answer 25

• Widely used one is a gene that causes fluorescence; originally gene first found in jellyfish and codes for the production of an enzyme that produces protein which causes it to fluoresce under UV light
• The gene for enzyme is inserted into plasmid and those that have taken it up are transformed organisms as will glow

Question 26

How do you produce human insulin from bacteria

Answer 26

• Approach involved extracting mRNA from the beta cells in Islets of Langerhans as are already specialised to produce insulin
• mRNA was then incubated with reverse transcriptase which makes DNA using mRNA as a template
• The single-stranded DNA then converted into double-stranded DNA which carried code for production of insulin

Question 27

What is reverse transcriptase

Answer 27

An enzyme that uses RNA to make a single-stranded molecule of complementary DNA

Question 28

What is a promoter

Answer 28

• Genes are switched on/off by promoters
• Bacteria contain many different genes which make many different proteins and so to get the gene we want, we have to insert the correct promoter for it to be switched on e.g. like the lac operon involving E.Coli

Question 29

What is electrophoresis

Answer 29

• The separation of fragments of DNA according to their lengths, by applying a voltage across them
• DNA is negatively charged and so moves to positive end with the shortest fragments moving fastest

Question 30

Describe the process of electrophoresis

Answer 30

1) A tank containing agarose gel is set up
2) A direct current is passed through the gel
3) DNA fragment=negatively charged and so move to anode); the shorter the fragments, the fast and further they travel
6) DNA drawn up
7) A DNA probe with a base sequence complementary to part of the wanted sequence is added; could be labelled with a fluorescent marker or radioactive isotope
8) DNA probe pairs up with DNA fragments on paper and so they can now be detected by shining UV light

Question 31

Define PCR

Answer 31

A method of making multiple copies of DNA molecules in a short space of time

Question 32

What are the stages of PCR

Answer 32

1) DNA is extracted with gene to be copied and heated at 95 degrees Celsius
2) Primers are added (which are complementary to nucleotides on DNA) when temp reduces to 55 degrees
3) Temperature increases to 72 degrees Celsius as is the optimum temperature for DNA polymerase to use free nucleotides to synthesise complementary DNA strands
4) From 1 double-stranded DNA molecule, 2 double-stranded DNA molecules are produced with copied gene
5) Cycle repeats again and so more copies are produced

Question 33

How is Golden Rice made

Answer 33

1) The genes, together with promoters, are inserted into plasmids
2) Plasmids inserted into bacteria called Agrobacterium tumefaciens
3) Bacteria mixed with rice embryos in Petri dishes and so some became ‘infected’ by bacteria carrying beta carotene
4) Rice embryos containing beta carotene are then grown into adult plants and these then produce sees containing beta carotene in their endosperm (part of seed which stores food for embryos)

Question 34

Why is golden rice needed

Answer 34

Helps improve diet for people in developing countries

Question 35

Give a disadvantage of using GM rice

Answer 35

• ethical concerns
• Although more Vitamin A has been produced, it’s still not enough for their diet
• concerns for future- effect unknown

Question 36

Symptoms of vitamin A deficiency

Answer 36

• Night blindness
• Weak bones
• Reduction in immune system

Question 37

Define xenotransplantation

Answer 37

The use of organs from a different species for transplant into humans

Question 38

Define gene therapy

Answer 38

Changing the DNA in some of a person’s cells e.g. to attempt to cure disease caused by faulty genes

Question 39

Describe cystic fibrosis

Answer 39

• A genetic disease caused by recessive allele of a gene that codes for a membrane protein responsible for passage of chloride ions out of cells
• Can be treated by somatic gene therapy
• Thick mucus produced in lungs and intestines

Question 40

What is the CFTR gene

Answer 40

• A protein that acts as a transporter for chloride ions
• If one of the alleles is recessive, it still means can make enough CFTR to remain healthy so gene therapy as just means need one dominant allele so can work properly

Question 41

What is somatic gene therapy

Answer 41

• Gene therapy in which body cells are transformed but not gametes
• Treatment is short-lived
• Gene IS NOT passed on to offspring
• Difficulty is getting gene into the correct target location
• Used to treat cystic fibrosis

Question 42

What is germline gene therapy

Answer 42

• Gene therapy that involves changing the DNA in cells; genetically modified cells are those which form gametes or embryonic stem cells
• Treatment is long-lived
• Gene IS passed on to offspring
• Method is illegal and ethically unacceptable in UK and US
• Effects of inserting new gene into genome is unknown

Question 43

List 3 vectors

Answer 43

• BAC (Bacterial Artificial Chromosome) which is a plasmid which infects bacteria
• Viruses- somatic and germ line gene therapy
• Lipsomes- somatic and germ line gene therapy
• Agrobacterium tumefaciens; in plants which express bacterial toxins that kill insects feeding on them
• Bacteriophages; viruses that infect bacterial cells