Gene Technology Techniques - A2

Question 1

What is meant by recombinant DNA technology?

Answer 1

The transfer of DNA fragments from one organism to another.

Question 2

Why does recombinant DNA technology work?

Answer 2

-The genetic code is universal
-Therefore transcription and translation occur by the same mechanism
- And result in same amino acid sequence across organisms

Question 3

Explain the process of using reverse transcriptase to produce DNA fragments.

Answer 3

• mRNA (no introns) complementary to target gene is used as a template
• it is mixed with free nucleotides which match up their base pairs
• reverse transcriptase occurs which forms the sugar-phosphate backbone
• creating cDNA (complementary DNA)

Question 4

Summarise the process of using enzymes to make DNA fragments.

Answer 4

• restriction endonuclease (REs) cut DNA at specific sequences
• difference REs cut at different points but 1 RE will always cut at the same sequence
• therefore using particular REs allows you to cut certain gene of interest

Question 5

In which 2 ways can we amplify DNA fragments?

Answer 5

in vitro - polymerase chain reaction (PCR) (lab)
in vivo - using host cells (in something living)

Question 6

describe the reaction mixture in the first stage of PCR.

Answer 6

• contains the DNA fragments to be amplified
• primers that are complimentary to the start of the fragment
• free nucleotides to match the exposed bases
• DNA polymerase to create the new DNA

Question 7

Summarise the process of amplifying DNA fragments using PCR. (in vitro)

Answer 7

1. heat DNA to 95 degrees celsius
2. breaks hydrogen bonds/separates strands
3. add primers
4. add nucleotides
5. cool to 50 degrees celsius
6. too allow binding of nucleotides/primers
7. add DNA polymerase
8. heat to 75 degrees celsius
9. DNA polymerase joins nucleotides together
10. repeat cycle many times - new DNA acts as a template for next cycle

Question 8

summarise the process of inserting a DNA fragment into a vector. (in vivo)

Answer 8

• a plasmid is used as the vector
• and is cut using the same restriction enzymes as DNA so that ends are complementary
• DNA ligase join the fragments and plasmid together

Question 9

Summarise the process of inserting a vector into a host cell.

Answer 9

• known as cell transformation
• the host cells (bacteria) are mixed with vectors in ice cold solution
• then heat shocked to encourage cells to take up vectors
• the cells can then be grown and the DNA fragment will be cloned

Question 10

Summarise the process of identifying transformed cells.

Answer 10

• marker genes eg. coding for fluorescence can also be inserted into vectors along with DNA
• when cells begin to grow, UV lights can be used to identify which have taken up the vector and which have not

Question 11

How can DNA probes be used to locate specific alleles?

Answer 11

• the probe is designed so its sequence is complementary to the specific allele
• they are labelled
• amplified using PCR
• then added to a sample of single stranded DNA
• the probe will bind if the allele is present

Question 12

Give some applications of DNA probes.

Answer 12

1. to screen someone’s DNA for a particular heritage health condition
2. to identify a gene for use in genetic engineering
3. to predict how someone will respond to a drug

Question 13

What is the purpose of DNA hybridisation?

Answer 13

• to measure the degree of difference between 2 strands of DNA
• can be used to compare someone’s DNA to a certain gene to see if they have it

Question 14

Summarise the process of DNA hybridisation.

Answer 14

• one DNA strand is labelled and mixed with an unlabelled comparison strands
• the more similar the strands, the more strongly they will bind
• and more energy will be required to break the strands apart

Question 15

What are the benefits of genetic profiling?

Answer 15

• can identify heritage diseases very early
• so can begin to treat them before symptoms develop, reducing impact on individual
• treatment can also be personalised so more chance of being effective

Question 16

What is genetic fingerprinting?

Answer 16

a technique used to compare 2 DNA samples and determine whether they came from the same individual

Question 17

How does genetic fingerprinting work?

Answer 17

• every organisms genome contains non-coding regions called variable number tandem repeats (VNTR)
• the probability of 2 people having the same VNTR is very low
• so we can compare these areas and see if the 2 DNA samples came from the same person

Question 18

Summarise the process of genetic fingerprinting analysis.

Answer 18

• DNA extracted from sample
• DNA cut into segments using restriction endonuclease
• must leave mini satellites intact
• DNA fragments separated using electrophoresis
• mixture put on wells in gel and electric current passed through
• immerse gel in alkaline solution / 2 DNA strands separated
• southern blotting / absorbent paper to absorb DNA
• DNA fixed to nylon/membrane using UV light
• radioactive marker / probe added - complementary to mini satellites
• areas with probe identified using x-ray film

Question 19

Outline a method for in vivo cell cloning.

Answer 19

1. cut desired gene from DNA of desired organism
2. using restriction endonuclease
3. make artificial DNA with correct sequence of bases
4. using DNA polymerase
5. cut plasmids open
6. with same restriction endonuclease
7. sticky ends/unpaired bases attach
8. use DNA ligase to join
9. return plasmids to bacterial cells

Question 20

How does gel electrophoresis work?

Answer 20

• DNA fragments are placed at one end of a slab of gel
• an electrical current is applied causing the DNA fragments to move to the other end of the gel
• shorted fragments travel further
• the pattern of bands created is unique to every individual

Question 21

Give some applications of genetic fingerprinting.

Answer 21

1. forensics - to identify suspects
2. medical diagnosis - to identify type of haemoglobin produces by an individual to diagnose sickle cell anaemia
3. animals and plant breeding eg. breed out harmful alleles, ensure pedigree

Question 22

How can target genes be isolated using restriction enzymes?

Answer 22

-DNA contains specific pallindromic sites called restriction sites
-Restriction enzymes can cut DNA at these restriction sites (active site complementary to specific base sequence of DNA)
-If there is is a restriction site either side of the target gene
-Restriction enzymes can be used to cut the target gene out
-Restriction enzymes leave the DNA with sticky ends (unpaired bases)

Question 23

How does genetic engineering occur?

Answer 23

1. DNA extracted from donor cell and cut into fragments by restriction endonuclease
2. fragments separated using gel electrophoresis
3. DNA fragment containing gene for insertion is identified using DNA probe
4. vector (plasmid) extracted from cell using same restriction endonucleases
5. bacterial plasmids and DNA fragments are mixed
6. some plasmids take up foreign DNA fragment
7. DNA ligase used to join plasmid and DNA fragment together forming recombinant plasmid
8. plasmids are mixed with bacteria, some bacteria take up by RP
9. these bacteria are selected and cultured to produce many bacteria containing RP-DNA
10. using marker gene such as antibiotic resistance
11. bacteria expresses new gene and protein is extracted

Question 24

Benefits of genetic engineering.

Answer 24

Agriculture - crops can be GE to give higher yield, more nutrients and pest resistance + resistance to higher temps and drought - helps meet growing populations food demands
Industry - GE organisms produced to make large n. of enzymes quickly and cheaply for industrial processes
Medicine - GE organisms can make large quantities of drugs or vaccines quickly and cheaply

Question 25

Concerns of genetic engineering.

Answer 25

1. use of antibiotic resistance marker genes within GE could lead to transfer of these genes to pathogens 2. inserting new genes into plants could alter normal genes function and create toxic products within GE food sources 3. introducing herbicide resistance genes to crop plants could transfer to wild species when interbreeding, producing weeds resistant to herbicides

Question 26

Gene therapy.

Answer 26

Insert a gene of functioning protein into the cells of person with genetic disease

Question 27

What is Somatic gene therapy?

Answer 27

- DNA transfers to our normal body tissue - cannot be inherited - mutation in tumour only eg. breast however - not all cells take up new DNA/express allele - only some tissue types accessible and need to be repeated (when cells replace themselves, faulty DNA back) - body can produce immune response to vector

Question 28

Germ line gene therapy.

Answer 28

-DNA transferred to sex cells (sperm and egg) - allows correction of disease causing mutation that are certain to be passed on (passes on to offspring) however - effects on gene transfer is unpredictable (could defect the embryo) - denial of human rights, offspring would have no say in whether their genetic material would be modified - potential abuse - could enhance favourable characteristics and supress others - could result in eugenics (manipulation of genetic properties of a population).

Question 29

Why are primers added in PCR?

Answer 29

- to prevent strands from re-joining each other - create starting point for DNA polymerase to add new DNA nucleotides

Question 30

What is meant by agglutination?

Answer 30

antibody sticks to antigen

Question 31

Describe how bacteria containing the insulin gene are used to obtain sufficient insulin for commercial use?

Answer 31

- use of ferments - provides nutrients plus suitable conditions for optimum growth - reproduction of bacteria - insulin accumulates and is extracted

Question 32

Give 2 advantages of using a gene therapy.

Answer 32

1. can target specific cells 2. can replicate into cells