3.8.2 Recombinant DNA Technology

Question 1

What are the stages of in vitro gene cloning

Answer 1

Isolation, Insertion, Transformation, Identification, Growth

Question 2

How is cDNA produced

Answer 2

Reverse transcriptase is used to covert mRNA into double stranded cDNA.

Question 3

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Question 4

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Question 5

How is cDNA different to the original nuclear gene and why might this be a good thing

Answer 5

cDNA would not contain any introns. THis is necessary if the gene is to be inserted into a prokaryote which does not have the ability to splice out introns

Question 6

State three methods that could be used to identify recombinant bacteria from non-recombinant

Answer 6

Antibiotic resistance markers, fluorescent markers, enzyme markers

Question 7

What is a restriction enzyme?

Answer 7

Enzymesused to cut double stranded DNA at a specific recognition site

Question 8

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Question 9

How are restriction enzymes useful in producing recombinant plasmids

Answer 9

If the same restriction enzyme is used to cut the plamid and target gene then 2 pieces of DNA with complementary sticky ends will be produced. The overhanging or exposed bases can join up via complementary base pairing

Question 10

How is DNA ligase used in DNA technology

Answer 10

used to join the sugar phosphate backbone after target gene and plamid anneal together

Question 11

Explain how 2 antibiotic resistance genes on one plamid can be used to identify recombinant bacteria

Answer 11

If two resistance genes are used the target gene is inserted into one of these genes. All bacteria that have taken up a plasmid will be resistant to one antibiotic (ampicillin usually) but bacteria that DONT have resistance to the second antibiotic (tetracycline usually) have taken up the recombinant plasmid. They can be identified using replica plating

Question 12

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Question 13

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Question 14

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Question 15

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Question 16

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Question 17

What is the function of PCR?

Answer 17

Used to make copies of DNA fragments, increasing the size of the sample (amplify the DNA)

Question 18

What is in the reaction mixture for PCR?

Answer 18

DNA, buffer, primers, free nucleotides, DNA polymerase

Question 19

Describe the process of PCR

Answer 19

1)95 deg C to separate the strands, 55 to anneal the primers, 72 for DNA polymerase to join the nucleotides

Question 20

What is a DNA primer

Answer 20

Short, single-stranded DNA sequence complementary to the ‘end’ of a DNA fragment

Question 21

Why are primers used in PCR

Answer 21

The DNA polymerase enzyme cannot bind to single stranded DNA but must “extend” or lengthen the

Question 22

What is genetic engineering?

Answer 22

Genes from one organism transferred into genome of another

Question 23

How are organisms genetically modified to produce drugs ?

Answer 23

The gene for the protein is isolated using restriction enzymes. The gene is copied using PCR. Copies are inserted into plasmids. Plasmids are transferred into microorganisms. the modified microorganisms are grown in large containers so they divide and produce lots of the useful protein, from the inserted gene. the protein is purified and used as a drug.

Question 24

Give an example of a drug that is produced from genetically modifided bacteria

Answer 24

human insulin (type 1 diabetes) and human blood clotting factors (haemophilia)

Question 25

What are the benefits of GMO?

Answer 25

Modify agricultural crops to give higher crops or to be more nutritious. reduces risk of famine and malnutrition. Modify crops to have pest resistance, so fewer pesticides are needed. this reduces costs and reduces any environmental probs. dont need to refrigerate vaccines produced in plant tissue. so vaccines are available to more people

Question 26

What are the risks of GMO?

Answer 26

Transmission of genetic material between plants/ could lead to herbicide resistant weeds or crops containing drugs/Long-term impacts - unforeseen consequences/Increased chemical use in plants/Wrong to genetically modify animals purely for human benefit

Question 27

State three methods that could be used to identify recombinant bacteria from non-recombinant

Answer 27

Antibiotic resistance markers, fluorescent markers, enzyme markers

Question 28

Explain why DNA must be treated with restriction enzymes before being inserted into a plasmid.

Answer 28

So the DNA and plasmid have complementary sticky ends

Question 29

Define ‘DNA probe’

Answer 29

Short, single stranded, labelled piece of DNA that is complementary to a specific sequence

Question 30

what are the 2 most common types of DNA probe

Answer 30

Radioactive (normally using 32-P) or fluorescent

Question 31

What do we mean by ‘hybridisation’?

Answer 31

When two strands of DNA from different sources join together (for example a DNA probe and a gene)

Question 32

When and why would PCR be used in genetic fingerprinting?

Answer 32

To amplify the DNA before it is digested and separated

Question 33

Name enzymes involved in genetic engineering and say how they are used

Answer 33

Restriction endonuclease - to isolate DNA, DNA ligase - inserts sections of DNA into host/plasmid, DNA polymerase - used in PCR

Question 34

What happens when an electric current is passed through an electrophoresis gel?

Answer 34

DNA fragments are negatively charged so they move towards the positive electrode at the far end of the gel

Question 35

State the first step in gel electrophoresis

Answer 35

DNA is placed into a well and covered in a buffer solution that conducts electricity

Question 36

State 3 uses of DNA fingerprinting

Answer 36

Forensic science, medical diagnosis, plant and animal breeding, determining genetic variability in a population

Question 37

Why do the small DNA fragments move further in gel electrophoresis?

Answer 37

They move faster as the current is applied

Question 38

Give stages of Genetic fingerprinting

Answer 38

Extraction, digestion, separation, hybridisation, development