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Biology > 21. rDNA TECHNOLOGY > Flashcards

21. rDNA TECHNOLOGY Flashcards

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1
Q

Define what recombinant DNA is

A

Recombinant DNA contains DNA from two types of organisms

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2
Q

Which feature of DNA means an organism from one species will be able to transcribe and translate DNA from a different species?

A

DNA is universal

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3
Q

Describe the three ways the desired gene can be isolated

A

Using restriction endonucleases to cut existing DNA, using reverse transcriptase to convert mRNA into cDNA, and production of the gene using a gene machine

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4
Q

What are restriction endonucleases?

A

Enzymes that cut DNA at specific restriction sequences / recognition sites

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5
Q

Why would two different restriction endonucleases be required to isolate the desired gene from an existing genome?

A

Because the restriction site at the beginning and the end of the gene would be different

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6
Q

Restriction endonucleases leave sticky ends. What are sticky ends?

A

Sticky ends are short sequences of bases on the end of DNA that are not bonded to their complementary base pair

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7
Q

Why can you not isolate a gene to be inserted into a bacteria using restriction endonucleases?

A

Because the gene would include the introns, and bacteria cannot splice out introns.

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8
Q

What is reverse transcriptase?

A

An enzyme that produces DNA from mRNA

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9
Q

If using reverse transcriptase to produce DNA, which cells would you choose to use?

A

Cells already translating the protein, as they should contain lots of the mRNA for that gene.

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10
Q

Why is it better to isolate a gene to be inserted into bacteria using reverse transcriptase rather than restriction endonucleases?

A

Because mRNA does not contain introns, therefore the gene produced from it will not contain introns either

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11
Q

What is a gene machine?

A

A computer that can reverse engineer a gene from a known protein

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12
Q

Describe a benefit of isolating a gene using a gene machine

A

It’s faster

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13
Q

Which process is used to amplify (make more copies of) the isolated gene?

A

PCR (Polymerase Chain Reaction)

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14
Q

Describe the function of PCR

A

PCR is used to increase the amount of DNA when there is only a small amount in a sample

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15
Q

Apart from amplifying gene fragments for rDNA, describe another use of PCR

A

To amplify DNA from a crime scene to ensure there is enough to carry out genetic fingerprinting

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16
Q

Which type of cloning is PCR an example of?

A

In vitro

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17
Q

List the ingredients required for PCR

A

Sample of DNA to be amplified, primers, DNA Polymerase, DNA nucleotides

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18
Q

Name the machine that PCR is carried out in

A

A thermocycler

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19
Q

What are primers?

A

Short fragments of single-stranded DNA which have a specific base sequence which is complementary to a section of the gene to be amplified / copied

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20
Q

Describe the function of primers

A

They allow DNA polymerase to attach to and copy the correct gene

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21
Q

Why are two different primers required for PCR?

A

Because the sequences at the beginning and end of the gene are different

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22
Q

Describe the function of the DNA nucleotides in PCR

A

To form the new DNA strands

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23
Q

Why is the temperature of the thermocycler increased to 95 degrees?

A

To break the hydrogen bonds between the complementary base pairs. This separates the strands to produce single strands.

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24
Q

After the primers and nucleotides are added, the thermocycler is cooled down to 70 degrees. Explain why.

A

To allow the reformation of the hydrogen bonds, so the primers and free nucleotides can bond to their complementary base pairs

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25
Q

Describe the function of DNA polymerase

A

To catalyse the formation of phosphodiester bonds between adjacent nucleotides to produce a complementary strand of DNA

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26
Q

Why is a heat stable version of the enzymes used?

A

So they don’t denature at high temperatures

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27
Q

Why might the number of DNA molecules produced from PCR plateau, even though the thermocycler is still completing cycles?

A

The free nucleotides are used up, so no new DNA strands can be made

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28
Q

Which type of cloning is rDNA an example of?

A

In vivo

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29
Q

What is a vector?

A

Vectors are carriers of DNA into a host cell

30
Q

What are most commonly used as vectors to insert the desired gene into the host cell?

A

Plasmids

31
Q

When inserting the desired gene into a plasmid, why should the plasmid and the gene be cut with the same restriction endonucleases?

A

To ensure the gene and the vector (plasmid) have the same sticky ends that are complementary

32
Q

Describe the function of DNA ligase in the insertion of the desired gene into the plasmid

A

DNA ligase joins the complementary sticky ends of the vector and the gene by forming phosphodiester bonds

33
Q

Apart from the desired gene, what two other pieces of DNA might be added to the plasmid?

A

Promoters and markers

34
Q

Describe the function of promoters

A

Promoters ensure the protein is only translated in certain target cells

35
Q

When are promoters used?

A

When the rDNA is being inserted into a multicellular organism that contains different types of specialised cells.

36
Q

Explain why its important to ensure the desired gene is only expressed in target cells?

A

Translation of the gene in non-target cells could be dangerous

37
Q

Describe the function of markers

A

Markers are used to identify which host cells have been transformed

38
Q

Explain why its necessary to use markers to identify transformed cells

A

Not all plasmids will take up the desired gene, and not all host cells will take up a plasmid

39
Q

Describe the three possible outcomes of the mixing of plasmids and the desired gene

A

The desired gene is taken up into the plasmid.
The plasmid closes up without the desired gene being incorporated.
The gene fragments join together without being incorporated into a plasmid.

40
Q

Name the three most commonly used markers

A

A gene that codes for a GFP (green fluorescent protein)
A gene that codes for antibiotic resistance
A gene that codes for an enzyme

41
Q

Why is using the GFP gene as a marker efficient?

A

Because the transformed bacteria can be identified quickly using the UV light

42
Q

How would transformed bacteria be identified if the antibiotic gene marker was used?

A

The bacteria would be plated on agar containing the bacteria. The cells that don’t die are the transformed cells

43
Q

If the desired gene is being inserted into a multicellular organism, how would you ensure all the cells of the organism contain the gene?

A

Insert it into embryonic cells. Then when these divide by mitosis to produce genetically identical cells, they will contain the desired gene (as its also replicated)

44
Q

Probes can be used to identify if certain alleles exist in a persons’ genomes. Give three reasons why someone might want to see if their genome contains a certain allele.

A

To find out if they (or their partner) were a carrier of a genetic disease.
To find out if they have an oncogene.
To find out if they have an allele that would increase their chances of developing a genetic condition later in life.

45
Q

What is a probe?

A

A piece of single stranded DNA with bases complementary to a known allele

46
Q

What are the two ways probes can be labelled?

A

Under UV light. The sample will glow if the probe is present.

47
Q

How are radioactive probes labelled?

A

By using the sample to expose an X-Ray film. Dark bands will appear if the probe is present.

48
Q

What must be the bases on the probe be complementary to?

A

The allele you are trying to detect

49
Q

How are many copies of the probe produced?

A

PCR

50
Q

Why is the patient’s DNA heated?

A

To break the hydrogen bonds between complementary base pairs to produce single strands

51
Q

After the probes are added, why is the temperature reduced?

A

To allow the formation of hydrogen bonds between the probe and the allele on one strand of the patient’s DNA (if it exists)

52
Q

Why is the mixture washed?

A

To remove any unbonded probes

53
Q

A probe is used that is complementary to a dominant allele. How many probes should bind to a heterozygous person’s DNA compared to a homozygous dominant person’s DNA?

A

Half the amount of probe should bind to a heterozygous person’s DNA compared to a homozygous dominant person’s DNA

54
Q

What is gel electrolphoresis?

A

Gel electrophoresis is a method used to separate DNA fragments according to their size

55
Q

Describe three uses of gel electrophoresis

A

Identify the presence of a known gene in a genome.
Identify the strain of pathogen infecting a patient.
Genetic fingerprinting.

56
Q

Describe three uses of genetic fingerprinting

A

Paternity testing.
Identifying suspects in a crime.
To reduce inbreeding when breeding captive animals.

57
Q

What is used to cut the DNA sample before separation?

A

Restriction endonucleases

58
Q

What is passed over the DNA sample after it’s put into the wells in the gel?

A

An electric current

59
Q

Describe and explain how the DNA fragments will separate

A

The DNA fragments will separate according to size. This is because the negative DNA moves towards the positive electrode. Smaller pieces of DNA move further.

60
Q

What is added to the gel to make the DNA single stranded?

A

An alkali

61
Q

What is the DNA sample transferred onto?

A

A sheet of nylon

62
Q

Why is the DNA made to be single stranded?

A

So when the probes are added, they will bind to the DNA by complementary base pairing

63
Q

Genetic fingerprinting is carried out by analysing the number and size of what type of DNA in genomes?

A

VNTRs

64
Q

What are VNTRs?

A

Variable Number Tandem Repeats. They are repeats of short sections of bases that are found in the introns of DNA.

65
Q

When analysing how closely related two people are, why should you cut their DNA using the same restriction endonucleases?

A

So their DNA is cut at the same points (at the beginning of the same VNTRs)

66
Q

What will the probes used to visualise their DNA be complementary to?

A

The VNTRs

67
Q

How is relatedness ascertained using genetic fingerprinting?

A

The more similar the pattern (the more bands in common), the more closely related the two individuals are.

68
Q

How can paternity be decided using genetic fingerprinting?

A

50% of the child’s genetic fingerprint bands should be the same as the mother’s and the other 50% should be the same as the father’s.

69
Q

How can a suspect be identified from the DNA left at a crime scene?

A

The genetic fingerprint of the suspect should exactly match that found at the crime scene.

70
Q

How can genetic fingerprinting be used to reduce inbreeding when breeding captive animals in zoos?

A

The individuals should be as distantly related as possible, so their genetic fingerprints should have the fewest bands in common as possible.

Biology (26 decks)

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