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13 Biology > 3.8.4 rDNA technology, probes and genetic fingerprinting > Flashcards

3.8.4 rDNA technology, probes and genetic fingerprinting Flashcards

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1
Q

What is meant by the term ‘recombinant DNA technology’?

A

transferring DNA fragments from one organism or species to another.

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2
Q

Why does recombinant DNA technology work so well?

A

the genetic code is universal as are the processes transcription and translation mechanisms.

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3
Q

What does universal mean?

A

all organisms have the same amino acid coded for by the same DNA codon (triplet code).

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4
Q

How are DNA fragments obtained?

A
  • conversion of mRNA to DNA (cDNA) using reverse transcriptase.
  • cut out of DNA using restriction enzymes.
  • creating a new gene in a ‘gene macnine’.
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5
Q

Via which two methods can DNA be amplified?

A

in vivo and in vitro

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6
Q

What does in vivo mean?

A

carried out inside a living body

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7
Q

What does in vitro mean?

A

carried out outside of a living body e.g. in a test tube

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8
Q

What does PCR stand for?

A

polymerase chain reaction

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9
Q

Is PCR in vivo or in vitro?

A

in vitro

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10
Q

When obtaining DNA fragments using restriction enzymes what must be added to the start and end of the desired gene?

A

promoter and terminator regions

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11
Q

When obtaining DNA fragments using restriction enzymes what is the name of the item that transports the desired gene into the host cell?

A

vector (plasmids for bacterial host cells)

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12
Q

When obtaining DNA fragments using restriction enzymes what enzyme must be used to bind the phosphate-sugar framework of the desired gene into the DNA (preferable with ‘sticky ends’)?

A

DNA ligase

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13
Q

What is the name given to DNA which consists of genetic material from two different organisms?

A

recombinant DNA

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14
Q

What is the name given to an organism which contains genetic material from two different organisms?

A

GMO (genetically modified organism) and transgenic as it has genetic information from more than one sources.

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15
Q

What are the stages involved in making a GMO?

A
  1. isolation
  2. insertion
  3. transformation
  4. identification
  5. growth / cloning
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16
Q

When making a GMO explain what happens in stage 1. (isolation)

A

The gene is isolated using 1 of 2 methods:
1. reverse transcriptase is used to obtain DNA from mRNA
2. restriction endonuclease enzymes are used to cut the desired gene from the DNA

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17
Q

When making a GMO explain what happens in stage 2. (insertion)

A

RE’s cut at specific recognition sites to make either blunt or sticky ends. Sticky ends are better as there are bases which will H bond the bases together, as well as the phosphate-sugar connection (made by DNA ligase enzyme). Promoter and terminator bases sequences are also added to initiate start and end of the gene being transcribed. The desired gene is then inserted into a vector (most commonly a plasmid) which has had its DNA cut with the same RE so that there is a palindrome.

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18
Q

When making a GMO explain what happens in stage 3. (transformation)

A

once the desired gene has been successfully incorporated into the vector it must be reintroduced to the host cell (e.g. plasmid into bacteria). Calcium ions and temperature changes are used to make the membrane permeable to allow the vector to pass into the cell.

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19
Q

Why are all of the vectors not successfully taken up by the host cell?

A
  • only some bacterial cells take up the recombination DNA (e.g bacterial cells taking up the plasmid).
  • some vectors (e.g plasmids) have not taken up the desired gene.
  • sometimes multiple fragments join together to form their own plsmids
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20
Q

When making a GMO explain what happens in stage 4. (isolation)

A

marker genes are used to identify which vector has successfully taken up the desired gene, and which host cell has successfully taken up the vector. Markers used are anti-biotic resistance, enzyme and fluorescence.

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21
Q

How does stage 4 (identification) work with the anti-biotic resistance method?

A

replica plating:
-The cells that survived the first antibiotic have taken up the plasmid.
-Those cells are cultured by spreading them thinly on nutrient agar plates.
-Each cell on the plate will become a culture of clones.
-A small sample from each colony is transferred to another plats (the replica plate) and placed in the same position as on the original plate.
-The replica plate contains the second antibiotic against which the bacterial cells will have no resistance if they have the plasmid with the DNA fragment (as the resistance gene will have been removed).
-Colonies killed on the replica plate are the ones with the required gene.
-The same ones on the original plate are alive and can be cultures further.

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22
Q

How does stage 4 (identification) work with the enzyme method?

A

-the enzyme gene marker codes for the production of the protein enzyme lactase.
-Lactase turns a certain substance from clear to blue.
-the desired gene is placed in the centre of the lactase coding gene.
-If the plasmid with the required gene is present in the bacterial cells the colony grown from it will not produce lactase and therefore not cause the colour change to blue whilst the undesired cells will cause a colour change.

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23
Q

How does stage 4 (identification) work with the fluorescence method?

A

-A more recent and faster method is the transference from a jellyfish plasmid which produces a green fluorescent protein (GFP).
-The desired gene to be cloned is transferred to the centre of the GFP gene so any bacterial cells which have taken up the desired gene will not express the GFP so they will not fluoresce whilst the other cells without the desired gene will.
-There is no need for replica plating as the desired cells have not been harmed. All is required is to retain the cells which do not fluoresce by viewing them under a microscope.

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24
Q

Where is the reverse transcriptase used in obtaining a DNA fragment (stage 1. isolation) come from?

A

Retrovisuses are human viruses of which human immunodeficiency virus (HIV) is the best known.
They have RNA as their genetic information but they can synthesise DNA from the RNA using REVERSE TRANSCRIPTASE (RT) (an enzyme).

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25
Q

How does using reverse transcriptase to isolate a single gene work?

A

-A cell that produced the protein is selected.
(e.g. Beta-cells of the islets of Langerhans in the pancreas produce insulin)
-The mRNA is extracted from the cells.
-RT is used to make DNA from RNA. This is known as COMPLIMENTARY DNA (cDNA) as the DNA bases are complimentary to the RNA bases.
-To make the other strand of DNA the enzyme DNA POLYMERASE is used to build up the complimentary nucleotides on the cDNA template.
-The resulting double strand of DNA is the required gene!!!

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26
Q

Why do bacterial cells have restriction endonuclease enzymes naturally?

A

Bacteria are often invaded by viruses (bacteriophages) which inject foreign DNA into their cells and so the bacteria have enzymes which cut up the viral DNA. These are RESTRICTION ENDONUCLEASES (RE).
There are many different RE’s. Each cuts the DNA at a different sequence of bases called the recognition sequence.

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27
Q

What is another name for the recognition site that the restriction endonuclease cut at?

A

cleavage site

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28
Q

Once all of the stages for producing a GMO with recombinant DNA have been completed, there is only one thing left to do. Grow or clone the new organisms. If your GMO is a bacteria what must you do to ensure maximum growth?

A

Ensure the conditions for bacterial growth are optimum (e.g. suitable temperature, water availability, Oxygen and a nutrient source).

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29
Q

What is ‘nick name’ for the machine used to create genes in the laboratory?

A

the gene machine

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30
Q

Before we started to use gene technology to create new organisms, how did breeders and farmers alter their livestock?

A

selective breeding over many generations.

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31
Q

Give some uses of DNA technology

A

examples:
Increased yield of farm animals and crops.
Improved nutrient content of food.
Introducing disease and pest resistance to animals and plants.
Making crop plants herbicide tolerant.
Developing tolerance to environmental conditions.
Making vaccines.
Producing medicines and treatment of disease.

32
Q

What items/apparatus are needed to perform PCR?

A

-The DNA fragment to be copied.
-DNA polymerase to join together the nucleotides in a hot environment.
-Primers with short, set nucleotides complementary to those on the ends of the fragment to join to the ends of the DNA fragment. 15-20 bases long.
-Nucleotides of all ATC and G to bind to complementary bases.
-Thermocycler to control temperature changes during PCR.

33
Q

What temperature is needed to separate the DNA strands in PCR?

A

Heat the contents of the thermocycler to 95°C to separate the DNA strands.

34
Q

What temperature is needed to for primers to anneal in PCR?

A

Cool the thermocycler contents to 55°C. This causes the annealing primers to attach (anneal) to the start and end complementary bases on the DNA strands. (this also keeps the strands apart).

35
Q

What temperature is needed for DNA to bind new nucleotides in PCR?

A

Heat up to 72°C. This is the optimum temperature for DNA polymerase to cause new nucleotides to bind to their complementary base pairs.

36
Q

Why do we use PCR?

A

PCR is the basis of Genetic Fingerprinting, where a sample form a known source can be compared against a sample.
It allows even a tiny sample of skin, blood, a hair follicle or semen to contain enough DNA to be amplified in PCR.

37
Q

Why is a thermocycler used in PCR?

A

PCR uses a computer controlled machine which allows temperatures to be controlled and timed accurately,
The whole temperature cycle takes around 2 minutes so over 1 million copies of the DNA can be made in 25 cycles, and 100 billion can be made in a few hours.

38
Q

Where do we get thermostable enzymes from to use in PCR?

A

thermostable enzymes are from thermophilic bacteria (e.g. Thermus aquaticus) which survive in hot springs which join nucleotides together.

39
Q

What are the advantages of in vivo coning?

A

-Can add a gene to another organism via vectors and plasmids.
-Low risk of contamination as the same RE is used to cut the same codes for all DNA involved so no stray DNA is present.
-Only desired genes are cut out.
-Produces transformed bacteria to produce many products (e.g. insulin).
-No prior knowledge of bases sequences is needed.

40
Q

What are the advantages of in vitro coning?

A

-Quick. Can make billions of copies from a small sample in a few hours.
-No living cells are needed.

41
Q

What are the disadvantages of in vitro coning?

A

-Cannot add genes to other organisms.
-A very pure sample is needed as contaminants can also be multiplied giving false results.
-The whole DNA fragments are copied.
-Only magnifies DNA sample.
-Prior knowledge is needed to synthesise primers.

42
Q

What are the advantages of in vivo coning?

A

-Takes days or weeks to make billions of copies from a small sample.
-Requires living cells and growing cultures.

43
Q

What are primers and how are they used in PCR?

A

Short pieces of DNA with set bases complementary to those on DNA sample. They attach to the start and end of the DNA to provide start and stop instructions for PCR. DNA polymerase can only attach nucleotides at the end of an existing chain. Primers also keep the 2 strands apart.

44
Q

Why are 2 different primers needed in PCR?

A

The sequences at the 2 ends of the strand are different

45
Q

What bond is broken when DNA strands are separated in PCR?

A

H Bonds

46
Q

What could happen if contaminant biological material exists in the sample?

A

Any contaminant would also be copied.

47
Q

What are the two methods that can be used in gene therapy?

A

germ line therapy and somatic cell therapy

48
Q

How does germ line gene therapy work?

A

Replacing or supplementing the defective gene in the fertilised egg. This means all of the developing cells will be normal, as will the cells of the next generation. The solution is permanently but currently prohibited for ethical reasons

49
Q

How does somatic cell gene therapy work?

A

Targets the affected tissues (such as the lungs). This does not pass to the next generation as it is not in the gametes. The treatment is also short lived for the patient as the lung cells are continually dying and being replaced. The treatment therefore needs to be repeated every few days in some cases.

50
Q

Which two vectors can be used to get a healthy gene into somatic cell of a patient?

A

-Using a harmless virus
-Wrapping the gene in lipid molecules

51
Q

How might a healthy CFTR gene be delivered into a CF patients lung cells using a virus?

A

-The adenovirus is made harmless by interfering with their genes involved in replication.
-These are then grown in epithelial cells in the lab with plasmids holding the CFTR gene (which has been artificially inserted).
-The CFTR gene becomes incorporated into the adenovirus DNA.
-The adenoviruses are isolated from the epithelial cell and purified.
-Adenoviruses with the CFTR gene are introduced into the nostrils of CF patients.
-The adenovirus inject their DNA (with the CFTR gene) into the epithelial cells of the lungs.

52
Q

How might a healthy CFTR gene be delivered to lung cells of a CF patient using lipids?

A

-CFTR genes are isolated from healthy human tissue and inserted into bacterial plasmid vectors.
-The plasmid vectors are reintroduced into their bacterial host cells and gene markers are added to identify the bacterial cells which have successfully taken up the CFTR gene.
-These bacteria are cloned.
-The plasmids from the clones are extracted and -wrapped in lipid molecules to form liposomes.
-The CFTR gene carrying liposomes are sprayed into the nostrils of the patient using an aerosol and inhaled into the lungs.
-The liposomes pass across the membrane into the lung epithelial cells.

53
Q

Why might gene therapy be unsuccessful?

A

-Adenoviruses may cause infection.
-Patients may develop immunity to the adenoviruses.
-The liposome aerosol may not be fine enough to pass through the bronchioles in the lungs.
-The CFTR gene may not be expressed even when successfully delivered to the epithelial cells.

54
Q

What is a DNA probe?

A

The DNA probe is a short, single stranded section of DNA that has an identifiable label attached to it.

55
Q

What are the two types of DNA probes and how do they work?

A

-Radioactively labelled probes – made of nucleotides with the isotope 32P. A photographic plate exposed to -adioactivity is used to identify the probe.
-Fluorescently labelled probes – emit light under certain conditions.

56
Q

How do DNA probes result in a scientist being able to identify a mutant allele in a patient?

A

A probe with bases complementary to the bases on the portion of the gene we want to find is made.
The DNA being tested is treated to separate the strands.
The separated strands are mixed with the probe which binds the complementary bases on the strands. This is DNA HYBRIDISATION.
The site at which the probe binds can be identified by radiation or fluorescence emitted.

57
Q

What is the point of genetic screening?

A

Genetic screening is carried out on individuals who may carry the mutant gene, this is determined by looking at their family history.
Screening can allow a couple to find out the chances of having a child with a genetic disorder. Couple as risk can then be referred to a genetic Councillor for advice.

58
Q

If a positive gene test result reveals that a seemingly healthy individual carries or has a genetic mutation associated with a specific disorder, what would a ‘negative gene test result’ be? How about a ‘false negative gene test result’?

A

A negative gene test result reveals that the faulty gene being looked for is not there. A false negative result means that further testing is probably needed as there was a problem with the procedure.

59
Q

Why are introns (not exons) used in genetic fingerprinting?

A

Genetic fingerprinting is based on the fact that DNA the genome of any organism has many repetitive non-coding bases – INTRONS (e.g. 95% of human DNA does not code for any characteristic).
Introns contain repetitive sequences of DNA called core sequences which differ in everyone (except identical twins).

60
Q

What are the stages of DNA fingerprinting?

A

Extraction, Digestion, Separation, Hybridisation, Development

61
Q

In DNA fingerprinting what happens in Extraction?

A

extract DNA from the sample (however small) by separating it from the rest of the cell. PCR can be used to make more if necessary

62
Q

In DNA fingerprinting what happens in Digestion?

A

the DNA is cut into fragments using restriction endonucleases (chosen as they cut close to but not inside core sequences).

63
Q

In DNA fingerprinting what happens in Separation?

A

gel electrophoresis separates the DNA fragments according to size. The gel is then immersed in an alkali to separate the 2 DNA strands. The single strands are then transferred onto a nylon membrane by a technique called southern blotting

64
Q

In DNA fingerprinting what happens in Hybridisation?

A

labelled DNA probes with complementary sequences are used to bind to the core sequences. The temp and pH must be correct for binding to occur. Different probes are used to bind different core sequences.

65
Q

In DNA fingerprinting what happens in Development?

A

an X-ray film is put over the nylon membrane and the film is exposed by the radiation in the probes (fluorescence probes are seen visually). A series of bars are revealed in a pattern of bars unique to the individual.

66
Q

How does southern blotting work in the separation stage of DNA fingerprinting?

A

-A nylon membrane is laid over the gel and several sheets of absorbent paper are placed on top which draws up the liquid containing the DNA via capillary action.
-The DNA fragments are transferred to they nylon in the same position.
-The fragments are fixed in place on the membrane using UV light.

67
Q

How are genetic fingerprint results interpreted?

A

A DNA fingerprint is compared for 2 or more samples of blood from the scene of a crime for example compared to the suspect(s). They are then visually checked.
If there seems to be a match each fingerprint is passed through an automated scanning machine which calculates the length of the fragments from the bands (done via measuring the distance travelled by known lengths of DNA). Finally the odds are calculated of someone having an identical fingerprint .
The closer the match between the two samples the more likely they came from the same person.

68
Q

Give several uses of genetic fingerprints.

A

Forensic science.
Check immigration applications.
Confirm animal pedigrees / ownership.
Check evolutionary relationships.
To determine genetic variability within a population.
Paternity testing. Any bars which are not from mum will be from dad

69
Q

Which method is easier, gene supplementation or gene replacement? why?

A

Gene supplementation, as the faulty gene does not need to be removed (it may be recessive and therefore not expressed if the healthy dominant gene is present).

70
Q

What does VNTR stand for in genetic fingerprinting?

A

variable number tandem repeats

71
Q

role of DNA polymerase

A

joins nucleotides together in DNA (complementary strand)

72
Q

name 2 enzymes involved in inserting DNA fragments into plasmids

A

ligase
restriction endonucleases

73
Q

describe the role of ligase

A

joins gene/DNA into the plasmid/vector

74
Q

describe the role of restriction endonuclease when used to add a piece of DNA into a plasmid

A

cuts the plasmid

75
Q

Describe 2 features of proteins what would allow different proteins to be separated by gel electrophoresis

A
  1. mass/length of AA
  2. charge
  3. R group differs

13 Biology (25 decks)

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