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biology paper 2 > 3.8.4 gene technologies > Flashcards

3.8.4 gene technologies Flashcards

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1
Q

what is the issue with recombinant DNA?

A

entire molecules of DNA are too large to be used in gene technology

molecular biologists use fragments which contain the genes or genes they are interested in

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2
Q

how is DNA isolated?

A

detergent is used to isolate the DNA from a sample as it breaks down the CM

protein may need to be removed using digestive enzymes

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3
Q

what are fragments of DNA produced by?

A
  1. conversion of mRNA to cDNA, using reverse transcriptase
  2. cutting DNA at specific palindromic recognition sequences using restriction endocleases
  3. gene machine
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4
Q

what is the process of the conversion of mRNA to cDNA, using reverse transcriptase?

A

isolate the gene = a cell that readily produces the protein you want to copy is chosen

cell will contain lots of relevant mRNA

found in specialised cells

mRNA is extracted

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5
Q

how is reverse transcriptase used in the converse of mRNA to cDNA?

A
  1. incubate with reverse tranship to synthesise cDNA strand
  2. When cDNA strand is completed, hydrolyse RNA strand
  3. incubate with DNA polymerase to synthesise second DNA strand
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6
Q

what is the process for cutting DNA at specific palindromic recognition sequences using restriction endonucleases?

A

we need to isolate the gene that is required from DNA

enzymes can be used to cut the DNA strand (restriction endonucleases)

the enzyme cuts the DNA backbone twice

different restriction enzymes cut the DNA at different points

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7
Q

what are the three types of restriction enzymes that produce different sticky ends?

A
  1. EcoRI = Ecoli
  2. Hindlll = H. influenzae
  3. Bam H1 = B. amyloliquefaciens
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8
Q

what is the process of a gene machine?

A

if the primary structure of a protein is known, then it is possible to synthesise the gene

amino acid sequence is entered into a computer

triplet code for each amino acid is used to work out DNA sequence

computer controls the machine and required DNA is made

no introns = genes can be transcribed and translated in prokaryotic cells

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9
Q

what is recombinant technology?

A
  1. multiple copies of the desired gene are produced
  2. the gene is inserted into a vector and transferred into host cells
  3. the host cells have successfully taken up the gene, identified using a marker
  4. the host cells are allowed to multiply/cloned
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10
Q

what are promoter and terminator regions?

A

additional lengths of DNA are added

DNA polymerase must attach to the promoter region of a gene, along with a transcriptional factor

promoter region must be added for transcription to take place

terminator region needs to be added to DNA fragment to stop transcription

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11
Q

how is DNA inserted into a vector?

A

Once an appropriate fragment of DNA has been cut from the rest of the DNA and the promoter and terminator regions added, the next task is to join it into a carrying unit, known as a vector.

This vector is used to transport the DNA into the host cell.

There are different types of vector but the most commonly used is the plasmid. Plasmids are circular lengths of DNA, found in bacteria, which are separate from the main bacterial DNA. Plasmids almost always contain genes for antibiotic resistance, and restriction endonucleases are used at one of these antibiotic-resistance genes to break the plasmid loop.

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12
Q

describe viruses as vectors

A

transfer genetic material into host cells, as they need to host viral proteins to allow replication

genetic material causes virulence must be removed from virus
desired gene can be added into viral genome

virus inflects the target cells, inserting its genome

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13
Q

how is DNA identified?

A

if a restriction enzyme is used, antibiotic-resistance genes can be used as markers

ampicillin is not affected by the new gene

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14
Q

what are enzyme markers?

A

gene marker is the gene that produces lactase
lactase is present, it turns a substrate blue

required gene can be transplanted into the lactase gene, using a specific restriction endonuclease

if successful the substrate will not turn blue when colonies are present

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15
Q

what factors does genetic modification increase?

A

yield
nutrient content
resistance to disease/pests
tolerant to herbicide
tolerant to environment eg temperature

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16
Q

what are genetically modified microorganisms?

A

antibiotics: produced very quickly by bacteria, not increased quality

hormones: insulin is made by bacteria

enzymes: food industry eg amalyse for beer production

gene is inserted (complimentary) to gene

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17
Q

what are the benefits of modifications?

A

herbicide resistance
disease resistance
pest resistance
plants that produce plastics

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18
Q

what are genetically modified animals?

A

disease resistance between animals

fast growing animus

production of rare proteins for medicine

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19
Q

what are the advantages of genetic engineering?

A

Bacteria can make human medicines and hormones

Improve the growth rates of plants and animals

Improve the food value of crops

Reduce the fat levels in meat

Produce plants that make their own pesticide chemicals

Crop plants give off a blue light so farmers know when to spray with pesticides

Possible cures for genetic diseases

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20
Q

what are the disadvantages of genetic engineering?

A

Insects may become pesticide-resistant if they eat a constant diet of pesticide-forming plants

Effect on human health of GM unknown

Genes from GM plants might spread to wildlife/environment

GM crops are often infertile so farmers would have to buy new seeds each year

People may want to manipulate the genes of their own children

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21
Q

what is gene therapy?

A

treatment of a genetic disease by providing the sufferer of a corrected copy of the defected gene

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22
Q

what is transfaction?

A

inserting a corrected gene into a cell

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23
Q

what is somatic cell therapy?

A

copies of the corrected gene are inserted directly into the somatic or body cell to the sufferers

does not prevent the disease from occurring in the next generation

doesn’t affect sperm and egg cells

has to be replicated many times as the effects do not last very long

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24
Q

what is germ line therapy?

A

the corrected gene is inserted into a fertilised egg produced via IVF

all cells of the embryo will contain the corrected gene when divided by mitosis

permanent and ensure offspring inherit corrected gene

illegal

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25
Q

how is cystic fibrosis related to gene therapy?

A

caused by a mutant recessive allele

caused by a deletion mutation of 3 bases (AAA) in CFTR gene which encodes the CFTR protein

role of the CFTR is to transport CL- ions across epithelial cell membranes
water follows by osmosis

CFTR is non-functional so water is retained, making membranes dry

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26
Q

what vector are used in gene therapy?

A
  1. harmless virus: adenovirus/ adeno associated virus
  2. liposomes
27
Q

describe adenovirus (as a vector)

A

viral vector

infect both dividing and non-dividing cells very effectively.
possible to target specific cells by engineering proteins on the virus surface

maximum length of DNA that can be inserted is 7,500 base pairs

genes activated in the nucleus

does not integrate into the host cell’s genome

causes an immune response in the patient

28
Q

describe liposomes (as a vector)

A

viral vector

not specific to any type of cell and entire cells far less effectively compared to others

no maximum DNA insertion length

DNA plasmid is transported to the cells nucleus but does not integrate into the cells genome

does not generate an immune response

29
Q

how are liposomes used as vectors?

A
  1. normal CFTR gene is isolated from human cells and inserted into plasmids
  2. recombinant plasmids are placed into bacteria - gene markers pick up cells that contain plasmids with CFTR gene
  3. bacteria is cloned
  4. plasmids are extracted and wrapped in lipid molecules (forming a liposome)
  5. liposomes are sprayed into patients airways via a nasal aerosol
  6. liposomes pass easily through the plasma membrane of cells and nucleus
30
Q

what are the limitations of gene therapy?

A

new technology - lots of potential but there has only been limited success

liposomes may not be small enough to pass into the lung through bronchioles

poor expression of CFTR gene

adenoviruses may cause infection

adenoviruses may trigger immune response/patients may develop immunity

patients have to undergo multiple rounds of gene therapy as short lived

31
Q

describe gene therapy as a treatment for SCID

A

normal ADA has been trialed to treat SCID

ADA gene therapy was trialled using retroviruses which transfect the patients WBC

trails have shown success but there is an increased risk of leukaemia

32
Q

what are polymerase chain reactions (PCR)?

A

technique for the amplification of DNA

in vitro = outside the body eg IVF

invented by Kary B.Mullis

Mullis conceptualised in 1983, partly due to a creative experience one weekend with a luminous racoon

without PCR, there would be insufficient DNA for the human genome project

33
Q

what are the 3 features of a PCR?

A

primer x2 = piece of single stranded DNA which is complimentary to the specific target sequence at the 3’ end of each DNA replicated strand)

nucleotides (free to make amplified DNA)

tag polymerase (stable at high temperatures)

DNA

34
Q

describe the PCR process

A
  1. DNA is heated 94-96 degrees
    - hydrogen bonds break between chains
    - separates into two strands
  2. Mixture cooled to 50-65 degrees
    - allows primers to ANNEAL to each 3’ end of each strand
  3. Heated to 72 degrees for DNA polymerase to attach nucleotides
    - heat tolerant DNA polymerase then replicates the region of DNA
    - takes longer for polymerisation of nucleotides
  4. Repeated cycles of heating and cooling amplify this region of DNA by thermal cycler (around 30 times)
35
Q

describe in vitro technology

A

Speed - 100 billion copies in a few hours

Valuable when only a small amount of DNA is available

Does not require living cells

Only makes the fragment

Requires a VERY pure sample or there will be contamination

More likely for errors to occur (20% at the start).
Which will then be amplified

Focus on whole sample

Only makes the fragment

36
Q

describe in vivo technology

A

Many days or weeks to produce the same amount

More intensive (culturing, time, effort)

When we want to introduce to a new organism e.g. gene therapy

No risk of contamination (same restriction endonuclease)

VERY Accurate

Can focus on one gene

Produces transformed bacteria - used in large quantities of gene products e.g. insulin

37
Q

what are probes?

A

DNA probes are short, single stranded fragments of DNA that is complimentary to a specific sequence

DNA florescent labelling allows detection

38
Q

what are DNA arrays?

A

an array is an ordered arrangement

arrays of DNA probes are used to detect the presence of specific sequences in samples of DNA

can be used in the detection the gene in cystic fibrosis

39
Q

what are the medical uses for PCR, DNA probes and DNA arrays?

A

by screening a cell sample from a patient for:

  • presence/abscence for a particular sequence
  • diagnosis of genetic disease status

risk of disease onset can be made

DNA profiling allows the identification of individuals through comparison of regions of the genome

40
Q

what is the forensic use of PCR technology?

A

in non-coding uses, there are highly repetitive sequences which are unique to each individual

scattered across the whole genome

allows the identification of individuals through comparison of regions of the genome

41
Q

describe the DNA profiling process

A
  1. Biological matter
  2. Isolate Nuclei
  3. Isolate and purify DNA
  4. Determine quantity and quality (yield gel)
  5. Digest DNA with restrictive enzyme
  6. Test gel = to determine completeness of digestion
  7. Separate DNA fragments by gel electrophoresis
  8. Southern transfer of DNA onto nylon membrane
  9. Hybridisation to labelled DNA probes
  10. Wash membranes
  11. Autoradiography of DNA patterns
  12. Visual and computer analysis of DNA probes
42
Q

what are probes and hybridisation?

A

a DNA probe is a short-stranded section of complementary DNA that has a label attached to it

radio labelled with 32P and identified with a photographic plate

fluorescently labelled and emits light

43
Q

why is genetic screening important?

A

Many genetic disorders are the result of a mutation

If the mutation occurs in the dominant allele, all individuals who possesses this allele will have the disorder

if recessive, individuals that are homozygous will exhibit the disorder

heterozygous will be carriers

important to screen individuals who have a family history because they may possess a mutant allele

once screened the probability of having children with the disorder can be determined

44
Q

why do we screen for oncogenes?

A

screening can be used to detect mutations that cause cancer

screening can be helpful at identifying people at risk and therefore ensure they cut down on mutagens

45
Q

describe the process of genetic screening

A

1 The order of nucleotides on the mutated gene is determined by DNA sequencing. Genetic libraries now store the DNA sequences of many of the genes responsible for common genetic diseases.

2 Fragment of DNA with complementary bases to the mutated portion of the gen is produced.

3 DNA probe is formed by radioactively labelling the DNA fragment.

4 PCR techniques are used to produce multiple copies of the DNA probe.

5 Probe is added to single-stranded
DNA fragments from the person being screened.

6 If the donor has the mutated gene, some donor DNA fragments will have a nucleotide sequence that is complementary to the probe and the probe will bind to its complementary bases on the donor DNA.

7 These DNA fragments will now be labelled with the probe and can be distinguished from the rest of the DNA fragments by the use of X-ray film.

8 If complementary fragments are present, the DNA probe will be taken up and the X-ray film will be exposed.

9 If complementary fragments are not present, the DNA probe will not be taken up and the X-ray film will be unexposed.

46
Q

what is genetic screening?

A

a form of social work where advice is given to parents to enable them to make person decisions about themselves

family history of a genetic disease is often used to advise parents of the likelihood of their children having the disease

allows lifestyle changes, diagnosis and treatment

47
Q

what factors affect cancer diagnosis and treatment?

A

oncogene mutations = determines the type of cancer, so can advise the best type of drug/radiotherapy

gene changes = predicts whether patients are more likely to benefit from certain treatments

mutations of the tumour suppressor alleles = can advise on lifestyle changes to reduce cancer risk

48
Q

what does personalised medicine rely on?

A

uses genotype to provide advice and healthcare

genes can affect the efficacy of drugs

saves money by not prescribing ineffective drugs
eg some painkillers need a specific enzyme

49
Q

how is diabetes treated through personalised medicine?

A

vitamin E reduces the risk of CVD for some genotypes but increases the risk for others

could be given to those who it reduces the risk for

50
Q

what is genetic fingerprinting?

A

a technique used by scientists which is based on the fact DNA of every individual is unique
identical twins is an exception

99% of human DNA does not code for any characteristics

51
Q

what are introns and exons?

A

introns:
non coding regions
contain blocks of repeated nucleotides called core sequences

exons:
regions of chromosomes that code for proteins

52
Q

what are variable number tandem repeats?

A

number of times that core sequences are repeated to produce the variations in individuals

more closely related the two individuals are the more similar the core sequences

53
Q

describe stage 1 of genetic fingerprinting

A

EXTRACTION

DNA is extracted from a sample (blood, hair, semen, skin)

if only a small amount of DNA is available it can be amplified using PCR
or
cells broken down to release DNA

54
Q

describe stage 2 of genetic fingerprinting

A

DIGESTION

DNA is cut into millions of small fragments using restriction endonucleases

restriction endonucleases are chosen for their ability to cut close to but not within the core sequence

sections of DNA that are cut out = restriction fragments

DNA yield thousands of restriction fragments of different sizes because the base sequence being cut may be far apart/close together

55
Q

describe stage 3 of genetic fingerprinting

A

SEPERATION

fragments are separated on the basis of size using gel electrophoresis

DNA fragments are injected into wells and an electrical current is applied along the gel

DNA is negatively charged so is attracted to the positive end of the gel

the smaller the fragment the faster it moves

gel is immersed in alkali in order to separate the double strands into single strands

the pattern of fragments are transferred to a nylon membrane by southern blotting

56
Q

what is southern blotting?

A

a thin nylon membrane is laid over the gel

membrane is covered with several sheets of absorbent paper, which is drawn up by the liquid containing DNA by capillary action
this transfers the DNA fragments to the nylon membrane in precisely the same relative positions they occupied on the gel

DNA fragments are then fixed to the membrane using UV light

57
Q

describe stage 4 of genetic fingerprinting

A

HYBRIDISATION

radioactive probes are used to attach to the VNTRs (hybridisation)

probes have base sequences which are complimentary to the core sequences

any probes not bound are washed off

membrane is dried

58
Q

describe stage 5 of genetic fingerprinting

A

DEVELOPMENT

the nylon sheet is placed under Xray film

the radioactive probes on the DNA fragment expose the film
produces visible pattern of light and dark bands which is unique to every individual

the pattern of fragment distribution is then analysed
patterns of the band is unique to every individual

59
Q

describe stage 6 of genetic fingerprinting

A

ANALYSISATION

the results can be analysed and interpreted

genetic fingerprinting is used to solve crimes and medical problems

DNA profile of each individual is highly specific
chances of two people having exactly the same DNA profile is 30,000 million to 1

60
Q

why is genetic fingerprinting used to solve crimes?

A

the pattern of the DNA profile is compared with those of the victim and suspect

if the profile matches the suspect it provides strong evidence that the suspect was present at the crime scene
may have been left at another time

if the profile doesn’t match the suspect then that suspect may be eliminated from enquiry

61
Q

why is genetic fingerprinting used to solve medical problems?

A

DNA profiles can be used to determine whether a particular person is the parent of a child
child’s paternity (father) and maternity (mother) can be determined

information can be used in:
paternity suits
inheritance cases
immigration cases

62
Q

how does genetic fingerprinting show genetic variability within a population?

A

A population, whose members have very similar genetic fingerprints has little genetic diversity

A population, whose members have a greater variety of genetic fingerprints has greater genetic diversity

63
Q

How is genetic fingerprinting used to give a medical diagnosis?

A

eg Huntinton’s

Results from ACG repetition at one end of a gene on chromosome 4

<30 reports unlikely

38-49 repeats almost certain

50+ early onset

64
Q

how can genetic fingerprinting be used in plant and animal breeding?

A

prevent inbreeding (farms and zoos)

highlights desirable characteristics

paternity - establishing degree

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