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A2 Biology > Topic 8B - Genome projects and Gene technologies > Flashcards

Topic 8B - Genome projects and Gene technologies Flashcards

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1
Q

What have improvements in technology allowed us to do? (in regards to genomes)

A

They have allowed us to sequence the genomes of a variety of organisms, from bacteria to humans.

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2
Q

What is the only condition when using gene sequencing methods?

A

They can only be used on FRAGMENTS of DNA so if you want to sequence the entire genome of an organism, you must chop it up into smaller pieces first. The smaller pieces would be sequenced and then put back in order to give the sequence of the whole genome.

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3
Q

What is the human genome project?

A

A major sequencing project, completed in 2003, that mapped the entire sequence of the human genome for the first time.

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4
Q

Why is it easy to determine the proteome of simple organisms such as bacteria?

A

Bacteria doesn’t contain much non-coding DNA, so much of the DNA would code for proteins. This means by reading the DNA sequence of the bacteria’s genome, we can determine the proteins they make.

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5
Q

How is determining the proteome of bacteria useful?

A

It is useful in medical research and development e.g. It allows us to identify the protein antigens of disease-causing bacteria/viruses, and this can help in the development of vaccines.

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6
Q

Why is it harder to determine the proteome of complex organisms?

A

More complex organisms contain larger sections of non-coding DNA and regulatory genes, which determine when the genes that code for particular proteins are switched on and off.
- This makes it difficult to find the bits that code for proteins among the non-coding and regulatory DNA.

Nonetheless, work is being done on the human proteome. The codes for more than 30,000 human proteins have been identified so far.

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7
Q

Describe how sequencing methods have evolved since the past?

A

In the past, they used to be labour-intensive, expensive and could only be done on a small-scale. Now the techniques are automated, more cost-effective and could be done on a large-scale.

E.g. pyrosequencing is a recently developed technique that can sequence around 400 million bases in a 10 hour period (which is super fast compared to older techniques). Techniques such as this allows scientists to sequence whole genomes much more quickly.

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8
Q

What does recombinant DNA technology involve?

A

It involves transferring a fragment of DNA from 1 organism to another.

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9
Q

Why can transferred DNA still code for a protein?

A

Because the genetic code is universal and transcription and translation mechanisms are similar in all living things

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10
Q

What do you call organisms that contain transferred DNA?

A

transgenic organisms

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11
Q

What are the 3 main ways in which DNA fragments can be made?

A
  1. Using Reverse Transcriptase
  2. Using Restriction Endonuclease Enzymes
  3. Using gene machines
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12
Q

How does reverse transcriptase work?

A
  1. Most cells only have 2 copies of a gene, making it hard to obtain a fragment containing target gene. But they can contain mRNA molecules that are complementary to the gene. so mRNA is easier to get.
  2. mRNA used as templates to make lots of DNA. Reverse Transcriptase makes DNA from RNA template. DNA produces is called complementary DNA.
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13
Q

How is cDNA made from insulin mRNA (using reverse transcriptase method)?

A

mRNA first isolated from cells, and then mixed with free floating nucleotides and reverse transcriptase. The reverse transcriptase uses the mRNA as a template to make the new strand of CDNA.

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14
Q

What are palindromic sequences?

A

Sequences that consist of base pairs that can be read the same in the opposite directions/antiparallel pairs.

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15
Q

How do restriction endonuclease enzymes work?

A
  1. They recognise specific palindromic sequences and cut/digest the DNA at these places.
  2. Difference restriction enzymes cut at different specific recognition sequence as shape of enzymes active site is complementary to recognition sequences shape.
  3. DNA sample is incubated with the specific restriction enzyme, which cuts the DNA fragment out via a hydrolysis reaction .
    - This cut may leave sticky ends - small tails of unpaired bases at each end of the fragment. Sticky ends can be used to bind/anneal the DNA fragment to another piece of DNA that has sticky ends with complementary sequences.
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16
Q

What is the main difference between gene machines and the other methods of making DNA fragments?

A

Pre-existing DNA templates aren’t needed when using gene machines. Instead, a database contains the necessary info to produce the DNA fragment, allowing any sequence to be made.

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17
Q

How do gene machines work?

A
  1. Sequence required is designed
  2. 1st nucleotide in sequence is fixed to some form of support e.g. a bead.
  3. Nucleotides are added step by step in the correct order, in a cycle of processes that includes adding protecting groups. Protecting groups make sure that nucleotides are joined at the right points, to prevent unwanted branching.
  4. Shorter sections of DNA called oligonucleotides, roughly 20 nucleotides long, are produced. Once these are complete, they are broken off from support and all protecting groups are removed. The oligonucleotides are then joined together to make longer DNA fragments.
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18
Q

What do you do after you’ve made your DNA fragments?

A

You amplify it so you have loads of copies to work with.

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19
Q

Name 2 methods of amplifications and give a brief sentence stating what they involve?

A
  • In vivo amlification: Involves transforming host cells

- In vitro amplification: Uses the Polymer Chain Reaction

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20
Q

What is a vector? (name a few examples)

A

Something that’s used to transfer DNA into a cell. They can be plasmids or bacteriophages (viruses that infect bacteria)

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21
Q

Describe step 1 of in vivo amplification

DIAGRAM

A

DNA FRAGMENTS INSERTED INTO VECTOR

  1. Vector cut open using same restriction enzyme used to isolate DNA fragment containing target gene, so sticky ends of vector are complementary to stick end of DNA fragment containing target gene.
  2. Vector DNA and DNA fragment mixed together with ligase. Ligase joins sticky ends in process called ligation.
  3. New combination of bases in DNA (vector DNA + DNA fragment) is recombinant DNA.
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22
Q

Describe step 2 of in vivo amplification

A

VECTOR TRANSFERS DNA FRAGMENT INTO HOST CELLS

  1. If plasmid vector is used, host cells have to be persuaded to take in the plasmid vector and its DNA.
  2. With a bacteriophage vector, bacteriophage infects host bacterium by injecting its DNA into it. The DNA with target gene in it then integrates into bacterial DNA.
  3. Host cells that take up of vectors containing target gene are said to be ‘transformed’.
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23
Q

Describe step 3 of in vivo amplification.

A

TRANSFORMED HOST CELLS ARE CLONED AND IDENTIFIED

  1. Marker genes inserted into vectors at same time as gene to be cloned, so host cells take in gene to be cloned and marker gene.
  2. Transformed host cells grown on agar plates. Each cell divides and replicates its DNA, creating colony of cloned cells that contain cloned gene and marker gene.
  3. Marker gene can code for antibiotic resistance - host cells grown on agar plates containing the specfic antibiotic, so only transformed cells that have the marker gene will survive and grow.
    - Or it can code for fluorescence so when agar plate is placed under a UV light, only transformed cells will fluoresce.
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24
Q

Why is it important to be able to identify which cells have been transformed?

A

As not all host cells will take up the vector and its DNA (only about 5% will take it up)

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25
Q

What do transformed host cells need to produce proteins and why?

A

Promoter and terminator regions

- These may be present in the vector DNA or they may be added along with the fragment.

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26
Q

What are primers?

A

Primers: Short pieces of DNA that are complementary to the bases at the start of the fragment you want

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27
Q

Describe step 1 of in vitro cloning/PCR

A
  1. Reaction mixture set up, containing DNA sample, free nucleotides, primers and DNA polymerase.
  2. DNA mixture heated to 95 to break H2 bonds between strands.
  3. Mixture then cooled to 55 so that primers can bind to strands.
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28
Q

Describe step 2 of in vitro cloning/PCR.

A
  1. Reaction mixture heated to 72 for polymerase to work
  2. DNA polymerase lines up free nucleotides alongside template strand. Specific base pairing means new complementary strands are formed.
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29
Q

Describe step 3 of in vitro cloning/PCR.

A
  1. As 2 new copies of DNA fragment are formed, 1 cycle of PCR is complete.
  2. Cycle repeats, with mixture being heated to 95 and this time all 4 strands act as templates.
  3. Each PCR cycle doubles amount of DNA each time.
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30
Q

What is genetic engineering?

A

Tranforming micro-organisms, plants and animals using recombinant DNA technology.

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31
Q

Briefly describe how proteins are made from transformed MICRO-ORGANISMS give an example?

A

Using same technology as in vivo cloning.

- Foreign DNA is inserted into micro-organisms to produce lots of useful protein e.g. insulin.

32
Q

Briefly describe how proteins are made from transformed PLANTS?

A

A gene that codes for a desirable protein is inserted into a plasmid. The plasmid is added to a bacterium and the bacterium is used as a vector to get the gene into the plant cells.

33
Q

Briefly describe how proteins are made from transformed animals?

A

A gene that codes for a desirable protein is inserted into an early human embryo or into female egg cells.

  • If gene is inserted into a very early human embryo, all of the body cells of the resulting transformed animal will end up containing the gene.
  • Inserting into the egg cell means that when the female reproduces, all the cells of her offspring will contain the gene,
34
Q

What important regions do we need to make proteins ?

A

Promoter regions and terminator regions

35
Q

What do promoter regions and terminator regions do?

A

Promoter regions tell the enzyme RNA polymerase when to start producing mRNA. Terminator regions tell it when to stop.

36
Q

Name a property of certain promoter regions and how this benefits us?

A

Property: Some promoter regions are only activated in specific cell types.

Benefit:

  • This means they can be used to control exactly which of the animals body cells a protein is produced in, so that the protein can be harvested more easily.
  • Producing the protein in the wrong cells could also damage the organisms so promoter regions would prevent this.
37
Q

Name 3 main domains in which transformed organisms are used in?

A
  1. Agriculture
  2. Industry
  3. Medicine
38
Q

Describe how transformed organisms are used in agriculture and give an example.

A

They’re used to give higher/more nutritious crop yields to reduce risk of famine and malnutrition. Crops can also be transformed to have pest resistance so that fewer pesticides are needed. This reduces cost and any environmental problems related to using pesticides.

e.g. Golden rice contains gene from maize plant and gene from soil bacterium, which allow plant to produce beta-carotene. Beta-carotene is used by our bodies to produce vitamin A. This rice is being produced in areas with shortage of dietary vitamin A e.g. south asia, Africa.

39
Q

Describe how transformed organisms are used in industry and give an example.

A

Industrial processes often use enzymes, produced from transformed organisms, so they can be produced quickly in large quantities for less money.

E.g. Rennin is an enzyme used in cheese-making. It used to be made from rennet (a substance produced in cows stomach’s) but now it can be made by transformed organisms. This means it can be made in large quantities, relatively cheaply, and without killing any cows, making some cheese suitable for vegetarians.

40
Q

Describe how transformed organisms are used in medicine and give an example.

A

Many drugs/vaccines are produced by transformed organisms. This means they can be produced quickly in large quantities for less money.

E.g. Insulin is used to treat type 1 diabetes and used to come from animals (cow, horse, pig pancreases). As it wasn’t human insulin, it didn’t work as well. Human insulin is now made from transformed organisms, using a cloned human insulin gene.

41
Q

Describe how insulin is produced? (using recombinant DNA technology?

A
  1. DNA fragment containing insulin gene is isolated using either reverse transcriptase/restriction enzymes.
  2. DNA fragment is inserted into a plasmid vector.
  3. Plasmid containing recombinant DNA is transferred into a bacterium.
  4. Transformed bacteria are identified and grown.
  5. The insulin produced from the cloned gene is extracted and purified.
42
Q

Describe some agricultural concerns about the use of recombinant DNA technology

A
  1. May lead to monoculture: Crops would be genetically identical so their biodiversity would decrease and plants would show little resistance to diseases.
  2. Superweeds (weeds resistant to herbicides): These could occur if transformed crops interbreed with wild plants. This could also lead to uncontrolled spread of recombinant DNA, with unknown consequences.
  3. Contamination: Organic farmers may have crops contaminated by wind-blown seeds from nearby genetically modified crops. This means they can’t sell their crop as organic anymore and may lose income.
43
Q

Describe some industrial concerns about the use of recombinant DNA technology

A
  1. Globalisation: As use of this technology increases, Large biotechnology companies that control genetic engineering become more powerful and put smaller companies out of business.
  2. Some GM crops are not labelled, so people aren’t given a choice.
  3. Some consumer markers, e.g. the EU, wont import GM foods and products. This can cause an economic loss to producers who have traditionally sold to those markets.
44
Q

Describe some medical concerns about the use of recombinant DNA technology

A
  1. Genetic engineering technologies used for GM crops, should be used to save lives.
  2. Whilst it is illegal, some worry this technology could be used to make designer babies (babies that have characteristics chosen by parents).
45
Q

Describe some ownership concerns about the use of recombinant DNA technology

A
  1. Debate about who owns genetic material from humans once it has been removed from the body - the donor or researcher. Some argue that human has right to own genetic information, and others argue that research is valuable in medicine and diagnosis.
  2. Small number of large corporations own patents to particular seeds. They can charge high prices, and can require farmers to repurchase seeds every year. If non-GM crops are contaminated by GM crops, farmers can be sued for breaching the patent law. **
46
Q

Describe how gene therapy works

A

Defective genes/mutated alleles in cells are altered to treat genetic disorders and cancers. How this is done depends on whether the disorder is caused by a mutated dominant allele or 2 mutated recessive alleles.

  • If it’s caused by 2 mutated recessive alleles, you can add a working dominant allele to make up for them (you ‘supplement’ the faulty ones).
  • It it’s caused by a mutated dominant allele, you can ‘silence’ the dominant allele (e.g. by sticking a bit of DNA in the middle of the allele so it doesn’t work anymore).
47
Q

Describe how you get the new allele (DNA) inside the cell during gene therapy

A

Using different kinds of vectors ,e.g. altered viruses, plasmids, or liposomes (spheres made of lipid)

48
Q

Name the 2 kinds of gene therapy

A
  1. Somatic therapy.

2. Germ line therapy.

49
Q

What does somatic therapy involve?

A

It involves altering the alleles in body cells, particularly the cells that are the most affected by the disorder.
- Somatic therapy doesn’t doesn’t affect the individual’s sex cells though, so any offspring could still inherit the disease.

50
Q

What does germ line therapy involve?

A

This involves altering the alleles in sex cells. This means that every cell of any offspring produced from these cells will be affected by the gene therapy and will not inherit a disease (however, it is currently illegal)

51
Q

What are the ethical issues regarding gene therapy?

A
  1. Technology can be used in ways other than medical treatment, e.g. treating cosmetic effects of aging.
  2. Risk of harm: May lead to over expression of genes - gene produces too much of the missing protein.
52
Q

What are DNA probes used for? (state in a brief sentence)

A

They are used to locate specific alleles of genes (e.g. on chromosomes) or to see if a person’s DNA contains a mutated allele that causes a genetic disorder

53
Q

What are DNA probes?

A

They are short strands of DNA that have a specific base sequence that’s complementary to the base sequence of part of the target allele.

54
Q

What does a DNA probe have attached to it and why?

A

It has a radioactive or fluorescent label attached to it so that it can be detected.

55
Q

Describe the process that explains how specific alleles are located.

(NON-micro array method)

A
  1. Sample of DNA is digested into fragments using restriction enzymes and separated using electrophoresis.
  2. Separated fragments transferred to nylon membrane and incubated with fluorescent labelled probe.
  3. If allele is present, DNA probe will hybridise (bind) to it.
  4. Membrane exposed to UV light and if gene is present, there’ll be a fluorescent band.
56
Q

What is a DNA microarray?

A

It is a glass slide with microscopic spots of different DNA probes attached to it in rows.

57
Q

Describe how a DNA microarray can be used to locate specific alleles.

A
  1. Sample of fluorescent labelled human DNA is washed over array.
  2. If labelled human DNA contains any DNA sequences that match any of the probes, it will stick to the array.
  3. Array is washed to remove any labelled DNA that hasn’t stuck.
  4. Array is then visualised under UV light - any labelled DNA attached to a probe will fluoresce.
  5. Any spot that fluoresces means that the person’s DNA contains that specific allele.
58
Q

How do you produce DNA probes?

A

You sequence the allele that you want to screen for, then use PCR to produce multiple complementary copies of part of the allele.

59
Q

Name 3 uses of screening using DNA probes.

A
  1. To help identify inherited conditions e.g. Huntington’s
  2. To help prescribe the right drugs that target specific receptors associated with mutated allele.
  3. To help identify health risks so people can make choices to reduce risk of disease developing.
60
Q

What is an argument against genetic screening?

A
  1. Discrimination by employers/insurance companies if people are known to have a high risk of a disease.
  2. May lead to prolonged stress that can affect daily life.
  3. May lead to pressure to not have a child
61
Q

What can the results of screening be used for?

A
  1. Genetic counselling

2. Personalised medicine

62
Q

What does genetic counselling involve?

A

It involves:

  • advising people about screening (It can take place before screening begins)
  • explaining results of a screening
  • Advising patients and their relatives about the risks of genetic disorders and the options of prevention or treatment available.
63
Q

What does personalised medicine involve?

A

Different people will respond to the same drug in different ways so doctors will tailor a drug to an individual’s DNA

64
Q

What are VNTR’s?

A

Variable number tandem repeats (VNTR’s) are repeats that don’t code for proteins and repeat next to each other over and over again.

65
Q

Do the number of times sequences are repeated differ or stay the same from person to person?

(what does this tell us about the length of nucleotide sequences then?)

A 
They differ (most of the time) 
- this tells us that the length of nucleotide sequences differ too
66
Q

What is genetic fingerprinting?

A

A method that involves comparing the number of times a sequence is repeated (and so the number of nucleotides) at specific places in the genomes of different individuals.

67
Q

What is the probability of 2 individuals have the same genetic fingerprint?

A

Very unlikely

68
Q

Name the technique that allows us to make a genetic fingerprint?

A

Electrophoresis

69
Q

Describe how a DNA fragment is prepared for electrophoresis.

A
  1. A sample of DNA is obtained (from blood, saliva)
  2. PCR used to make many copies of areas of DNA that contains VNTRs
  3. You end up with fragments where length corresponds to number of repeats the person has at each specific position.
  4. A fluorescent tag is added to all of the DNA fragments so they can be viewed under UV light.
70
Q

Describe the process of electrophoresis

A
  1. DNA mixture is placed into a well in a slab of gel and covered in a buffer solution that conducts electricity.
  2. An electrical current is passed through gel - DNA fragments are (-) charged so they move towards positive electrode at far end of gel.
  3. Smaller DNA fragments move faster and travel further through gel, so DNA fragments separate according to size.
  4. DNA fragments are viewed as bands under UV light - this is the genetic fingerprint.
71
Q

How do you evaluate your results from electrophoresis?

A

You compare the genetic fingerprints of the different individuals - e.g. if both fingerprints have a band at same location on the gel, it means they have the same number of nucleotides and so the same number of VNTSr at that place.

72
Q

Describe 2 uses of genetic fingerprinting

A
  1. Determining genetic relationships - We inherit VNTR base sequences from our parents. Roughly half of the sequences come from each parent. This means the more bands on a genetic fingerprint that match, the more closely related 2 people are.
  2. Determining genetic variability within population - The greater the number of bands that don’t match on
    genetic fingerprint, the more genetically different people are. This means you can compare the number of repeats at several places in genome for a population. to find out how genetically varied that population is.
73
Q

Name 3 fields that use genetic fingerprinting

A
  1. Forensic science
  2. Medical diagnosis
  3. Animal and plant breeding
74
Q

How is genetic fingerprinting used in forensic science?

A

It can be used to compare samples of DNA froma crime scene with samples of DNA from possible suspects.

  1. DNA is isolated from crime scenes and suspects
  2. Each sample is replicated using PCR.
  3. products run on electrophoresis gel.
  4. If samples match, it links a person to a crime scene.
75
Q

How is genetic fingerprinting used in medical diagnosis?

give an example

A

In medical diagnosis, a genetic fingerprint can refer to a unique pattern of several alleles.
- It can be used to diagnose genetic disorders and cancer. It’s useful when specific mutation isn’t known or where several mutations could’ve caused the disorder, as it identifies a broader, altered genetic pattern.

E.G. PGH screens embryos created by IVF for genetic disorders before they’re implanted into uterus. The faulty regions of parents DNA are used to produce genetic fingerprints, which are compared to genetic fingerprint of embryo. If they match, embryo inherits disorder and appropriate treatment is prescribed.

76
Q

How is genetic fingerprinting used in animal and plant breeding?

A

Genetic fingerprinting can be used to identify how closely-related individuals. The least related individuals would be bred together to prevent inbreeding, and increase the gene pool.

77
Q

Why is PCR necessary in genetic fingerprinting?

A

It amplifies the DNA, so enough is produced for it to be seen on the gel.

A2 Biology (11 decks)

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