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topic 7 Flashcards

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1
Q

7.1 [using gene sequencing]

Genome ?

A

All the genetic information within an organism

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2
Q

Exons ?
Introns ?

DNA/Gene sequencing ?

A

Exons - coding regions of DNA

Introns - non-coding regions of DNA that are removed

DNA/Gene sequencing - analysis of the base sequences along a DNA strand

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3
Q

What is PCR (polymerase chain reaction) ?

Amplified?

Why is PCR needed?

A

PCR - reaction used to AMPLIFY a section of DNA + make more copies of it in vitro (lab)

Amplify - When DNA is replicated repeatedly to produce a much bigger sample using a PCR

  • PCR provides sufficient material for investigation, such as gene sequencing and DNA profiling
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4
Q

How does PCR work?

A
  1. Reactants mixed:
    - DNA sample
    - DNA Primers (short sequences of DNA)
    - 4 nucleotide bases
    - heat-stable DNA polymerase enzymes (Taq DNA polymerase)
    - pH buffer
  2. Separate the 2 polynucleotide strands (unzip DNA) by heating at 90-95 oC for 30 seconds to break H+ bonds
  3. Cool to 50-55 oC for 20 seconds = bind the primers (anneal) to single-stranded DNA
    [The primers provide a starting sequence for DNA replication]
  4. Temp is increased to about 72 degrees with polymerase = the optimum temp DNA polymerase works at = to replicate DNA
  • DNA polymerase creates a copy of the sample by complementary base pairing using the free nucleotides.
  1. This cycle is repeated around 30x = gives rise to an amount of DNA sufficient to create a DNA profile.

= Amplified samples can be used in DNA sequencing + DNA profiling

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5
Q

Why is Taq DNA polymerase (heat-stable DNA polymerase enzyme) added ?

A
  • when heated to 90 to unzip DNA = DNA polymerase denatured = DNA can’t replicate

Taq polymerase can withstand high temp as its produced from bacteria which lives in hot conditions = won’t denature / destroy

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6
Q

In Vitro ?

In Vivo ?

A

In Vitro - outside the body (DNA replication in test tube)

In Vivo - inside the body (DNA replication in nucleus)

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7
Q

What can these amplified samples of DNA be used for?

A

1. Gene sequencing (analysis of the base sequences along a DNA strand) :

  • To predict the amino acid sequence of proteins + possible links to genetically determined conditions

2. DNA profiling (comparing DNA sequences) :

  • In forensic science, to identify criminals + to test paternity
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8
Q

How does Gene sequencing work ?

A

● DNA sample is divided into 4 separate sequencing reactions which contain
- all 4 standard nucleotides
-DNA polymerase
- primers required for replication
- terminator nucleotides (fluorescently labelled for ease of identification)

● When a terminator nucleotide is incorporated into a growing chain, replication is terminated (stop the production of a DNA molecule)

● DNA fragments of different lengths are produced across the reaction vessels

● High-resolution gel electrophoresis is used to separate the fragments by size

● The fragments are visualised under UV light = enabling the base sequence to be read from the boom of the gel upwards

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9
Q

ways in which DNA sequencing is moving scientific knowledge + understanding forward?

A
  1. Predicting amino acid sequences
    - used to predict proteins produced from particular genes
    - know the proteins that the sequence of genes code for, for certain diseases = can find ways to manage these disease
  2. Links to disease managements
    - allows us to identify faulty genes, see which bass have changed + understand how changes in DNA affect which proteins are made = how changes in proteins affect symptoms of conditions
  3. DNA sequencing allowed for discoveries
    - eg shown that certain genetic combinations greatly increase chance of heart attack
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10
Q

DNA profiling?

A

Identification of repeating patterns in the non-coding regions of DNA (introns)

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11
Q

Where are Micro-satellite + Mini-satellite found ?

A

within introns are short sections of DNA which are repeated many times to form:

  • Mini-satellite 10-100 base sequence will be repeated 50- several 100 times
  • Micro-satellite 2-6 bases repeated 5-100 times

The number of repeats for each satellite will vary as different patterns may be inherited from mother + father

= The more closely related two people/species are, the more similar the repeats are

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12
Q

Process of DNA profiling ?

A
  1. Fragments of DNA are cut with restriction endonuclease enzymes (either side of satellites)
  2. Gel electrophoresis
    (Fragments are separated + visualised using gel electrophoresis)
  3. Southern Blot
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13
Q
  1. Gel electrophoresis
A
  1. Fragments are separated + visualised using gel electrophoresis
  • fragments placed in wells in agarose gels + dyed with ethidium bromide so they fluoresce under UV light.
  • A current is then applied to the gel. DNA is negative + fragments of different sizes move at different speeds according to mass so ‘bands’ appear
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14
Q
  1. Southern Blot
A
  1. Southern Blot
  • Alkaline buffer solution added + nylon filter placed over it
  • This dry absorbent material draws solution containing DNA fragments to the filter
    = fragments appear as ‘blots’ on filter
  • Gene probes (labelled complementary sequences that fluoresce or are radioactive) are added + bind with DNA (hybridisation)
  • ‘Blots’ compared + the number of satellites visualised as a graph = more closely related 2 people/species are = more similar the repeats are
  • Southern Blot also denatures the DNA fragments so the strands separate + the base sequences are exposed
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15
Q

How does DNA profiling help identify individuals for Paternity tests + forensic science (criminals) ?

A
  • Compare number of microsatellite repeats from 2 DNA samples

= The more closely related two people/species are, the more similar the repeats are

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16
Q

7.2 [ factors affecting gene expression]

what is meant by the term ‘gene expression’ ?

A
  • when the protein coded for by a gene is produced / synthesised (via transcription & translation)
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17
Q

Different types of cells from the same organism have the same genotype.

Explain how this is possible in terms of ‘gene expression’

A

Although they have the same genes in the nucleus.

Not all genes are switched on (expressed) or off (repressed) at the same time.

Cells differ from each other because each cell type expresses a different group of genes.

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18
Q

What is ‘cell differentiation’ ?

What are housekeeping proteins?

A
  • the process by which certain groups of genes are activated to produce those proteins that are specific to that particular cell type
  • Different proteins control cellular activity, e.g. enzymes.
  • ALL cells have housekeeping proteins - basic proteins needed to survive
    = There are extra proteins in each cells based on differentiation
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19
Q

What are transcription factors?

A
  • Proteins that bind to DNA to regulate gene expression
  • transcription factors either smulate or prevent transcription of the gene.
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20
Q

Why are transcription factors mostly used rather than translation?

A

= controlling gene expression through controlling either transcription / translation

Mostly transcription factors used,
to stop DNA → mRNA as mRNA strand can be used to form hundreds of proteins so they would be more effective in stopping more proteins formed whereas stopping translation will only stop the formation of that 1 protein.

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21
Q

Transcription factors can bind to 2 regions…

A

Promoter Sequences

  • Found upstream (5’) of the gene they act on
  • enable the binding of RNA polymerase + therefore promote transcription

Enhancer Sequences

  • Regulate DNA activity by changing chromatin structure
  • making it more / less open to RNA polymerase
  1. OPEN chromatin → gene expressed
    = more accessible to RNA polymerase = more transcription can occur
  2. CLOSED chromatin → gene not expressed / gene inactivity
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22
Q

How does RNA splicing (post-transcriptional modification of mRNA) explain how eukaryotes produce more proteins than they have genes ?

A
  • RNA splicing = results in different products from a single gene
  1. Gene is transcribed which results in pre-mRNA (the transcript of the whole gene)
  2. All introns (non-coding regions) + some exons (coding regions) are removed
  3. The remaining genes are joined together by enzyme complexes called spliceosomes
  4. The same exons can be joined in a variety of ways to produce several different versions of mature functional RNA
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23
Q

Gene expression can be changed by epigenetics such as..
(epigenetic modifications)

A

Epigenetics = heritable + reversible modifications that DO NOT involve changes to the base sequence of DNA, affecting gene expression

  1. DNA methylation
  2. Histone Modification
  3. Non-Coding RNA (ncRNA)
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24
Q
  1. DNA methylation
A

= The addition of a methyl (CH3) group to a cytosine in the DNA molecule next to a guanine in the DNA chain

  • It inactivates the gene by reduced binding of transcription factors + reducing transcription of a gene, lowering gene expression
  • Methylation will change the shape of cytosine, preventing normal action of the enzyme RNA polymerase
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25
Q

what happens when DNA is demethylated ?

A

The removal of the methyl group enables genes to become active so they can be transcribed.

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26
Q
  1. Histone Modification
A

Histone Modification :

  1. Acetylation
  • addition of an acetyl (COCH3) group- activates chromatin = allows transcription
  • addition of COCH3 to lysine in histone = loose packing of nucleosomes = transcription factors can bind to DNA = genes expressed
  1. Methylation
  • addition of a methyl group (-CH3)- can cause activation/inactivation of chromatin depending on the position of the lysine
  • addition of -CH3 to lysine in histone = nucleosomes pack tightly together = transcription factors cannot bind to DNA = genes not expressed
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27
Q
  1. Non-Coding RNA (ncRNA)
A
  • Some non-coding RNA can modify the products of transcription
    = preventing translation = silencing the gene
  • Some non-coding RNA can act like transcription factors + inactivate genes + chromosomes = preventing gene expression
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28
Q

Why is epigenetic modification important in ensuring cell differentiation ?

A

Cell differentiation requires unspecialised cells to switch different genes off or on.

Epigenetic changes lead to this differential expression of genes.

During this process some genes are activated and others are silenced.

It is the combination of these particular gene products that results in the different characteristics of the fully differentiated mature cells.

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29
Q

How are epigenetic changes are different from mutations of DNA?

A
  1. Epigenetic changes do not alter DNA base sequences but mutations do.
  2. The DNA retains correct information on how to produce a polypeptide with epigenetic changes, whereas mutation may lead to different polypeptides being produced.
  3. Epigenetic changes just alter the degree to which a gene is expressed.
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30
Q

7.3 [stem cells]

What are stem cells?

What are the 3 types of stem cells?

A

undifferentiated cells which have the ability to differentiate into many different cell types by dividing by mitosis

  1. Totipotent stem cells (early embryonic)
  2. Pluripotent stem cells (late embryonic)
  3. Multipotent stem cells (adult)
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31
Q

What’s the difference between cell determination + cell differentiation ?

A

Cell determination:

  • Cells in the early embryo become predestined to form particular cell types + tissues as a result of their position in the embryo

Cell differentiation:

  • Is the process by which different genes are switched on / off within the nucleus resulting in very specific types of cells, making specific proteins for the jobs they need to do (specialised)
32
Q

Internal v External cues affecting what the stem cells differentiate into :

A

Internal cues:

  • Transcription factors present in different quantities in a zygote - at start of differentiation

External cues:

  • cell signalling from other cells (proximity)
  • temperature
33
Q
  1. Totipotent stem cells
  • potency of stem cell?
  • Source of stem cell?
A
  • Undifferentiated cells that can (replicate itself) to form ANY type of cell needed for an entire new organism
    (including placental cells = can form new embryo)

SOURCE:
- Very early embryo = zygote
( just a few cells are present)

34
Q
  1. Pluripotent stem cells
  • potency of stem cell?
  • Source of stem cell?
A
  • Undifferentiated cells that can form MOST of the cell types needed for an entire new organism

(but NOT placental cells = cannot form new embryo)

SOURCE:
- Inner cell mass of a later embryo, called a blastocyst

35
Q
  1. Multipotent stem cells
  • potency of stem cell?
  • Source of stem cell?
A
  • Cells that can form a very LIMITED range of differentiated cells within a mature organism
  • produce limited range of related cell types, such as all red + white blood cells

SOURCE:
- Adult stem cells - somatic
- in bone marrow, skin, liver, muscles

36
Q

Which type of stem cell is mostly used for medical advancement + Why ?

A

Pluripotent stem cells = most useful source of stem cells for medical use

  • have the potential to produce a wide range of cells
  • They are more plentiful than totipotent cells
  • Genes only found in totipotent cells are not relevant to treatment (development of embryonic membranes)
  • They are easier to obtain than adult stem cells
37
Q

Evaluate the use of pluripotent stem cells from embryos for medical purposes

+ pros :

A
  • Ability to produce almost all cell types = giving the potential to grow replacement organs + tissues
  • Provision of cells for drug testing replaces the need for animal testing during drug development
  • Embryos would die anyway (if donated by couples when they are no longer needed for IVF)
  • Potential benefits outweigh harm
38
Q
  • Cons :
A
  • Possible rejection of cells by the immune system of the recipient = cell recognised as ‘non-self’
  • Some religions consider human life + rights begin at conception
  • Consent cannot be given by an embryo
  • Some feel that killing an embryo is no different from killing an adult = both life
  • The embryo has potential to develop into a human being
39
Q

How epigenetic modifications can result in totipotent stem cells in the embryo developing into pluripotent cells in the blastocyst, and finally into fully differentiated cells ?

Totipotent –> Pluripotent –> differentiated somatic cells

A
  • The change is at least partly caused by epigenetic modification (which controls gene expression) by epigenetic mechanisms such as DNA methylation, histone modification + ncRNAs
  • Some genes are activated + others are silenced
  • As development progresses more genes are silenced in each cell
  • It is the combination of the particular genes that are activated for silencing that results in the different characteristics of fully differentiated mature cells
40
Q

Arguments for using adult stem cells instead of embryonic stem cells ?

A
  • Own cells less likely to be rejected by the immune system = if recognised as ‘self’
  • Do not need a source of human embryos = more ethical
41
Q

problems associated with the use of adult stem cells ?

A
  • still chance of rejection by the immune system
  • In bone marrow transplants the implanted white blood cells may damage the recipient
  • Many adult stem cell types are difficult to grow + prepare for transfer
  • Not all cell types have adult stem cells
42
Q

Induced pluripotent stem cells (iPS cells) ?

A

Adult cells that have been reprogrammed by the introduction of new genes to become pluripotent again

43
Q

How can differentiated fibroblasts be reprogrammed to form induced pluripotent stem cells (iPS) by the artificial introduction of named genes ?

A

1.Fibroblasts (connective tissue) taken from skin samples

2.They can be treated with introduced 4 genes for transcription factors (by vectors - eg virus)
= that encode transcription factors that allow genes that had been switched off to be expressed again (activate specific genes in adult cell to produce pluripotent cells)

3.= This can reverse differentiation = so they regain the properties of pluripotent stem cells

( factors activate specific genes in adult cells to produce pluripotent cells )

44
Q
  1. Why were certain genes are added to the cells?
  2. Why are the patient’s own cells used in the treatment?
A
  1. Adding certain genes converts the cells into an induced pluripotent stem cell (iPS), which can differentiate into almost any cell type.
  2. Cells respond exactly as patient cells
    Avoids testing ineffective treatments on patient
    not rejected
45
Q

Why is the use of iPS stem cells less problematic than the use of embryonic stem cells ?

A
  • No ethical concerns about the use of embryos as they are obtained from adult tissues
  • Made from a patient’s own fibroblasts = injection of these iPS cells would avoid rejection by the patient’s immune system.
46
Q

Problems associated with the use of iPS cells ?

A
  • The genes used can promote cancer development if start replicating by mitosis uncontrollably + form a tumor
  • Sometimes the cells are changed in a way that still causes rejection by the immune system
47
Q

Therapeutic cloning ?

potential issue?

A

Experimental technique used to produce embryonic stem cells from an adult cell donor

Potential issue - can be taken further in future to make cloned artificial designer babies ?

48
Q

7.4 [gene technology]

What’s recombinant DNA?

A

New DNA produced by genetic engineering technology that combines genes from the DNA of one organism with the DNA of another organism

49
Q

What’s genetic engineering / modification ?

A

The insertion of genes from one organism into the genetic material of another organism or changing the genetic material of an organism

50
Q

What 3 enzymes are commonly used in gene technology ?

A
  • reverse transcriptase
  • restriction endonucleases
  • DNA ligase
51
Q

How is recombinant DNA produced ?

A
  1. Isolation of the gene:
    - Restriction endonuclease enzyme - Cuts out a section of DNA with the gene of interest
  • Each restriction endonuclease enzyme cuts at a specific (restricted) site in the DNA sequence

= This leaves ‘sticky ends’ - staggered complementary exposed bases

  • Use reverse transcriptase enzymes to make DNA from mRNA
  1. Cut plasmid’s DNA with the same restriction endonuclease enzyme to leave complementary sticky ends
  2. DNA ligase anneals (joins/seals) the 2 DNA structures from the plasmid + gene together
    = to form recombinant DNA
52
Q

Role of

a. Restriction endoclunucleous enzyme

b. DNA ligase

c. The plasmid

A

a. Restriction endoclunucleous enzyme:
- Cuts out a section of DNA with the gene of interest
- at a specific (restricted) site in the DNA sequence
- to leave ‘sticky ends’ ( staggered complementary exposed bases)

b. DNA ligase:
- Anneals (joins/seals) the 2 DNA structures from the plasmid + gene together = forms recombinant DNA

c. Plasmid acts as a vector to transfer the recombinant DNA

53
Q

variety of vectors can be used to transfer recombinant DNA into another organism
(target cell)

4 ways the recombinant DNA be inserted into other cells ?

A
  1. Viral vectors
  2. Liposome wrapping
  3. Gene guns
  4. Microinjection
54
Q

What do you call the organism the recombinant DNA is inserted into?

A

transforming the recipient= becomes a genetically modified organism (GMO)
+ a transgenic organism

55
Q
  1. Viral vectors
A
  • A harmless virus can be engineered to carry a desirable gene
  • and then to infect the animal’s cells
  • carrying the DNA with it
  • introduce this DNA into the DNA of the host cell during latency
56
Q
  1. Liposome wrapping
A
  • The gene to be inserted is wrapped in liposomes (spheres formed from a lipid bilayer)
  • These fuse with the cell membrane + can pass through it to deliver the DNA into the cytoplasm of host cell
57
Q
  1. Gene guns
A
  • DNA is shot into the cell at high speed carried on minute gold / tungsten pellets
  • Some cells survive this treatment + accept the DNA as part of the genetic material
58
Q
  1. Microinjections
A
  • DNA is injected into a cell through a very fine micropipette

(manipulated using a micromanipulator, because the steadiest hand would tremble enough to destroy the cell)

  • So many cells have to be injected before 1 takes up the DNA successfully
  • but it is the method that has resulted in most successful transgenic animals
59
Q

Identifying recombinant cells

  • Antibiotic resistance marker genes & replica plating
A

Gene markers are used to show where a foreign gene has been inserted.

Fluorescence + antibiotic resistance combined with replica plating can be used as gene markers.

Bacteria are transferred from a ‘master plate’ onto plates with antibiotics using a sterile block - if they don’t grow, the gene was inserted successfully and the colonies are still on the ‘master plate’.

60
Q

Define what replica plating is?

A

Process used to identify recombinant cells that involves growing identical patterns of bacterial colonies on plates with different media.

61
Q

How antibiotic resistance marker genes & replica plating is used to identify recombinant cells :

A

Bacteria containing a gene of interest (transformed bacteria) on a plasmid can be identified if the plasmid also contains 2 antibiotic resistance/marker genes

  • Bacterial colonies are grown on an agar plate containing 1 of those antibiotics
  • some transferred to a second plate containing both antibiotics.
  • If they have the first antibiotic (i.e. the plasmid) there will be colonies on both agar plates
  • if they have the gene of interest inserted into + therefore disrupting the second antibiotic gene those bacteria will be absent on the second plate.

= Therefore, bacteria containing the inserted gene can be identified.

62
Q

Explain why replica plating is necessary

A

Replica plating provides two identical agar plates with the colonies in the same positions so they can be identified.

Each colony grows from a single original cell.

One plate will contain antibiotic 1 only, the second plate will contain antibiotic 2.

Only those sensitive to the second antibiotic but also resistant to the first will be selected but will only be found as they are missing on the second plate.

Therefore, the first plate is needed as a comparison + to select the colonies from

63
Q

Describe how bacterial cells modified with recombinant DNA can be identified from a culture

A
  • Recombinant DNA is used that includes antibiotic resistance marker genes
  • Bacteria with modified DNA do not survive exposure to antibiotic
  • Replica plating preserves living colonies of identified bacteria
64
Q

Explain how culturing could be used to obtain recombinant bacteria

A
  • the gene of interest is inserted into plasmids (1)
  • this plasmid has a marker gene for antibiotic resistance (1)
  • the recombinant bacteria will be resistant to this antibiotic (1)
  • the bacteria are grown on agar containing that
    antibiotic (1)
  • only the recombinant bacteria will grow (1)
  • use of replica plating to transfer recombinant bacteria to fresh medium (1)
65
Q

1 What are ‘knockout’ mice / organisms ?

2 Why are they important ?

A
  1. Knockout mice are mice with 1 or more genes silenced (knocked out) so they no longer function, via the insertion of a similar gene that makes the original gene impossible to read

2.
- The existence of many genes is known but their function is not obvious

  • To discover their function = use ‘knockout’ mice = gene has been deliberately damaged = lose its function + observe the effect
66
Q

What are the uses of knockout mice?

A
  1. Help to understand gene function
  2. Used as a model to test possible treatments for genetic disorders
  • Genes that are non-functional in humans are knocked out in mice to create a model of the disease
  • study genetic conditions where in humans mutations have caused genes to be inactivated and protein function to be lost
67
Q

Why can’t’ all genes be investigated using
knockout mice ?

A
  • Some genes control functions essential for life.

= When no functioning gene is present the mouse cannot develop or survive

  • It will not be possible to determine the specific role of these genes.
68
Q

Explain how u can test to see if a particular gene may be associated with cancer using knockout mice

A
  • Gene being studied is inactivated
  • Gene is inactivated in embryonic stem cells of mice (by inserting an additional nucleotide sequence)
  • Knockout stem cells are fused with embryos to create mice that lack a functional version of studied gene
  • Phenotypes of knockout mice + normal mice can then be compared in terms of the incidence of cancer
69
Q

Explain genetic modification of plants:

Transgenic plants - plants which contain genetic material from an unrelated organism

A

Genetic modification using Agrobacterium tumefaciens :

  1. Ti plasmid (transfers bacterial genetic information directly to plant DNA) extracted
  2. Bacterial genes are inserted into plasmid via genetic modification
  3. Plasmid is returned to a bacterium (vector)
  4. Plant is infected with the bacterium
  5. Plant grows a crown gall - the cells of the crown gall contain the inserted gene
  6. These cells can be isolated + cultured to grow whole new transgenic plants
70
Q

What GM soya plants produce?

A

GM soya plants have been produced that are:

  1. Resistant to common herbicides
    = yield increases
  2. Resistant to fungal pathogens
  3. Drought + flood resistant
  4. Pesticide production (make their own pesticides via leaves)

5.Changing the nutrient value of plants + altering balance of faty acids:

  • Linoleic acid (polyunsaturated) is replaced by oleic acid (monounsaturated) = oxidises less easily = doesn’t go off as quickly + is also healthier
  • Linoleic acid = produced less
  • oleic acid = produced more
71
Q

Explain what ‘human gene therapy’ is and give one example of a genetic disease that this could be used for.

A

Gene therapy is the therapeutic delivery of nucleic acids into a patient’s cells to treat disease.

  • The healthynopy of the gene is used by the cells to produce a healthy functioning version of the protein.

= EG: cystic fibrosis sufferers could receive a healthy version of the CFTR gene into their lung cells

72
Q

advantages + disadvantages of gene therapy

  1. ADV
A
  1. Treat genetic diseases by introducing a healthy copy of the gene into affected cells. (CFTR eg)
  2. Germ line gene therapy could be used to
    introduce the healthy genes into an early
    embryo so that the genetic disease is not
    passed on = can cause permanent cure
73
Q
  1. DIADV
A
  1. If germ line gene therapy is used = no
    idea of the long-term health implications of
    introducing genes into the early embryo
  2. ETHICAL:
    Germ line gene therapy technology allows the
    possibility to produce ‘designer babies’ not just
    treat disease
74
Q

Genetic modification caused public debate (crops etc)

-Benefits of recombinant gene technology

A
  1. GM crops can improve the nutrition + health + last longer for producers + consumers
  2. Agricultural productivity can be improved while using less artificial pesticides / fertilisers = helping the environment.
    - GM crops can grow on previously unsuitable soil / in previously unsuitable climates = more yield
  3. Medicines + drugs can be produced safely in large quantities from microbes rather than from slaughtered animals.
    - These medicines benefit humans + can spare animal suffering as well.
75
Q
  • Negatives of recombinant gene technology
A
  1. Genetic modification of an organism may have unforeseen genetic effects on that organism + its offspring
  2. Environmental concern about gene transfer from GM plants + animals to wild species
  3. Developing GMOs is expensive = only available for rich countries + not farmers who need it
  4. Risks that marker genes (include antibiotic resistance) getting into wild plants = issues with build up of antibiotic resistance
  5. GMOs may continue to reduce the genetic biodiversity already occurring due to selective breeding.

bio (alevel) (10 decks)

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