Control Of Gene Expression Flashcards
1
Q
What is a gene mutation?
A
● A change in the base sequence of DNA
● Can arise spontaneously during DNA replication (interphase)
2
Q
What is a mutagenic agent?
A
A factor that increases rate of mutation, eg. ultraviolet (UV) light or alpha particles.
3
Q
Explain how a gene mutation can lead to the production of a non-functional
protein or enzyme (general)
A
- Changes sequence of base triplets in DNA so changes sequence of codons on mRNA
- So changes sequence of amino acids in the encoded polypeptide
- So changes position of hydrogen / ionic / disulphide bonds (between amino acids)
- So changes tertiary structure (shape) of protein
- Enzymes- active site changes shape so substrate can’t bind, enzyme-substrate complex can’t form
4
Q
Substitution
A
A base / nucleotide is replaced by a different base / nucleotide in DNA
5
Q
Addition
A
1 or more bases / nucleotides are added to the DNA base sequence
6
Q
Deletion
A
1 or more bases / nucleotides are lost from the DNA base sequence
7
Q
Duplication
A
A sequence of DNA bases / nucleotides is repeated / copied
8
Q
Inversion
A
A sequence of bases / nucleotides detaches from the DNA sequence,
then rejoins at the same position in the reverse order
polypeptide.
9
Q
Translocation
A
A sequence of DNA bases / nucleotides detaches and is inserted at a
different location within the same or a different chromosome
10
Q
Explain why not all gene mutations affect the order of amino acids
A
● Some substitutions change only 1 triplet code / codon which could still code for the same amino acid
○ As the genetic code is degenerate (an amino acid can be coded for by more than one triplet)
● Some occur in introns which do not code for amino acids
11
Q
Explain why a change in amino acid sequence is not always harmful
A
● May not change tertiary structure of protein (if position of ionic / disulphide / H bonds don’t change)
● May positively change the properties of the protein, giving the organism a selective advantage
12
Q
Explain what is meant by a frameshift
A
● A frameshift occurs when gene mutations (eg. addition,deletion,duplication or translocation) change the number of nucleotides /
bases by any number not divisible by 3
● This shifts the way the genetic code is read, so all the DNA triplets
/ mRNA codons downstream from the mutation change
● The sequence of amino acids encoded changes accordingly and
the effects on the encoded polypeptide are significant
If a multiple of 3 bases is added / removed there won’t be a frameshift, but extra / less triplets will result in extra /
less amino acids in the encoded polypeptide.
A frameshift could also lead to production of a stop codon (that doesn’t code for amino acids so terminates
translation), resulting in a shorter polypeptide.
13
Q
What are stem cells
A
Undifferentiated / unspecialised cells capable of:
1. Dividing (by mitosis) to replace themselves indefinitely
2. Differentiating into other types of (specialised) cell
14
Q
Describe how stem cells become specialised during development
A
● Stimuli lead to activation of some genes (due to transcription factors- see 8.2.2)
● So mRNA is transcribed only from these genes and then translated to form proteins
● These proteins modify cells permanently and determine cell structure / function
15
Q
Describe totipotent cells
A
● Occur for a limited time in early mammalian embryos
● Can divide AND differentiate into any type of body cell (including extra-embryonic cells eg. placenta)
16
Q
Describe pluripotent cells
A
● Found in mammalian embryos (after first few cell divisions)
● Can divide AND differentiate into most cell types (every cell type in the body but not placental cells)
17
Q
Describe unipotent cells, using an example
A
● Found in mature mammals
● Can divide AND differentiate into just one cell
- Example: unipotent cells in the heart can divide and differentiate into cardiomyocytes (cardiac muscle cells)
18
Q
Explain how stem cells can be used in the treatment of human disorders
A
● Transplanted into patients to divide in unlimited numbers
● Then differentiate into required healthy cells (to replace faulty / damaged cells)
19
Q
Example of how stem cells can be used to treat human disorders
A
● Potential treatment of Type 1 diabetes by creating healthy islet cells that produce insulin
● Bone marrow stem cell transplant for blood cancers
Destroy patient’s bone marrow before treatment → so no faulty cells are produced
Transplant stem cells from healthy person → divide and differentiate into healthy cells
20
Q
Explain how induced pluripotent stem (iPS) cells are produced
A
- Obtain adult somatic (body) cells (non-pluripotent cells or fibroblasts) from patient
- Add specific protein transcription factors associated with pluripotency to cells so they express
genes associated with pluripotency (reprogramming)
○ Transcription factors attach to promoter regions of DNA, stimulating or inhibiting transcription - Culture cells to allow them to divide by mitosis
21
Q
Once made iPS cells can divide and differentiate …..
A
into healthy cells to be transplanted into the same patient
22
Q
Arguments for using stem cells to treat human disorders
A
✓ Can divide and differentiate into required healthy cells, so could relieve human suffering by
saving lives and improving quality of life
✓ Embryos are often left over from IVF and so would otherwise be destroyed
✓ iPS cells unlikely to be rejected by patient’s immune system as made with patient’s own cells
✓ iPS cells can be made without destruction of embryo and adult can give permission
23
Q
Arguments against using stem cells in treating human disorders
A
𝖷 Ethical issues with embryonic stem cells as obtaining them requires destruction of an
embryo and potential life (embryo cannot consent)
𝖷 Immune system could reject cells and immunosuppressant drugs are required
𝖷 Cells could divide out of control, leading to formation of tumours / cancer
24
Q
What are transcription factors?
A
● Proteins which regulate (stimulate or inhibit) transcription of specific target genes in eukaryotes
● By binding to a specific DNA base sequence on a promoter region
25
Describe how transcription can be regulated using transcription factors
Transcription factors move from cytoplasm to nucleus
Bind to DNA at a specific DNA base sequence on a promoter region (before / upstream of target gene)
This stimulates or inhibits transcription (production of mRNA) of target gene(s) by helping or preventing RNA polymerase binding
26
Explain how oestrogen affects transcription
1)Oestrogen is a lipid-soluble steroid hormone so diffuses into cell across the phospholipid bilayer
2)In cytoplasm, oestrogen binds to its receptor, an inactive transcription factor, forming an oestrogen-receptor complex
3)This changes the shape of the inactive transcription factor, forming an active transcription factor
4)The complex diffuses from cytoplasm into the nucleus
5)Then binds to a specific DNA base sequence on the promoter region of a target gene
6) Stimulating transcription of target genes forming mRNA by helping RNA polymerase to bind
27
Explain why oestrogen only affects target cells
Other cells do not have oestrogen receptors.
28
Describe what is meant by epigenetics
● Heritable changes in gene expression without changes to the base sequence of DNA
● Caused by changes in the environment (eg. diet, stress, toxins)
29
Describe what is meant by epigenome
All chemical modification of DNA and histone proteins - methyl groups on DNA and acetyl groups on histones.
30
What 2 things happen for epigenetic control of gene expression to inhibit transcription
Methylation of DNA is increased
Acetylation of histones is decreased
So dna not very tightly wound up, chromatin open
31
What 2 things happen for epigenetic control of gene expression to allow transcription
Methylation of DNA is decreased
Acetylation of histones is increased
So dna very tightly wound up, chromatin closed
32
How does increased methylation of dna inhibit transcription
1. Increased methylation of DNA - methyl
groups added to cytosine bases in DNA
2. So nucleosomes (DNA wrapped around
histone) pack more tightly together
3. Preventing transcription factors and
RNA polymerase binding to promoter
33
How does decreased acetylation of histones inhibit transcription
1. Decreased acetylation of histones
increases positive charge of histones
2. So histones bind DNA (negatively
charged) more tightly
3. Preventing transcription factors and
RNA polymerase binding to promoter
34
Explain the relevance of epigenetics on disease development and treatment
● Environmental factors (eg. diet, stress, toxins) can lead to epigenetic changes
● These can stimulate / inhibit expression of certain genes that can lead to disease development
○ Increased methylation of DNA OR decreased acetylation of histones inhibits transcription
○ Decreased methylation of DNA OR increased acetylation of histones stimulates transcription
● Diagnostic tests can be developed that detect these epigenetic changes before symptoms present
● Drugs can be developed to reverse these epigenetic changes
35
What is RNA interference (RNAi)?
● Inhibition of translation of mRNA produced from target genes, by RNA molecules eg. siRNA, miRNA
● This inhibits expression of (silencing) a target gene
36
RNA interference happens in…
This happens in eukaryotes and some prokaryotes.
37
Describe the regulation of translation by RNA interference
1. Small interfering RNA (siRNA) or micro-RNA (miRNA) is incorporated into binds to a protein, forming an RNA-induced silencing complex (RISC)
○ siRNA synthesised as double-stranded RNA → 1 strand incorporated
○ miRNA synthesised as a double-stranded hairpin bend of RNA →
both strands incorporated
2. Single-stranded miRNA / siRNA within RISC binds to target mRNA with a
complementary base sequence
3. This leads to hydrolysis of mRNA into fragments which are then
degraded OR prevents ribosomes binding
4. Reducing / preventing translation of target mRNA into protein
38
Describe how tumours and cancers form
● Mutations in DNA / genes controlling mitosis can lead to
uncontrolled cell division
● Tumour formed if this results in mass of abnormal cells
○ Malignant tumour = cancerous, can spread by metastasis
○ Benign tumour = non-cancerous
39
Compare characteristics of benign and malignant tumours
- benign grow slowly with cells dividing less often vs malignant which grow quicker with cells dividing more often
- benign tumours have cells which are well differentiated and specialised vs malignant tumours have cells which become poorly differentiated and unspecialised
- benign have normal regular nuclei vs malignant tumours that have irregular larger and darker nuclei
- benign tumours have well defines borders and are often surrounded by a capsule so dont invade surrounding tissue vs malignant Poorly defined borders and not encapsulated so can
invade surrounding tissues (growing projections)
- benign do not spread by metastasis (as cell adhesion molecules stick cells together) vs malignant Spread by metastasis- cells break off and spread to other parts of the body, forming secondary tumours
(due to lack of adhesion molecules)
- benign can normally be removed by surgery
and they rarely return vs malignant can normally be removed by surgery combined with
radiotherapy / chemotherapy but they often return
40
Describe the function of tumour suppressor genes
Code for proteins that:
● Inhibit / slow cell cycle (eg. if DNA damage detected)
● OR cause self-destruction (apoptosis) of potential
tumour cells (eg. if damaged DNA can’t be repaired)
41
Explain the role of tumour suppressor genes in the development of tumours
● Mutation in DNA base sequence → production of non-functional protein
○ By leading to change in amino acid sequence which changes protein tertiary structure
● Decreased histone acetylation OR increased DNA methylation → prevents production of protein
○ By preventing binding of RNA polymerase to promoter region, inhibiting transcription
● Both lead to uncontrolled cell division (cell division cannot be slowed)
42
Describe the function of proto-oncogenes
Code for proteins that stimulate cell division
(eg. through involvement in signalling pathways
that control cell responses to growth factors)
43
An oncogene is a …
An oncogene is a mutated / abnormally expressed form of the corresponding proto-oncogene.
44
Explain the role of oncogenes in the development of tumours
● Mutation in DNA base sequence → overproduction of protein OR permanently activated protein
○ By leading to change in amino acid sequence which changes protein tertiary structure
● Decreased DNA methylation OR increased histone acetylation → increases production of protein
○ By stimulating binding of RNA polymerase to promoter region, stimulating transcription
● Both lead to uncontrolled cell division (cell division is permanently stimulated)
45
Suggest why tumours require mutations in both alleles of a tumour
suppressor gene but only one allele of an oncogene
● One functional allele of a tumour suppressor gene can produce enough protein to slow the cell cycle
OR cause self-destruction of potential tumour cells → cell division is controlled
● One mutated oncogene allele can produce enough protein to lead to rapid / uncontrolled cell division
46
Explain the relevance of epigenetics in cancer treatment
Drugs could reverse epigenetic changes that caused cancer, preventing uncontrolled cell division. For example:
● Increasing DNA methylation OR decreasing histone acetylation of oncogene
○ To inhibit transcription / expression
● Decreasing DNA methylation OR increasing histone acetylation of tumour suppressor gene
○ To stimulate transcription / expression
47
Explain the role of increased oestrogen concentrations in the development
of some (oestrogen receptor-positive) breast cancers
1. Some breast cancers cells have oestrogen receptors, which are inactive transcription factors
2. If oestrogen concentration is increased, more oestrogen binds to oestrogen receptors,
forming more oestrogen-receptor complexes which are active transcription factors
3. These bind to promoter regions of genes that code for proteins stimulating cell division
4. This increases transcription / expression of these genes, increasing the rate of cell division
48
Suggest how drugs that have a similar structure to oestrogen help treat
oestrogen receptor-positive breast cancers
Suggest how drugs that have a similar structure to oestrogen help treat
oestrogen receptor-positive breast cancers
● Drugs bind to oestrogen receptors (inactive transcription factors), preventing binding of oestrogen
● So no / fewer transcription factors bind to promoter regions of genes that stimulate the cell cycle
49
Define genome
the complete set of genes in a cell
50
Define proteome
The full range of proteins that a cell can produce (coded for by the cells DNA/genome)
51
What is genome sequencing and why is it important?
● Identifying the DNA base sequence of an organism’s genome
● So amino acid sequences of proteins that derive from an organism’s genetic code can be determined
52
Explain how determining the genome of a pathogen could allow vaccines to be developed
● Could identify the pathogen’s proteome
● So could identify potential antigens (proteins that stimulate an immune response) to use in the vaccine
53
Suggest some other potential applications of genome sequencing projects
● Identification of genes associated with genetic diseases
○ New targeted drugs / gene therapy can be developed
○ Can screen patients, allowing early prevention / personalised medicine
● Identification of species and evolutionary relationships
54
Explain why the genome cannot be directly translated into the proteome in
complex organisms
● Presence of non-coding DNA (eg. introns within genes do not code for polypeptides)
● Presence of regulatory genes (which regulate expression of other genes, eg. by coding for miRNA)
55
Describe how sequencing methods are changing
● They have become automated (so are faster, more cost-effective and can be done on a larger scale)
● They are continuously updated
56
What is recombinant DNA technology?
Transfer of DNA fragments from one organism or species, to another.
57
Explain why transferred DNA can be translated within cells of recipient
(transgenic) organisms
1. Genetic code is universal
2. Transcription and translation mechanisms are universal
58
Describe how DNA fragments can be produced
using restriction enzymes
1.Restriction enzymes cut DNA at specific base ‘recognition
sequences’ either side of the desired gene
○ Shape of recognition site complementary to active site
2. Many cut in a staggered fashion forming‘sticky ends’
(single stranded overhang)
59
Describe how DNA fragments can be produced from mRNA
1. Isolate mRNA from a cell that readily synthesises the protein coded for by the desired gene
2. Mix mRNA with DNA nucleotides and reverse transcriptase → reverse transcriptase uses
mRNA as a template to synthesise a single strand of complementary DNA (cDNA)
3.DNA polymerase can form a second strand of DNA using cDNA as a template
60
Suggest two advantages of obtaining genes from mRNA rather than directly
from the DNA removed from cells
● Much more mRNA in cells making the protein than DNA → easily extracted
● In mRNA, introns have been removed by splicing (in eukaryotes) whereas DNA contains introns
○ So can be transcribed & translated by prokaryotes who can’t remove introns by splicing
61
Describe how fragments of DNA can be produced using a gene machine
● Synthesises fragments of DNA quickly & accurately from scratch without need for a DNA template
○ Amino acid sequence of protein determined, allowing base sequence to be established
● These do not contain introns so can be transcribed & translated by prokaryotes
62
Name an in vitro and in vivo technique used to amplify DNA fragments
● In vitro (outside a living organism) - polymerase chain reaction
● In vivo (inside a living organism) - culturing transformed host cells eg. bacteria
63
What is in reaction mixture when DNA fragments are amplified by PCR
Reaction mixture: DNA fragment, DNA polymerase (eg. taq polymerase), primers and DNA nucleotides
64
1st step of dna fragments being amplified by PCR
1. Mixture
heated to 95oC
● This separates DNA strands
● Breaking hydrogen bonds between bases
65
2nd step of dna fragments being amplified by PCR
2. Mixture
cooled to 55oC
● This allows primers to bind to DNA fragment
template strand
● By forming hydrogen bonds between
complementary base
66
3rd step of dna fragments being amplified by PCR
3. Mixture
heated to 72oC
● Nucleotides align next to complementary
exposed bases
● DNA polymerase joins adjacent DNA
nucleotides, forming phosphodiester bonds
67
What’s important about the cycle of amplfiying dNA w PCR
This cycle is repeated. In every cycle, the amount of DNA doubles causing an exponential increase (2^n)
68
Explain the role of primers in PCR
Explain the role of primers in PCR
● Primers are short, single stranded DNA fragments
● Complementary to DNA base sequence at edges of region to be copied / start of desired gene
● Allowing DNA polymerase to bind to start synthesis (can only add nucleotides onto pre-existing 3’ end)
● Two different primers (forward and reverse) are required (as base sequences at ends are different)
69
Suggest one reason why DNA replication eventually stops in PCR
There are a limited number of primers and nucleotides which are eventually used up.
70
Summarise the steps involved in amplifying DNA fragments in vivo
1. Add promoter and terminator regions to DNA fragments
2. Insert DNA fragments & marker genes into vectors (eg. plasmids) using
restriction enzymes and ligases
3. Transform host cells (eg. bacteria) by inserting these vectors
4. Detect genetically modified (GM) / transformed cells / organisms by identifying
those containing the marker gene (eg. that codes for a fluorescent protein)
5. Culture these transformed host cells, allowing them to divide and form clones
Following this, DNA can be extracted from the host cells if needed or the host cells can produce
a protein coded for by a gene in the DNA fragment.
71
Explain why promoter regions are added to DNA fragments
that are used to genetically modify organisms
Allow transcription to start by allowing RNA polymerase to bind to DNA
Can be selected to ensure gene expression happens only in specific cell types
○ Eg. in gland cells of a mammal so the protein can be easily harvested
72
Explain why terminator regions are added to DNA fragments
that are used to genetically modify organisms
Ensure transcription stops at the end of a gene, by stopping RNA polymerase
73
What are the role of vectors in recombinant DNA technology?
To transfer DNA into host cells / organisms eg. plasmids or viruses (bacteriophage)
74
Explain the role of enzymes in inserting DNA fragments into vectors
1.Restriction endonucleases / enzymes cut vector DNA
○ Same enzyme used that cut the gene out so vector DNA & fragments
have sticky ends that can join by complementary base pairing
2. DNA ligase joins DNA fragment to vector DNA
○ Forming phosphodiester bonds between adjacent nucleotides
75
Describe how host cells are transformed using vectors
● Plasmids enter cells (eg. following heat shock in a calcium ion solution)
● Viruses inject their DNA into cells which is then integrated into host DNA
76
Explain why marker genes are inserted into vectors
Explain why marker genes are inserted into vectors
● To allow detection of genetically modified / transgenic cells / organisms
○ If marker gene codes for antibiotic resistance, cells that survive antibiotic exposure = transformed
○ If marker gene codes for fluorescent proteins, cells that fluoresce under UV light = transformed
● As not all cells / organisms will take up the vector and be transformed
77
how recombinant DNA technology can be useful in medicine
GM bacteria produce human proteins (eg. insulin for type 1 diabetes)→ more ethically
acceptable than using animal proteins and less likely to cause allergic reactions
GM animals / plants produce pharmaceuticals (‘pharming’)→ cheaper
Gene therapy
78
how recombinant DNA technology can be useful in Agriculture
GM crops resistant to herbicides → only weeds killed when crop sprayed with herbicide
GM crops resistant to insect attack → reduce use of insecticide
GM crops with added nutritional value (eg. Golden rice has a precursor of vitamin A)
GM animals with increased growth hormone production (eg. Salmon)
79
how recombinant DNA technology can be useful in Industry
GM bacteria produce enzymes used in industrial processes and food production
80
Describe gene therapy
● Introduction of new DNA into cells, often containing healthy / functional alleles
● To overcome effect of faulty / non-functional alleles in people with genetic disorders eg. cystic fibrosis
Note- if body cells are altered, changes are not heritable. Gene therapy in gametes is currently illegal.
Students should be able to relate recombinant DNA technology to gene therapy.
81
Suggest some issues associated with gene therapy
● Effect is short lived as modified cells (eg. T cells) have a limited lifespan → requires regular treatment
● Immune response against genetically modified cells or viruses due to recognition of antigens
● Long term effect not known - side effects eg. could cause cancer
○ DNA may be inserted into other genes, disrupting them → interfering with gene expression
82
Suggest why humanitarians might support recombinant DNA technology
● GM crops increase yields → increased global food production → reduced risk of famine / malnutrition
● Gene therapy has potential to cure many genetic disorders
● ‘Pharming’ makes medicines available to more people as medicines cheaper
83
Suggest why environmentalists and anti-globalisation activists might
oppose recombinant DNA technology
● Recombinant DNA may be transferred to other plants → potential herbicide resistant ‘superweeds’
● Potential effects on food webs eg. affect wild insects → reduce biodiversity
● Large biotech companies may control the technology and own patents
●
84
What are DNA probes?
● Short, single stranded pieces of DNA
● With a base sequence complementary to bases on part of a target allele / region
● Usually labelled with a fluorescent or radioactive tag for identification
85
Suggest why DNA probes are longer than just a few bases
Suggest why DNA probes are longer than just a few bases
● A sequence of a few bases would occur at many places throughout the genome
● Longer sequences are only likely to occur in target allele
86
What is DNA hybridisation?
● Binding of a single stranded DNA probe
to a complementary single strand of DNA
● Forming hydrogen bonds / base pairs
87
Explain how genetic screening can be used to locate specific alleles of genes
1. Extract DNA and amplify by PCR
2. Cut DNA at specific base sequences (either side of target gene) using restriction enzymes
3. Separate DNA fragments / alleles (according to length) using gel electrophoresis
4. Transfer to a nylon membrane and treat to form single strands with exposed bases
5. Add labelled DNA probes which hybridise / bind with target alleles (& wash to remove unbound probe)
6. To show bound probe, expose membrane to UV light if a fluorescently labelled probe was used
OR use autoradiography (expose to X-ray film) if a radioactive probe was used
88
What is gel electrophoresis?
● A method used to separate nucleic acid (DNA / RNA) fragments OR proteins
● According to length / mass (number of bases / amino acids) AND charge (DNA is negatively charged due
to phosphate groups and protein charge varies based on amino acid R groups)
89
Explain how gel electrophoresis can be used to separate DNA fragments
1. DNA samples loaded into wells in a porous gel and
covered in buffer solution (which conducts electricity)
2.Electrical current passed through → DNA is negatively
charged so moves towards positive electrode
3. Shorter DNA fragments travel faster so travel further
90
How can data showing results of gel electrophoresis be interpreted
● Run a standard with DNA fragments / proteins of known lengths under the same conditions
● Compare to position of unknown DNA fragments / proteins to estimate their size
● Shorter DNA fragments/ proteins travel further / fasteR
91
Describe examples of the use of labelled DNA probes
● Screening patients for heritable conditions (eg. cystic fibrosis)
● Screening patients for drug responses (some alleles code for enzymes involved in drug
metabolism that enable better responses to certain drugs)
● Screening patients for health risks (some alleles predispose patients eg. to high blood cholesterol)
92
Describe the role of a genetic counsellor
Explain results of genetic screening, including consequences of a disease
Discuss treatments available for genetic condition
Discuss lifestyle choices / precautions that might reduce risk of a genetic
condition developing eg. regular screening for tumours or a mastectomy
Explain probability of condition / alleles being passed onto offspring →
enable patients to make informed decisions about having children
93
What is personalised medicine?
● Medicine tailored to an individual'
s
genotype / DNA
● Increasing effectiveness of treatment
eg. by identifying the particular
mutation / allele causing cancer and
treating it with tailored drugs
94
Arguments for the screening of individuals for genetically determined conditions
and drug responses
✓ Can enable people to make lifestyle choices to reduce chances of diseases developing
✓ Allows people to make informed decisions about having their own biological children
✓ Allows use of personalised medicines, increasing effectiveness of treatment
95
Arguments against the screening of individuals for genetically determined conditions
and drug responses
𝖷 Screening for incurable diseases or diseases that develop later in life (where nothing
positive can be done in response) may lead to depression
𝖷 Could lead to discrimination by insurance companies / employers
𝖷 May cause undue stress if patient does not develop the disease
96
Amplifying dna means
Cloning dna genes so that you have many copies
97
What are variable number tandem repeats (VNTRs)
● Repeating sequences of nucleotides / bases (eg. GATA)
● Found within non-coding sections of DNA at many sites throughout an organism’s genome
98
Why are VNTRs useful in genetic fingerprinting?
● Probability of two individuals having the same VNTRs is very low
● As an organism’s genome contains many VNTRs and lengths at each loci differ between individuals
99
Explain how genetic fingerprinting can be used to analyse DNA fragments
1. Extract DNA from sample (eg. blood cells) and amplify by PCR
2. Cut DNA at specific base sequences / recognition sites (either side of VNTRs) using restriction enzymes
3. Separate VNTR fragments according to length using gel electrophoresis (shorter ones travel further)
4. Transfer to a nylon membrane and treat to form single strands with exposed bases
5. Add labelled DNA probes which hybridise / bind with complementary VNTRs (& wash to remove
unbound probe)
6. To show bound probe, expose membrane to UV light if a fluorescently labelled probe was used
OR use autoradiography (expose to X-ray film) if a radioactive probe was used
100
Compare and contrast genetic fingerprinting with genetic screening
● Both use PCR to amplify DNA sample
● Both use electrophoresis to separate DNA fragments
● Both use labelled DNA probes to visualise specific DNA fragments
● Genetic fingerprinting analyses VNTRs whereas genetic screening analyses specific alleles of a gene
101
Explain how genetic fingerprinting can be used to determine genetic
relationships
● More closely related organisms have more similar VNTRs, so more similarities in genetic fingerprints
● Paternity testing- father should share around 50% of VNTRs / bands with child (due to inheritance)
●
102
Explain how genetic fingerprinting can be used to determine genetic
variability within a population
Differences in VNTRs arise from mutations, so more differences show greater diversity within a population.
103
Explain the use of genetic fingerprinting in the fields of forensic science,
Compare genetic fingerprint of suspects to genetic fingerprint of dna at crime scene
If many bands match the suspect was likely present at the crime scene
104
Explain the use of genetic fingerprinting in the fields of ,
medical diagnosis
● Some VNTR patterns are associated with an increased risk of certain genetic
disorders eg. Huntington
105
Explain the use of genetic fingerprinting in the fields of, animal and plant breeding
● Shows how closely related 2 individuals are, so that inbreeding can be avoided
- Breed pairs with dissimilar genetic fingerprints
