manipulating genomes Flashcards
what does a PCR allow scientists to do
produce a lot of DNA from tiniest original sample
(amplify the DNA)
from 1 million to 10 billion copies
requirements for PCR
DNA sample
excess triphosphate of the 4 bases
enzyme DNA polymerase
PCR machine (thermal cycler)
primers
Mg2+ cofactor for DNA polymerase
what are the excess triphosphate of the 4 bases called in PCR
dNTP’s (deoxynucleotide triphosphate)
what DNA polymerase enzyme is used in PCR & why
Tap polymerase (from thermophilic bacterium = Archaea)
what does Mg2+ cofactor allow for in PCR
enables tight binding between active site and substrate
what are primers and what are they used for in PCR
short sequences of bases
site of attachment for Taq polymerase to bind
stages of PCR
- denaturation of double stranded DNA
- annealing of primers
- elongation/synthesis of DNA
denaturation of double stranded DNA (step 1 of PCR) description
H bonds are broken between the 2 strands to form 2 separate strands (normally carried out in the body by helicase enzyme)
what temperature is denaturation of double stranded DNA (step 1 of PCR) carried out at
90-95C
annealing of primers (step 2 of PCR) description
primers bind to 3’ end of DNA
needed for DNA/Taq polymerase to attach and start replication
primers bind by H bonds
what temperature is annealing of primers (step 2 of PCR) carried out at
55-68C
elongation/synthesis of DNA (step 3 of PCR) description
Taq polymerase moves from 5’ to 3’ direction, forming phosphodiester bonds between nucleotides
complementary strand of DNA formed
what temperature is elongation (step 3 of PCR) carried out, why at and for how long
71-75C (optimum temperature for Taq polymerase)
for at least 1 minute
how many copies of DNA sample does 30 cycles of PCR give
about 1 billion
in PCR, how is size of DNA sample said to grow
exponentially (it is logarithmic)
how many fragments of DNA after 5 PCR cycles
2^5= 32
log32= 1.51
10^1.51 fragments
what is electrophoresis (general)
a technique used in laboratories in order to separate macromolecules (DNA or proteins) based on size
how accurate is electrophoresis
accurate enough to separate nucleic acid fragments that are different buy only 1 base in length
what is agarose
carbohydrate mesh compatible w DNA/protein in electrophoresis
what does electrophoresis use as medium
a gel ‘plate’ or ‘slab’ containing agarose which is covered in a buffer solution
purpose of buffer solution in electrophoresis
allows electrical current to travel across whole tank
what is attached at each end of gel in electrophoresis and why
electrodes
so a current can be passed through it
step by step basic procedure to separate DNA fragments in electrophoresis
- dna samples treated w restriction enzymes to cut large fragments to smaller fragments
- dna samples placed in wells cut in negative electrode (cathode) end of gel
- gel immersed in tank of buffer solution and an elec current passed through solution for fixed time period (usually 2 hr)
- DNA is -vely charged, so attracted to +ve electrode (so DNA fragments diffuse through gel towards +ve electrode end (anode))
- shorter lengths of DNA move faster and so move further in fixed time that current is passed through gel
- position of fragments can be shown using dyes that stain DNA molecules OR southern blotting can be used w radioactive probes if a particular sequence is being searched for
what is negative electrode called
cathode
what is positive electrode called
anode
why is DNA negatively charged
because of the many phosphoryl (phosphate) groups (-ve sugar phosphate backbone)
how can position of fragments be shown after electrophoresis
use dyes that stain DAN molecules
us Southern blotting and radioactive probes (if a particular sequence is being searched for)
why do different proteins move less/more far through gel in electrophoresis
proteins have different R groups so different 3D shapes (tertiary structures) and overall charges which would affect movement through gel
method used to cancel out charges of protein R groups
SDS PAGE
how does SDS page work?
when a protein mixture is heated in presence of SDS, the protein is denatured (reverts to its primary structure) and so charges and hydrophobic regions are exposed
the SDS detergent wraps around the polypeptide backbone so that the intrinsic charges of polypeptides become negligible when compares to the -ve charges contributed by SDS
SDS binds to proteins in proportion to what
what does this result in
their RMM
a molecule with a uniform mass:charge ratio
this means they can be separated on the basis of their size
results of SDS PAGE to proteins
all proteins now linear (straight chains of amino acids)
all molecules negatively charged so all move in same direction in gel (attracted to anode)
small molecule move through gel faster (bc lower RMM)
standards of known mass run in adjacent lanes for comparison (control/baseline)
proteins obtained from gel for identification
uses of SDS PAGE
analysis of haemoglobin for diagnosis of sickle cell anaemia (missense mutation w 1 amino acid change)
urine protein electrophoresis (proteins w/ an MR >69000 are able to pass into BC)
analysis of plasma proteins for diagnosis
how much DNA is used when an individuals DNA is profiled
short sections of non-coding DNA (satellite DNA, does not code for proteins)
what does a human genome contain
simple repetitive sequences that are scattered throughout our 46 chromosomes which are called Tandem repeats
what are variable number tandem repeats
tandem repeats which are highly variable in length
why is every person’s DNA is unique
due to the variable length of their tandem repeats
this can be used to identify them
where do tandem repeats occur
at more than 1000 locations in the genome
what is a DNA probe
short sequence of DNA that binds complementary to certain sequences
stages involved in DNA profiling
extraction
digestion
separation (gel electrophoresis)
separation (southern blotting)
hybridisation
development
describe stage 1 extraction in dna profiling
dna extracted from sample semen, blood, skin cells, hair roots, saliva
PCR used to amplify DNA
describe stage 2 digestion of DNA profiling
strands of DNA are cut into small fragments using restriction endonuclease
different enzymes cut DNA at specific nucleotide sequences
all restriction enzymes make 2 cuts: one through each strand of DNA (cuts leave VNTRs intact)
describe stage 3 Gel electrophoresis of DNA profiling
cut fragments separated on basis of charged particles moving through an agarose gel under the influence of an electric current
-ve charged PO4^3- groups cause DNA to move to anode
smaller fragments move further
gel immersed in alkali to separate DNA double helix into single strands
describe stage 4 southern blotting of DNA profiling
single stranded DNA transferred to nylon membrane which is placed over gel
membrane covered w several sheets of dry absorbent paper drawing alkaline solution containing DNA through membranes
DNA unable to pass through membrane and is transferred to same relative position on membrane as in gel
DNA is fixed
describe stage 5 hybridisation of DNA profiling
radioactive or fluorescent probes are added in excess to DNA fragments
DNA probes are short DNA or RNA sequences complementary to known DNA sequence and it binds to it
probes identify VNTRs
describe stage 6 development of DNA profiling
radioactive probes-> x ray image taken -> autoradiograph
fluorescent probes -> UV light and they glow
fragments give a pattern of bars called a DNA profile
unique profile for each person except identical twins
technique is v sensitive and even a trace of DNA left when someone touched an object can produce results
uses of DNA profiling
forensic science
maternity and paternity cases
species identification
identifying individuals at risk of developing particular diseases
uses of DNA profiling: forensic science
criminal convictions:
DNA traces obtained from blood, semen, saliva, hair roots and skin cells. DNA profile compared to sample taken from suspect/criminal database
identification:
victims body parts after air crashes etc
match profiles from descendants of those lost in WW1 w unidentified remains of soldiers
uses of DNA profiling: maternity and paternity cases
half genetic material from mum and half from dad
all bars from child’s profile not matched in 1 parent must be matched in other parent’s profile
uses of DNA profiling: species identificaiton
used to demonstrate evolutionary relationship between different species
uses of DNA profiling: identifying individuals at risk of developing particular diseases
certain non-coding VNTRs have been found to be associated with an increased risk of a particular disease eg various cancers and heart diseases
2 examples of DNA sequencing
Sanger sequencing
pyrosequencing
define the term DNA sequencing
finding the order/sequence of bases/nucleotides in DNA
step by step Sanger sequencing
DNA chopped into fragments and each fragment is sequenced 5’ to 3’ (similar to DNA rep)
DNA for sequencing is mixed w a primer, DNA polymerase and an excess of normal free activated nucleotide and terminator bases
mixture placed in thermal cycler that rapidly changes temp in programmed intervals in a repeated cycle (96C, 50C, 60C)
each time a terminator base is incorporated instead of normal nucleotide, synthesis of DNA stops. (these r present in lower amounts and are added at random, resulting in many DNA fragments of diff lengths)
after many cycles, all possible length DNA chains will be produced
DNA chains separated according to length by capillary sequencing (works like gel electrophoresis in minute capillary tubes and shortest lengths travel fastest)
fluorescent markers on terminator bases used to identify final base of each fragment- lasers detect diff coloured tags and thus order of bases in sequence (of new complementary strand- use to build up sequence in OG DNA strand)
data fed into computer that reassembles genome by comparing all fragments and finding areas of overlap between them
what are terminator bases (used in Sanger sequencing)
modified version of the 4 nucleotides
called ddNTPs (dideoxynucleotide triphosphate)
have H instead of OH on C3
inhibit DNA polymerase
stop DNA synthesis when they are included
what are terminator bases given in Sanger sequencing
different coloured fluorescent tags or radioactive labels
what is a primer (used in Sanger sequencing)
short sequence of DNA that binds complementary to the DNA sample (allows DNA polymerase to attach)
Sanger sequencing DNA polymerase
must be thermostable eg. Taq
must withstand 96C
describe what happened at each temp in Sanger sequencing
96C: double strand of DNA separates to single strands (denaturation)
50C: primer anneals (binds to) DNA strand
60C: DNA polymerase starts to build up new DNA strand by adding nucleotides w complementary bases to ss template DNA
what is pyrosequencing also known as
high throughput sequencing
2 uses of DNA sequencing
bioinformatics
computational biology
what is bioinformatics w/ example use
creating online databases that solve global issues
it allows rapid access to large volumes of data which is universally available
format is the same across all countries
could be used to identify a source of disease outbreak, target most vulnerable individuals and start appropriate treatment
what is computational biology
making comparisons between DNA sequences, which allows comparison of newly discovered sequences and previously discovered sequences
why are bioinformatics and computational biology useful:
facilitate access to large amounts of data
format of information s inuversal
computational biology allows rapid comparison of stored sequences and new sequences eg. can analyse 3000 genes in 100 samples in minutes
genes can be put into clusters which show the same pattern of green expression
can perform statistical analyses
describe genome-wide comparisons between individuals: 2 types
human genome project
analysing genomes of pathogens
how many genes/base pairs does the human genome contain
24000 genes
3 billion base pairs
what is genomics
changing epidemiology (study of distribution and determinants of disease)
what do computerised comparisons between genomes of people with/without a disease allow for
detection of particular mutations that could be responsible for an increased risk of disease
example of using human genome project in epidemiology
mutations of BRCA1 gene linked to breast cancer
what are places where substitutions occur called
effects?
single nucleotide polymorphisms or SNPs
silent (no effect on protein), missense/nonsense (alter protein or way RNA regulates expression of another gene in some way)
what is methylation
adding a methyl group to certain chemical groups (cytosine and adenine) in DNA
plays a major role in regulating gene expression in eukaryotic cells
what does acetylation do
increase gene transcription
what does methylation in gene promoter region of DNA do
represses gene transcription (DNA wraps more tightly around histones)
what is epigenetics
control of gene expression by modification of DNA (switching genes on/off)
what can methods to map the methylation of whole human genomes help w
helps researchers understand the development of certain diseases
e.g. certain types of cancer & why they may not develop in genetically similar individuals
what does sequencing genomes of pathogens (fast and cheap) allow doctors to do?
find source of an infection eg MRSA
identify antibiotic resistant strains ensuring antibiotics only used when they will be effective (allows selection of a narrow-spectrum antibiotic), which is useful for bacteria that are slow to culture
monitor potenital epidemics e.g. covid 19
many pathogens eg viruses have a high mutation rate and so many strains exist (variants- antigens changing shape)
sequencing DNA allows doctors to identify them and then implement specific treatment plans: give examples
identify targets in the development of drugs
identify genetic markers coding for proteins which act as antigens which can be used in vaccines (allow recognition by immune system)
test to identify who is infected so they can self isolate to decrease transmission. tests look for pathogen antigen in body e.g. lateral flow tests for covid 19
how does bioinformatics allow for species identification
there are particular sections of the genome that are common to all species but vary between them, so comparisons can be made
scientists can determine which species an organism belongs to by comparison to a standard sequence for different species
species identification in animals
uses cytochrome c oxidase (evidence for evolution- look at amino acid sequence)
short section so can be sequenced quickly and cheaply, yet varies enough to give clear differences between species
fewer differences= more recent common ancestor
species identification in plants
cytochrome c oxidase region of DNA does not evolve quickly enough to show differences between species
2 region of DNA in chloroplasts are used
is species identification by bioinformatics available for fungi/bacteria yet
no suitable regions of DNA suitable yet
bioinformatics to find evolutionary relationships (phylogeny)
DNA sequences of diff organisms can be compared bc basic mutation rate of DNA can be calculated
scientists can work out how long ago 2 species diverged from a common ancestor
how do spliceosomes join same exons
single gene may produce several different versions of functional mRNA
coding for different sequences of amino acids- primary structure
resulting in different proteins
resulting in different phenotypes
what is synthetic biology
using GMOs to produce drugs/medicines/useful molecules
OR
synthesis of new genes
what is personalised medicine
the choice/development of a drug is linked to the genotype of the individual
examples of synthetic biology
information storage
production of medicines
novel protein
genetic engineering
use of biological system in industrial contexts
synthesis of new genes or replacement of faulty ones
synthesis of biosensors
food production
production of monoclonal antibodies for targeted drug deliveries
describe information storage (type of synthetic biology)
can encode vast amounts of digital info onto single strands of synthetic DNA
example: production of medicines (type of synthetic biology)
GM E.coli to make human insulin
describe genetic engineering (type of synthetic biology)
e.g. similar to Hb can bind to oxygen but not carbon monoxide
describe use of biological system in industrial contexts (type of synthetic biology)
‘cells’ (chemical cells) to hydrolyse cellulose -> sugars which can be used as liquid fuel
exampleof synthesis of new genes or replacement of faulty ones (type of synthetic biology)
eg treating cystic fibrosis (gene therapy)
example of synthesis of biosensors (type of synthetic biology)
eg GM bacteria that glow if air is polluted with eg petroleum pollutants
describe food production(type of synthetic biology)
decrease fertiliser use by engineering synthetic microbial communities
suggest how the interdisciplinary field of bioinformatics may be useful in determining whether a newly-sequence allele causes a genetic disease
base sequences of normal allele &known alternatives are held in database & amino acid sequences of known proteins
info held in universal format
computational analysis allows a rapid comparison between new sequences & previously known sequences
describe differences between DNA profiling and DNA sequencing
DNA profiling produces a fingerprint unique pattern (from specific section of DNA), sequencing doesnt
sequencing determines order of DNA bases, profiling doesnt
explain why only selected sections of non-coding DNA are used when profiling a human
in most people, genomes are very similar
so using coding sequences would not provide unique profiles
non-coding DNA contains short tandem repeats/variable number tandem repeats which vary between individuals
suggest why the binding of SDS to proteins is necessary for protein electrophoresis
standardise mass:charge ratio so fragments are separated out based on size/mass
SDS makes all protein negative so they can be separated like DNA (from -ve to +ve terminals)
what is a DNA probe
a short single-stranded piece of DNA (50-80 nucleotides long) that is complementary to a section of DNA being investigated
what is a DNA probe labelled by
using a radioactive marker (detected by exposure to x-ray)
using a fluorescent marker (emit colour when exposed to UV light)
what do DNA probes bind to
any fragment where a complementary base sequences is present
binding by complementary base pairing is called annealing (H bonds form)
uses of DNA probes
locates specific gene for genetic engineering
identify same gene in a variety of genomes e.g. separate species to show phylogeny
identify presence or absence of allele for a particular genetic disease (could inform genetic counselling)
used in electrophoresis
what do scientists use DNA microarrays for
to measure the expression levels of large numbers of genes simultaneously and reveal the presence of mutated alleles (expression level determined by presence of mRNA)
what does each DNA spot on a microarray contain
a specific DNA sequences (probe)
how does a microarray work
mature mRNA extracted from cells eg tumour and normal cell
mRNA converted to ss cDNA using reverse transcriptase
amplified using PCR
cDNA labelled with fluorescent markers
applied to DNA chip where it anneals to cDNA probes
reference (normal) and test (tumour) DNA samples are labelled w different fluorescent markers
where a test subject or reference marker binds to a particular probe the scan reveals the fluorescence of one colour, indicating the presence of a particular sequence in the DNA
where both bind with a particular probe the fluoresces of both colours is seen
MICROARRAY (red=tumour sample, green=normal sample):
what does red mean
gene highly expressed in tumour cell but not normal cell
MICROARRAY (red=tumour sample, green=normal sample):
what does green mean
gene highly expressed in normal cell but not in tumour cell
MICROARRAY (red=tumour sample, green=normal sample):
what does yellow mean
gene highly expressed in both tumour and normal cell
microarray: how is ratio of expression in tumour: normal cells calculated
the colour/intensity of the dots are converted to numbers and the ration is calculated
MICROARRAY:
what does a ratio greater than 1 mean
gene expression induced by tumour formation
this info can be used to target drugs to tumour cells
MICROARRAY:
what does a ratio less than 1 mean
gene expression repressed by tumour formation
MICROARRAY:
what does a ratio equal to 1 mean
tumour has no effect on gene expression
what is recombinant DNA
combing DNA from 2 different species (transgenic organism formed)
stages involved in genetic engineering
isolating desired gene
putting gene into vector
transferring vector into host cell
host cell expresses new gene
3 types of isolating desired gene in genetic engineering
directly from DNA
from mRNA
from the nucleotide sequence of the gene
step 1 of genetic engineering: isolating desired gene: directly from DNA
a DNA probe can be used to locate the gene within the genome and the gene can be cut out using a restriction endonuclease
these enzymes can be used to cut DNA at a specific sequence along the length of the DNA
some make a clean, blunt cut but many cut the 2 DNA strands unevenly leaving 1 strand longer than the other
what are regions of unpaired bases called
sticky ends
what do sticky ends allow for
tighter annealing between vector DNA and gene of interest (H bonds form as well as phosphodiester bonds)
step 1 of genetic engineering: isolating desired gene: from mRNA
mRNA is isolated for the desired gene from cells expressing the gene
e.g. pancreas cell for insulin gene
using the mRNA as a template, an enzyme called reverse transcriptase is used to make a singe strand of cDNA
primers are added and DNA polymerase can make this cDNA into a double stranded length (must be same as double-stranded plasmids)
why is isolating desired gene from mRNA rather than DNA advantageous
because introns have already been spliced out (post-transcriptional modification)
many copies of mRNA available
mRNA is only from gene coding for insulin (being expressed)
step 1 of genetic engineering: isolating desired gene: from the nucleotide sequence of the gene
if scientists know the nucleotide sequence of the gene, then the gene can be synthesised using an automated polynucleotide synthesiser (synthetic biology)
3 methods of putting gene into a vector in genetic engineering
using plasmids
using viruses
using liposomes
step 2 of genetic engineering: putting gene into a vector: using plasmids
plasmids are cut using the same restriction enzyme as was used to cut out the gene, so cut plasmid has complementary sticky ends
ligase enzyme catalyses the condensation reactions that form phosphodiester bonds between sugar and phosphate groups on 2 strands of DNA. H bonds form between complementary bases
this forms recombinant DNA
what is recombinant DNA
molecule created in vitro by joining foreign DNA with vector molecule e.g. plasmid and human gene
what type of genes do plasmids often have 2 of
marker genes
what are plasmid marker genes for and how can scientists use them
may have a gene for antibiotic resistance
scientists ca determine which genes have taken up the plasmid by growing on media containing antibiotics
however some plasmids will be modified but others won’t be
the 2nd marker gene gets cut when gene is inserted so no longer functions (used to determine which cells have taken up modified plasmid)
step 2 of genetic engineering: putting gene into a vector: using viruses
genes can be put into attenuated viruses (made harmless) that then carry gene into host cells
step 2 of genetic engineering: putting gene into a vector: using liposomes
liposomes consist of plasmid DNA surrounded by a lipid bilayer so it can fuse w the cell membrane of various cell types
liposomes can have monoclonal antibodies attached to their surface which means they target particular cell types (immunotherapy)
e.g. supplying genes to cancer cells to activate tumour suppressor genes
5 methods of transferring vector into host cell in genetic engineering
heat shock treatment
electroporation
electrofusion
transfection
using bacteria: Agrobacterium tumefaciens
step 3 of genetic engineering: transferring vector into host cell: heat shock treatment
bacteria subjected to alternation periods of 0C and 42C in presence of calcium chloride so walls and membranes become more permeable and allow DNA in
+ve calcium ions surround -vely charged parts of both DNA molecules and phospholipids in plasma membranes reducing repulsion between the 2
increases no. & size of pores which decreases need for channel/carrier proteins
step 3 of genetic engineering: transferring vector into host cell: electroporation
high voltage pulse applied to cell to disrupt membrane by inducing pores to form (by introducing recombinant DNA at same time as the electric field, the DNA is likely to be taken up)
used to get DNA plasmids into bacteria or DNA fragments into eukaryotic cells
step 3 of genetic engineering: transferring vector into host cell: electrofusion
tiny electric currents applied to membrane of 2 different cells
this fuses the cells and nuclear membranes of the 2 different cells together to forma. hybrid/ polyploid cell containing DNA from both
in plants, cell walls are removed using cellulase, followed by electrofusion, followed by use of hormones to stimulate growth of new cell wall
used to produce GM plants
used in somatic cell nuclear transfer
is electrofusion successful in animalss
no but it is important in the production of monoclonal antibodies
a monoclonal antibody is produced by a combination of a cell producing 1 single type of antibody w a tumour cell, which means it divides rapidly in culture
step 3 of genetic engineering: transferring vector into host cell: transfection
DNA can be packaged into a bacteriophage- virus that infects bacterial cells and transfers DNA into host bacterial cell
step 3 of genetic engineering: transferring vector into host cell: using bacterium Agrobacterium tumefaciens
plasmids are inserted into the bacterium which infects some plants and naturally inserts its genome into host cell genomes
(can introduce insecticide-resistant genes/ herbicide-resistant genes)
step 4 of genetic engineering: direct transfer of gene into recipient
small pieces of gold or tungsten are coated with the DNA and shot into plant cells = gene gun
differences between TLC and electrophoresis
TLC: separates by relative solubility, E: separates by relative size/length
TLC: separates uncharged particles, E: separates charged particles
TLC: buffer not used, E: buffer used
TLC: no dyes used, E: dyes used (fluorescence/ radioactive)
suggest why genome is fragmented before sequencing
genome is too large
fewer errors/greater accuracy
why is Taq polymerase used for PCR
it is thermostable i.e. does not denature at 95C during strand separation
PCR can cycle repeatedly without having to replace the enzyme
possible desired characteristics for plant GM
high yield
drought resistance
pesticide production
step by step genetic engineering in plats using agrobacterium tumefaciens/ particle gun
1a. desired gene is placed in Ti plasmid of bacteria along with a marker gene e.g. antibiotic resistance or fluorescence. desired gene carried directly into plant cell DNA as bacteria infects cell
1b. alternatively a gene gun is used to get the genes into plant cell
2. transgenic plant cells form a callus (mass of GM cells)
3. each cell in callus can be grown into a new transgenic plant using plant hormones to encourage root and shoot formation
what is gene therapy
inserting functional allele of a particular gene into a cell that contains mutant and non-functioning allele of that gene
if inserted allele is expressed, functioning protein will be produced
types of gene therapy
somatic gene therapy
germ line gene therapy
describe somatic gene therapy
only affects body cells (alteration to patients genome but these changed not inherited)
involves inserting functional allele into body cells
only temp. solution & when somatic cell dies, somatic cell replaced w stem cells that will have faulty allele (needs repetitive treatment)
faulty allele passed onto offspring
can only be used to treat recessive genetic disorders
dominant condition faulty protein produced and you can’t stop this and can’t remove dominant allele
describe germ line gene therapy
involves inserting cantonal alleles into gametes/zygotes
all cells of individual are altered (inherited by future generations and does not need to be repeated; is permanent)
has the potential to change genetic make-up of many people, the descendants original patient; none of whom could give consent)
concerns that genes may find their way into a location that could disrupt the expression & regulation of other genes (increased risk of cancer)
technology might eventually be used to enable people to choose desirable or cosmetic characteristics of their offspring
considered ethically impermissible for humans
successfully done on animal embryos
somatic vs gene line therapy permanence?
somatic= temporary, needs repetitive treatment
germ line= permanent, doesnt need to be repeated
how does germ line therapy increase the risk of cancer OR epigenetic changes
concerns that genes may find their way into a location that could disrupt the expression and regulation of other genes
gene could insert itself into a regulatory region
is gene transfer predictable
what does this depend on
no; unpredictable
depends on where the allele inserts
example of somatic gene therapy
cystic fibrosis
what is cystic fibrosis caused by
inheritance of 2 recessive alleles
describe cystic fibrosis
production of lots of very thick mucus as a result of a defective chloride ion channel
outward flow fo Cl- prevented which results in Na+ entering cell to balance charge
prevent water leaving cells resulting in thick mucus
how does somatic gene therapy treat cystic fibrosis
functional alleles (to synthesise functional protein= CFTR channel) can be packaged into virus/ liposomes which can then be inhaled (inhaler/nebuliser)
the functional alleles will get into some of the cells lining the respiratory tract and the host cell will produce functional CFTR protein (functional allele must pass through
nuclear membrane & integrate into chromosomes)
epithelial cells replace every 10-14 days so treatment must be repeated at regular intervals
problems with using a virus to insert gene (IN VIVO)
virus may still evoke an immune response
patient may become immune to virus so subsequent deliveries impossible
virus may insert allele in location that disrupts gene regulating cell division, increasing risk of cancer
virus may insert allele in location that disrupt regulation of gene expression of other gene
describe ex vivo somatic gene therapy
adult stem cells isolated from patient and propagated in lab and therapeutic gene introduced into cells
GM cells reintroduced into patient
produce cells w functional proteins
what does genetic manipulation refer to
changing structure of DNA in an organism
ethical issue with genetic manipulation
problem or situation that requires person/organisation to choose between alternatives that must be evaluated as right (ethical) or wrong (unethical)
general positive issues of genetic manipulation
benefit to human health (could improve symptoms of diseases)
decrease starvation, make life-saving drugs, patenting generates funds for research, technology transfer to LIDCs: improved QoL
reduced use of pesticide (decreased chance of bioaccumulation)
general negative issues of genetic manipulation
risk to human health (could cause frameshift mutations)
animal welfare; not right to treat animals poorly
GM animals reduced to commodities
patenting increases cost
GM crops encourage monoculture (increased susceptibility to disease, climate change etc.) and decrease biodiversity (decrease species diversity)
using organisms as models for disease deliberately causes them harm
examples of GM plants
insect resistant GM soya
herbicide resistant soya
nutritionally enhances GM rice: golden rice
positive ethical impacts of using insect resistant GM soya
increases yield, cheaper product
reduces starvation
reduced use of chemical pesticides
benefit to human health
negative ethical impacts of using insect resistant GM soya
pest may become resistant to toxin
engineered plant would be ineffective , pest number would increase and eat other crops
increases starvation
plant may produce toxins which are toxic to humans
risk to human health
Bt is toxic to some non-pest species, reducing biodiversity (reduced species diversity)
describe insect resistant GM soya
soya= good source of protein, vitamins and minerals
Bt toxin coded for= poisnonous to insect pest
binds to receptors of epithelial cells in larva gut, causing formation of pores/ion channels so WP imbalance, killing insect
describe herbicide resistant soya
modified plant expresses a gene from agrobacterium tumefaciens
allows plant to make essential amino acids even after herbicide spray
positive ethical impacts of herbicide resistant soya
use of herbicide kills competing weeds (decreased interspecific competition)
therefore increases yield
reduces starvation
negative ethical impacts of herbicide resistant soya
may encourage monoculture, decreasing biodiversity
encourages natural selection of super weeds (directional selection)
herbicide risk to human health bc carcinogenic
leaches into waterways, leading to eutrophication
describe golden rice
2 genes (1 from daffodils, 1 from soil bacteria) inserted into rice genome to activate production of beta carotene, a precursor for vitamin A which is needed to produce visual pigment rhodopsin (improves vision, particularly at night)
positive ethical impacts of golden rice
reduces blindness in potentially over 500,000 annually in India
negative ethical impacts of golden rice
potential costs issues for seeds but free humanitarian use licences offered to farmers so they can keep and replant seeds -> stunt to gain public acceptance of use of GM crops
reduce in biodiversity
safety of engineered rice?
risk to human health
GM rice could breed with wild type and contaminate these populations
describe pharming
GM animals to produce a human protein for use as a medicine
examples of pharming
gene inserted into fertilised sheep’s egg, along w a promotor sequence (specific location for RNA polymerase to bind to) so that the gene is expressed only in the mammary gland so protein can be harvested from the milk
transgenic sheep with human gene that coded for AAT (decreases emphysema): protein too large for production in bacterial cell
goats w gene for spider milk
creating animal models so they develop certain diseases e.g. mice
positive ethical impacts of pharming
(sheep milk protein production, goat spider silk, animal model diseases)
used to treat hereditary deficiency of AAT which leads to emphysema
benefit to human health
silk from goats used for sutures, artificial ligaments
animal models allow diseases to be studies and drugs tested
benefit to human health
negative ethical impacts of pharming
(sheep milk protein production, goat spider silk, animal model diseases)
inserting foreign alleles into another organism may cause them harm (ie could disrupt regulatory genes)
possible that trans gene would be activated in places other than mammary gland and resulting protein may be toxic to animal
inflicting unnecessary suffering on an animal (but they are valuable so very well looked after)
GM humans in gene therapy positive ethical impacts
benefit to human health
e.g. potential treatment of cystic fibrosis
negative ethical impacts of GM humans in gene therapy
potential problems of using viruses as vectors:
may insert allele in location that disrupts gene involved in regulating cell division, leading to cancer or disrupting expression of other genes
human germ line therapy is ethically impermissible (many generations of offspring cannot have given consent)
describe/examples GM pathogens for research
GM viruses with no virulence can be used to make vaccines
some tumour cells have receptors on membranes for poliovirus so poliovirus will recognise and attack them
poliovirus GM to inactivate genes that cause polio
positive ethical impacts of GM pathogens for research
(viruses w no virulence for vaccines, poliovirus gene inactivation)
reduces chance of vaccine making recipient ill and vaccines saves lives
possible treatment for some forms of brain cancer and saves human lives
negative ethical impacts of GM pathogens for research
(viruses w no virulence for vaccines, poliovirus gene inactivation)
researchers become infected with live pathogen (unlikely as more harmless viruses chosen), but potential mass outbreak of disease
GM virus reverts back to original form, leading to disease outbreak
use in biowarfare
positive ethical impacts of GM pathogens
GM bacteria makes human insulin: benefit to diabetics (T1 autoimmune)
GM viruses can also act as vectors in gene therapy
negative ethical impacts of GM pathogens
bacteria have antibiotic resistance genes and could transfer these to other bacteria by exchanging plasmids (modified so can’t grow outside lab)
allele may be inserted into genome in a way that increases risk of cancers or may interfere w gene regulation: risk to human health
describe patenting
legal protection for GM techniques or products
by law can control who uses product and how for a set period of time
positive ethical impacts of patenting
owner of patent gets money from product: used in further research, generates competition between companies carrying out GM, get GM products faster
patent allows public to hold owner of patent accoutnable
negative ethical impacts of patenting
patented products not affordable in 3rd world, so starvation increases
treats life as a commodity; patent undermines dignity by allowing ‘ownership’ of genes
positive ethical impact of technology transfer (sharing of GM knowledge, skills and tech)
globally GM products can be created at a faster rate
negative ethical impact of technology transfer (sharing of GM knowledge, skills and tech)
may be cost implications if patents involved which may limit who uses GM product
differences between somatic gene therapy and germ line gene therapy
S: insertion of functional allele into body cells, GL: insertion of functional allele into gamete/zygote
S: temporary, short term solution which needs repetitive treatment, GL: permanent long term solution, no further treatment required
S: cannot be inherited, GL: can be inherited
S: only some cells get allele, GL all cells get allele
“GM plants and animals should be classed as new species””
evidence for/against
use fertility as a basis
breed GM organisms with non-GM and observe whether they are fertile
is so, same species
or compare DNA band patterns by electrophoresis
explain why primers are needed for PCR but not for natural DNA replication
DNA polymerase cannot bind to ssDNA
which parts of the genome are compares in DNA profiling for forensics
non-coding regions (introns, STRs, VNTRs, minisatellites, micro satellites)
in gel electrophoresis, what are DNA markers
mixtures of DNA molecules of known size
run in 1 lane & used to estimate sizes of other DNA samples
explain how genome sequencing an help identify evolutionary relationships
closer % match of genome sequence means less time since the 2 species diverged from a common ancestor
what is epidemiology
study of incidence, distribution and possible control of disease (& other factors relating to health)
what is a restriction enzyme
enzyme that cuts a dsDNA fragment at a specific place (its recognition site)
what is meant when a restriction enzyme site is said to be ‘palindromic’
has same sequence on both strands (reading from 5’ to 3’)
what process could be used to describe the reverse of restriction digestion?
DNA ligation
name common vectors used in genetic engineering
cosmids
viruses
BACs
liposomes
what is the role of DNA ligase
joins DNA backbone/sugar phosphate backbone
makes phosphodiester bonds
how are restriction enzymes used in genetic modification
cut plasmid,
isolate gene
producing sticky ends
negative aspect of GM-ing plants
expensive to buy so farmers may be priced out
chance of gene transfer to create superweeds
create monocultures (susceptibel to extinction)
why is it harder to treat genetic disorders caused by dominant alleles than disorders caused by recessive alleles
recessive allele treatment needs addition of ‘correct’ allele anywhere in genome
treatment of dom. allele disorder requires that specific gene to be disrupted/silenced -> more specific placement of inserted DNA
similarities between DNA sequencing and DNA replication
both sequence 5’ to 3’
both use DNA polymerase
both require dNTPs
similarities between PCR and DNA replication
both copy DNA
both use DNA polymerase
both sequence 5’ to 3’
both form phosphodiester bonds between nucleotides
both require dNTPs
H bonds break between complementary strands in both
differences between DNA sequencing and DNA replication
S: involves electrophoresis to separate strands in mass order, R: no electrophoresis
S: whole new complementary fragment sequenced, R: each new DNA mol consists 1 original strand and 1 new strand (semi conservative)
S: sequencing of unknown fragments, R: replication of known sequences
S: requires addition of synthetic primers, R: no primers
S: requires thermal cycling, R: none
S: H bonds break due to high temps 96C, R: H bonds break due to helicase
S: no gyrase or ligase, R: uses gyrase and ligase
S: terminator bases (ddNTPs) involved, R: none
differences between PCR and DNA replication
P: exponential growth, R: linear growth
P: DNA polymerase has optimum temp 64 to 62C : Taq, R: optimum temp 37C
P: only short sequences can be rep, R: entire chromosome replicated
P: more copies of DNA forms, R: 1
P: requires addition of synthetic primers, R: none
P: H bonds broken by high temps, R: H bonds broken by helicase
P:thermal cycling, R: body temp
P: no gyrase or ligase, R: both
P: Mg ion cofactor, R: none
what is a clone w a couple examples
genetically identical organisms produced by asexual reproduction (mitosis)
e.g. yeast budding, bacteria by binary fission
advantages of cloning
if conditions favour the parents they will also favour the offspring
rapid process so can rapidly colonise new environment
only 1 parent needed
disadvantages of cloning
overcrowding so leads to intraspecific competition
limited genetic diversity (except mutations)
natural selection does not occur
environmental changes e.g. new disease or drastic climate change may wipe out a population if all are vulnerable
how are plant clones formed
by vegetative propagation (asexual reproduction in which new plants develop from meristematic regions (undifferentiated cells in vegetative organs of plant (stem, roots and leaves)) rather than specialised reproductive structures)
types of vegetative propagation
runners
rhizomes
tubers
suckers
bulbs
corms
leaves/plantlets
layering
what are runners
example
use in horticulture
horizontal stems on surface of ground that can form roots at certain points
strawberries
removing young plants from runners
what are rhizomes
example
use in horticulture
stout horizontal stems underground that can form roots at certain points
ginger
cutting up rhizomes
what are tubers
examples
use in horticulture
swollen end of underground stem/root
potatoes=stem, dahlias=root
removing tubers and planting separately
what are suckers
example
use in horticulture
new stems that grow from roots of plants
banana trees
dig out suckers and plant separately
what are corms
example
use in horticulture
underground solid stem w scaly or fleshy leaves
crocus
dividing up corms, remove baby corms, known as cormels or cormlets, attached to the bottom
what are leaves/plantlets
example
use in horticulture
immature plants grow on leaf margins, drop off and take root
Bryophyllum spider plants
remove immature plants and plant separately
what is layering
example
use in horticulture
portion of an aerial stem grows roots while still attached to the parent plant and then detaches as an independent plant
honeysuckle peg stem below ground to encourage root formation
describe how to take a cutting (4 marks)
cut a healthy shoot between 2 nodes at an angle
dip in rooting powder
plant in deep compost
remove lower leaves and cover with a transparent plastic bag
why would you remove a shoot for a stem cutting in the early morning
contains most water bc stomata not open overnigth
why a clean diagonal cut when taking a cutting
increased surface area for root growth and uptake of water and minerals
why remove lower leaves when taking a cutting
most energy can be channelled into root growth and less water loss by transpiration
why remove flowers and buds when taking a cutting
stop energy usage for flowers but increased energy for root growth
what does rooting powder contain when taking plant cutting
synthetic auxin
cytokinins
to increase root growth
why should compost for plant cutting be well watered and not too compressed
air spaces for O2
provides water for p/s
why cover plant with transparent plastic bag or cut off lemonade bottle when doing plant cutting
light can reach it
increases humidity to decrease water loss
why keep plant cutting warm but avoid direct sunlight
allows low levels of p/s
warm for enzymes
avoid damage to chloroplasrs
why mist leaves of plant cutting regularly
maintains high humidity
what is grafting
joining 2 plant stems together to produce hybrid variety w combined characteristics
grafting use
select for particular colour
select for disease resistance, pesticide production, heat tolerance
what is tissue culture
series of techniques used to grow cells or tissues or organs from a small sample of cells or tissue
carried out on a (agar) nutrient medium under sterile conditions
application of plant growth substances at the correct time can encourage the cells in growing tissue to differentiate
what is micropropagation
making large numbers of genetically identical offspring from a single parent plant
step by step micropropagation
suitable piece of plant taken and cut into small pieces (explant) e.g. leaf
meristematic tissue often used as this is usually free from virus infection e.g. apical bud
explants sterilised using dilute bleach/alcohol
explants placed in sterile growth medium. aseptic techniques used. sterile agar gel contains suitable nutrients e.g. amino acids, sucrose and a high conc of growth substances e.g. auxin, cytokinins to stim. mitosis for root/shoot growth
once a callus forms, split into many smaller calluses
clumps stimulated to grow, divide and differentiate into plant tissues by moving cells onto diff growth media (changing ratio at different times/stages of growth e.g. more auxins for root growth, less for shoot growth)
tiny platelets transferred to greenhouse to be grown in compost or damp soil and acclimatised to normal growing conditions (LI, temp, CO2 conc)
why are explants sterilised in micropropagation
kills any bacteria/fungi which would otherwise thrive in these conditions
examples of aseptic techniques used in micropropagation
wash hands
disinfect bench
windows closed
bunsen flame to create convection current
what is a callus
mass of undifferentiated totipotent (can differentiate into any cell type) identical cells
arguments in favour of micropropagation
allows rapid production of large number of plants w known genetic makeup and therefore display desirable characteristics
generally produces disease-free plants
can produce viable number of plants after GM or selective breeding
way of producing a large number of plants which are seedless (sterile) eg bananas and grapes
way of growing plants which are naturally relatively infertile or difficult to grow from seeds eg orchids
way of reliably increasing the no. of rare/ endangered plats
new plants are uniform in phenotype which makes them easier to grow and harvest
facilities can be set up anywhere in the world at any time so not dependent on climate
arguments against micropropagation
produces a monoculture so all plants may be susceptible to same disease/ change in env.
if source material is infected by a virus, all clones will be infected (explants and platelets are vulnerable to infection by moulds)
large numbers of plants can be lost during the process
loss of variation and genetic diversity
labour intensive: relatively expensive to maintain sterile conditions and requires skilled workers
why are plants more able to form natural reproductive clones than animals
have meristematic tissue and almost all stem cells are totipotent
plant specialised cells can undifferentiated and then re-differentiate
animals only have multipoint stem cells and tissue specific stem cells
cloning in invertebrate animals name and brief description
parthenogenesis
in greenflies and water fleas
female produces a diploid egg without fertilisation
describe parthenogenesis in aphids
summer growing season: female aphids produce diploid eggs by mitosis (genetically identical to each other and parent)
these eggs develop inside the body of the female, hatch and emerge as miniature adults (not fertilised by sperm to form zygotes)
some species produce winged females in summer months (probably triggered by low food quality or poor conditions for colony)
in autumn a change in photoperiod and temp (or lower food quality/quantity) causes females to produce females and males by parthenogenesis. sexual female and males mate, and females lay eggs that dev outisde of mother. eggs endure winter and emerge as winged or wingless females in following spring (genetic variation as a result of meiosis and random fertilisation)
examples of natural clones in animal species
parthenogenesis
fragmentaiton in flatworms
budding in hydra
natural identical (monozygotic) twins in mammals
describe fragmentation in flatworms
flatworm stretches itself to breaking point and each part forms a complete flatworm
describe budding in hydra
small ‘buds’ produced on side of adult body
form tentacles and separate from adult
describe natural identical (monozygotic) twins in mammals
fertilised egg (zygotę) or early embryo splits in 2
both the embryos that are formed implant in uterus and develop
3 per 1000 natural pregnancies in humans
rare in domestic cattle
what does artificial embryo splitting/twinning/cloning produce
2 or more individuals that are clones of each other but not of either parent
describe artificial embryo splitting/twinning/cloning in cow example
cows w desirable traits treated w hormones to super ovulate
treat female w hormones to ensure correct stage of menstrual cycle
ova may be fertilised naturally or by artificial insemination by a bull w good traits
early embryos fused out of uterus
around day 6 when cells of embryo are still totipotent (can still become cells of placenta/umbilical cord). embryos are split to produce several smaller embryos
each embryo grown in lab for few days and a single one implanted into each surrogate mother (female cows have single pregnancies)-> in pigs a number of cloned embryos are put into a surrogate mother (multiple piglets)
number of genetically identical cows born from different mothers (exact phenotype unknown until birth so no guarantee)
embryos may be frozen and transported around the world
what is super ovulating
producing many eggs
describe reproductive cloning by enucleation and somatic cell nuclear transfer (SCNT) using sheep as example
somatic cell taken from udder of female transgenic sheep and extract nucleus using micropipette
take an egg cells from a female of the same species and enucleate it
use electrofusion/electroporation/heat shock treatment to fuse somatic nucleus into empty egg cell:triggers cell division by mitosis
split embryo into many smaller embryos and implant each into a surrogate mother
requirement of surrogate organism for SCNT
must be of same species
most be of good health, free from disease, treated w hormones so at correct stage of menstrual cycle
offspring of SCNT DNA
mitochondrial DNA identical to that of egg donor (from enucleated egg cell)
nuclear DNA identical to that of nucleus donor
what is non-reproductive/ therapeutic cloning?
follows same initial stages as reproductive cloning but once embryo has formed the cells are removed and subdivided
these stem cells can grow into any type of cell
induced pluripotent stem cells formation and use in medicine
patients cells taken e.g. skin and treated w reprogramming factors and pluripotent stem cells formed that are genetically identical to patient so will not be rejected
SCNT: longevity of animals?
dolly wasp put down when’s he was 6 years old bc she suffered form arthritis and lung disease
techniques have been improved ad mice now cloned that have normal life expectancies
use of animal cloning in agriculture
producing many individuals that have same productive features
e.g. high milk yield
uses of animal cloning in medicine: subcategories?
- pharming
- transplants
- scientific research
uses of animal cloning in medicine: pharming
producing lots of genetically identical sheep that have been genetically engineered to produce human proteins
e.g. human factor VIII (blood clotting factor)
goats w spider silk genes (can produce silk in their milk, used for suturing)
uses of animal cloning in medicine: transplants
producing GM pigs which grow organs that have the potenital to be used in human transplants
pigs used, HOWEVER their organs are coated in carbohydrate molecule that triggers reaction reaction in humans (autoimmune response) SO pigs are GM using gene editing technologies e.g. make antigens less harmful to reduce risk of rejection
why are pigs used for transplants
pigs used bc anatomically similar to humans, large litters, reproduce rapidly
uses of animal cloning in medicine: scientific research
share findings with other scientists on the effects of medicinal drugs
argument for using embryo splitting as a type of animal cloning
produce max. number of offspring (many more than normal reproduction) from particularly good animals
argument against using embryo splitting as a type of animal cloning
not possible to predict exactly how productive animals produced by embryo cloning will be as not genetically identical to either parent (phenotype unknown)
arguments for using SCNT as type of animal cloning
produces genetically identical copies of v high value individuals (cloning successful racehorses)
allows specific animals to be cloned eg replacing specific pet
potenital to allow rare endangered or extinct animals to be reproduced (simlar to plants)
arguments against using SCNT as type of animal cloning
very inefficient (Dolly took 277 cell fusions): many cloned animals fail to develop and miscarry/ produce malformed offspring
many SCNT cloned animals have short lifespans
factors contributing to extra cost of micropropagation compared to traditional methods
maintaining sterile conditions is expensive
requires skilled workers/specialised training
labour intensive
high setup costs
how is supply of cow egg cells obtained for cloning
egges flushed out of oviduct
ways of setting up gene bank for animal
sperm banks
egg banks
embryo freezing
zoos/wildlife reserves
how can cloning help save an endangered species of mammal
increase rate of reproduction so population size rapidly increased
doesn’t require a fertile female bc uses surrogate
embryo can be subdivided (embryo splitting)
van use adult cells from all existing members of species to maximise genetic diversity
differences between Sanger sequencing and high throughput sequencing
high throughput sequences more DNA/bases per unit time
high throughput can sequence longer DNA sequences
Sanger uses terminator bases
Sanger=1 enzyme
Sanger involves electrophoresis
