Manipulating Genomes Flashcards
what is the genome
entire genetic makeup
what is the proteome
all proteins produced by the genome of an organism
what do dna sequences determine
therefore dna sequencing =
the precise order of nucleotides in a DNA molecule, allowing genes to be isolated and read
= working out the sequence of bases
why does the genome have to be fragmented over time before sequencing
- genome too large
- smaller fragments make sequences more accurate (less errors)
is sanger sequencing/chain sequencing new or old
old
laborious
but more reliable
Process of Sanger Sequencing
- Add single strand of DNA template, DNA polymerase, and free (deoxy)nucleotides
(A/T/G/C) - In each tube add 1 of 4 types of dideoxynucleotides (A/T/C/G)
- If deoxynucleotide added to DNA strand, DNA replication continues
- If dideoxynucleotide added to DNA strand, DNA replication is stopped
- This produces many complementary DNA strands of different lengths
- These developing strands are separated from the template DNA
- They are separated by length using gel electrophoresis
● Gel electrophoresis has 4 wells (one for each dideoxynucleotide)
● The shorter the length of DNA, the faster it moves down the gel
● Allows the base sequence of the developing strand to be built up one base at a time
Dideoxynucleotides = modified nucleotides. how?
● Lack -OH group on 3’ carbon of sugar ring
o -OH group cannot bind to next nucleotide
o No more nucleotides can be added to chain
● When added to strand, will stop replication
● Labelled with radioactive isotope/different colour/fluorescent dye
what is Sanger sequencing
sequencing by chain termination
High-throughput sequencing - NEW (much faster) does what
● Detects when new nucleotide is added to the chain
what is Pyrosequencing
sequencing by synthesis
● 1 million reads occur simultaneously
what is an activated nucleotide
nucleotide with 2 phosphate
groups added
what does apyrase do
degrades activated nucleotides which have not been added to the template strand (not complementary)
Process of high-throughput sequencing/pyrosequencing
- Single strand of DNA, sequencing primer,
enzymes (DNA polymerase, ATP sulfurylase,
luciferase, apyrase) and substrates (APS and
luciferin) and one of activated nucleotide (ATP,
TTP, CTP, GTP) all added - When a (complementary to template strand)
activated nucleotide added to strand of DNA - 2 phosphate groups released as pyrophosphate
(PPi) - APS + PPi → ATP (in presence of ATP sulfurylase)
- Luciferin → oxyluciferin (in presence of luciferase and ATP)
- Light is released
Process of high-throughput sequencing/pyrosequencing image
Uses of DNA sequencing
● Genome-wide comparisons between individuals and species
Sequence the DNA of a whole genome → determines the complete DNA sequence of
organism
what can we do using dna sequencing
Can analyse & compare genomes of many humans
● Epidemiology - the study of the distribution and determinants of diseases
● Reveals diseases to which individuals are vulnerable to & likelihood of developing
certain diseases
Can analyse genomes of pathogens
● Identify source of infection
● Identify antibiotic-resistant strains
● Track outbreaks of potential epidemics
Can search for evolutionary relationships between organisms
● Identify species
● Identify when 2 different species diverged
● Investigate genetic variation
o More differences in genome between individuals → more genetic variation
Can interpret genotype-phenotype relationships
● Stop expression (“knock out”) different genes → what effect does this have on the
organism’s phenotype
● Can target specific base sequences to stop the expression of certain genes
what is Bioinformatics
- development of software needed to analyse and store raw biological data in
a database
● Data such as: DNA/RNA/protein sequences
what is computational biology
- uses raw biological data to build theoretical models of biological
systems, using computational techniques
what can computational biology help us predict
Can predict what will happen in certain situations
Prediction of the sequences of amino acids in polypeptides
This means…
Can predict how protein folds into its tertiary structure
Can use this for synthetic biology
what is synthetic biology
Synthetic biology is the creation of new or redesigning existing biological systems and
molecules
● Large alterations to an organism’s genome → cells act in completely new ways
Application of synthetic biology:
Production of medicines
what does dna profiling determine
genetic identity (DNA characteristics)of an individual
Uses of DNA profiling:
● Paternity and maternity tests
● Proving/disproving family relationships
● Identifying species & evolutionary history of organism
● Identifying in forensics
● Identifying individual with high risk for particular disease
DNA Samples are compared to:
Saliva
Blood
Hair roots
Skin cells
The process of DNA profiling
- DNA obtained from individual
o Extracted from sample
o PCR used to give many copies of sample - DNA digested with restriction enzymes
o Restriction endonuclease enzymes cut the DNA at specific restriction sites
within introns
o DNA cut into fragments (vary in size between individuals)
o VNTRs (variable number tandem repeats) known as STRs (short tandem
repeats) found within introns
● Number of STRs varies from person to person - Fragments separated by gel electrophoresis
- Radioactive/fluorescent DNA probes added which act as markers
o Bind to complementary strands of DNA
o Different banding patterns can be seen - DNA to which the individual’s DNA is being compared with is treated with the same
restriction endonuclease enzymes & subjected to gel electrophoresis
what are we looking for in dna profiling
to have many fragments in common with the sample
Short, single-stranded RNA/DNA sequences complementary to known DNA
sequence
● Bind to complementary strands of DNA (is complementary to DNA which is being
investigated)
● Act as markers
o Labelled by radioactive or fluorescent markers
what are STRs (short tandem repeats)
- highly variable, short, repeating lengths of DNA (found in
introns - do not code for proteins)
The exact number of STRs vary from person to person
who are Short VNTRS (variable number tandem repeats) unique to
VNTRs unique to everyone, except identical twins
what is PCR
polymerase chain reaction
why do we use pcr / to produce what
to produce many (billions) identical copies of the DNA replicated from a sample
in lab (in vitro)
o Which is needed for DNA analysis
● Each cycle doubles the amount of DNA
o DNA is amplified in vitro
Components of PCR:
● Buffer
● PCR tube
● Heat-resistant Taq polymerase (from thermophilic bacteria found in hot springs - organisms adapted to living at hot temperatures and so will not denature at high temperatures - unlike our bacteria)
● DNA sample
● Primers (forwards and reverse primer)
● Free nucleotides
● Thermal cycler (contains all components)
Stages of PCR:
- Denaturation
● Temperature increased to 95 for 1 minute
● DNA denatures - hydrogen bonds between complementary base pairs broken
● Strands are separated - Annealing of primers
● Temperature decreased to 55-60 for 1 minute
● Primers bind to ends of DNA strand
o Needed to allow replication of strands to occur - Synthesis of DNA/Elongation/Extension
● Temperature increased 72-75 for at least 1 minute - 2 minutes
● Taq polymerase (heat tolerant) used to bind to primer, and add nucleotides
o 72 optimum temperature for Taq polymerase
● Strand is copied
Other applications of PCR:
● Detecting mutations (which could lead to genetic disease)
● Identifying the presence of viral genome amongst the host cell’s DNA → identifies a
viral infection
● DNA profiling - making many identical copies need to identify/compare DNA
● Monitor spread of infectious disease
● Detecting oncogenes (detection of type of mutation involved in specific patient’s
cancer → medication can be better tailored to patient)
what are oncogenes
Oncogenes - mutated form of proto-oncogenes (involved in normal cell growth and cell division)
● Too many copies made of proto-oncogene OR more active than normal proto-oncogene → called an oncogene
what does gel electrophoresis do
separates DNA fragments/proteins in order of size (by length) due to their overall electrical charge
Gel electrophoresis allows the base sequence to be built up and read
Components of gel electrophoresis:
● Positive electrode and negative electrode (electrical current is applied via a power
supply)
● Charged molecules
● Gel medium (through which the molecules move) covered in buffer solution
Separating DNA by gel electrophoresis:
● Negatively charged DNA (due to phosphate groups) moves towards the positive
electrode
● The smaller the fragment, the faster it moves
When separating proteins, typically carried out in the presence of a charged detergent e.g SDS (sodium dodecyl sulfate)
why?
Equalises surface charge on molecules, so proteins separate according to their
molecular mass
why do we use dna probes
● Locate specific gene needed for use in genetic engineering
● Identify same gene in different genomes when comparing
● Identify presence or absence of specific allele for genetic disease, or which gives
susceptibility to particular condition
what is genetic engineering/recombinant DNA technology/genetic modification
● the manipulation of DNA sequences of an organism
● Gene (for desirable characteristic) isolated in an organism and placed into another
organism using a suitable vector
● Forms genetically modified organisms containing the recombinant DNA
● Why is genetic engineering possible?
o DNA is universal - same codons code for the same amino acids in all living organisms ∴ genetic information is transferable
what is recombinant DNA
altered DNA with introduced nucleotides
what is a transgenic/genetically modified organism
contains nucleotide sequences from a different species/organism
o Transgenic bacteria have antibiotic resistance - do not want them to escape
beyond lab
Uses of genetic engineering:
● Genetic modification of crops to increase crop yield (through resistance to
drought/disease/pesticides) or increase nutritional value
● Genetic modification of bacteria to produce medicines
● Genetic modification of livestock to increase productivity or provide resistance to
disease/pests
Principles of genetic engineering
- Desired gene obtained & identified
- Copy of gene placed inside vector
- Vector carries gene to recipient cell
- Recipience cell expresses the novel gene
Steps of genetic engineering in more detail
- Desired gene is obtained & identified
o From cell where gene is being expressed
o From synthesis (by an automated polynucleotide synthesiser) if nucleotide
sequence of gene is known
● PCR used to amplify the gene
o Using DNA probe to locate the gene - Desired gene is isolated
● Method 1: restriction endonucleases
o cut required gene at restriction sites → fuses with plasmid (vector) by DNA
ligase
o Some cut strand unevenly at the ends to leave extended regions of unpaired
bases (sticky ends) → easier to insert DNA
o Some cut strand to leave blunt ends
● Method 2: reverse transcriptase
o mRNA for desired gene isolated → reverse transcriptase used to make singlestranded cDNA (complementary DNA) from mRNA template → primers and
DNA polymerase form double-stranded DNA from cDNA → fuses with plasmid
(vector) by DNA ligase
o Easier to identify very specific types of mRNA - Desired gene is multiplied
o By PCR - Desired gene is transferred by suitable vector into recipient cells
o Electroporation - high voltage pulse applied to cell to disrupt membrane &
make it more porous
● Encourages uptake of plasmid vectors by cell
● Too high electrical current → permanent damage / destruction of
membrane which can destroy whole cell
o Electrofusion - electrical fields used to introduce DNA into cells
o Transfection - DNA packaged into bacteriophage which can transfect host cell
● Transfect - infect cell with a free nucleic acid (DNA)
o Heat shock treatment - alternative periods of cold (0 degrees) and hot (42
degrees) in the presence of calcium chloride
● Bacteria walls and membranes become more porous
● Recombinant vector allowed in
● Calcium chloride - positive calcium ions surround negatively charged
parts of DNA molecule and phospholipid bilayer in cell membrane to
reduce repulsion between recombinant DNA & host cell membranes
o A. tumefaciens in plants - Cells with desired gene identified by a marker and cloned
- Recipient cells express the new gene
Genetic engineering: Vectors for plants - Agrobacterium tumefaciens (A. tumefaciens)
● Causes tumours in healthy plants
● Recombinant plasmids inserted into bacterium
● Bacterium infects some plants & naturally inserts its genome into host cell genomes
Genetic engineering: Direct introduction of gene into recipient how
Gene gun - small pieces of gold/tungsten coated with DNA and shot into plant cells (which
are not susceptible to A. tumefaciens)
Tools used in genetic engineering:
Reverse transcriptase - forms single-stranded (complementary) DNA from mRNA
● Ligase - joins together cut ends of DNA (by forming phosphodiester bonds)
● Restriction endonucleases - cuts genes at restriction sites
o Cut (genome) DNA into smaller fragments & cut vectors (plasmids)
o Restriction sites - specific DNA base sequences
o Sticky ends - exposed unpaired nucleotide bases
● Free nucleotide bases complementary to sticky ends added, gene &
plasmid will anneal - bind - catalysed by DNA ligase
● Plasmids - transfer DNA fragments into bacteria or yeast
● Vectors - transfer DNA fragments into cell
General ethical issues relating to genetic manipulation of organisms & microorganisms
Poor welfare of genetically modified organisms
Antibiotic resistant gene may transfer to pathogenic bacteria, which can cause a
mutation with unknown effects
how is gm soya modified
to be resistant to herbicide & produce Bt protein which
increases the yield!
o Bt protein toxic to pest animals
● Reduces amount of pesticide spraying
● Protects environment
● Helps poorer farmers
o Weeds competing with soya plants could be killed by herbicide
Perceived risks: of gm of plants and microorganisms
● Gene for herbicide/pesticide resistance could be passed into weeds/pests
● Non-pest insects/insect eating predators may be damaged by toxins in GM plants
● Reduced biodiversity if herbicides overused to kill weeds
GM pathogens for research: how are viruses used
● Genetically modified viruses made non virulent (weakened/dead) so they can be
used to make vaccines (still have specific antigens on outer surface)
● GM viruses can be used as vectors in gene therapy
what is “Pharming” - where GM animals to produce pharmaceuticals
Adding/removing genes of animals so they develop certain diseases and can act as
models for the development of new therapies for humans
Creating human pharmaceutical proteins and inserting them into animals so that the
gene can be expressed & product (IE milk) can be harvested
Genetic modification: Patenting & technology transfer issues
People in less economically developed countries prevented from using patented GM
crops (crops that no one else can use without payment)
● May not be able to afford what they need
what is golden rice
2nd generation GM crop bred to contain more beta carotene, the precursor to vitamin A
Reduces vitamin A deficiency in Asia/developing world/area where rice is a staple in diet
Reduces eye problems & blindness
perceived risks of gm golden rice
● Reduces genetic diversity of rice
● Seeds are expensive and need to be bought yearly - farmers may not be able to
afford
mark scheme steps to sequence dna
- extract samples of dna from cells
- cut dna into sections of varying length
- amplify the dna - many copies
- sequence short sections of dna
- place sections in order by matching overlapping regions
developments to increase dna sequencing speeds
- whole genome sequencing
- next generation sequencing
- use of luciferase in pyrosequencing
how bioinformatics would increase the effectiveness of a vaccination programme against ebola etc
- facilitates the access to lots of data on dna and proteins
- can identify vulnerable populations and the source of outbreaks to target certain areas and individuals
how dna sequencing would increase the effectiveness of a vaccination programme against ebola etc
can predict viral strains / mutations
makes sure vaccines contain the right antigen
why may we need booster / repeated vaccinations
memory cells may have REDUCED in number - not fully gone
why may pcr not have 100% yield
temp;late strands may rejoin rather than bond to primers
primers fail to anneal to fragment
lack of primers (free nucleotides)
temperature damage to fragments
extra things to improve electrophoresis
Why do we use taq polymerase?
thermostable
doesn’t denature @ 95 degrees, so PCR can be cycled repeatedly without stopping to reload with enzyme
how can dna be visualised after electrophoresis
uv tags & light, plus fluorescent markers
steps of protein electrophoresis
TLC vs electrophoresis
how does inserting a new gene into a chromosome affect the function of other genes in that chromosome
Frameshift -> altered triplet -> new gene could disable a functioning gene if disabled into it
Why can gene therapy be unlikely to work as a treatment for some diseases
Some are caused by dominant alleles
So that allele will still code for the protein to cause the disease
Why can gene therapy be unlikely to work as a treatment for some diseases
Some are caused by dominant alleles
So that allele will still code for the protein to cause the disease
