Module 6 Section 4: Manipulating Genomes Flashcards
What are the different techniques needed to study genes
Cutting out DNA fragments using restriction enzymes
Polymerase chain reaction (PCR)
DNA sequencing
Gel electrophoresis
How is PCR used
Can be used to select a fragment of DNA (containing the gene or area of DNA you’re interested in) and amplify it to produce millions of copies
Occurs in a few hours
Full process of PCR
A reaction mixture is set up that contains the DNA sample, free nucleotides, primers and DNA polymerase.
The DNA mixture is heated to 95 °C to break the hydrogen bonds between the two strands of DNA.
DNA polymerase doesn’t denature even at this high temperature
The mixture is then cooled to between
50 and 65 °C so that the primers can bind (anneal) to the strands.
The reaction mixture is heated to 72 °C so DNA polymerase can work.
The DNA polymerase lines up free DNA nucleotides alongside each template strand.
Complementary base pairing means new complementary strands are formed.
Two new copies of the fragment of DNA are formed and one cycle of PCR is complete.
The cycle starts again, with the mixture being heated to 95 °C and this time all four strands (two original and two new) are used as templates
How are primers and DNA polymerase used in PCR
Primers are short pieces of DNA that are complementary to the bases at the start of the fragment you want.
DNA polymerase is an enzyme that creates new DNA strands.
Why is it important that DNA polymerase doesn’t denature at the temperatures PCR uses
It means many cycles of PCR can be carried out without having to use new enzymes each time
How much DNA can a PCR cycle produce
Each PCR cycle doubles the amount of DNA,
e.g. 1st cycle = 2 × 2 = 4 DNA fragments, 2nd cycle = 4 × 2 = 8 DNA fragments, 3rd cycle = 8 × 2 = 16 DNA fragments
What is electrophoresis used for
Uses an electrical current to separate out DNA fragments, RNA fragments or proteins depending on their size
How to set up apparatus for electrophoresis
Pour agarose gel into a gel tray and leave to solidify.
A row of wells is created at one end of the gel.
Put the gel tray into a gel box (or tank).
Make sure end of the gel tray with the wells is closest to the negative electrode on the gel box.
Add buffer solution to the reservoirs at the sides of the gel box so that the surface of the gel becomes covered in the buffer solution.
How to load DNA samples into wells
Use micropipette to add the same volume of loading dye to DNA fragments (from PCR or restriction enzymes)
Loading dye helps samples sink to the bottom of the wells to make them easier to see
Add 10μl of DNA sample to first well
Make sure tip of micropipette is in buffer solution and just above opening of the well
Don’t stick tip too far into well as it may pierce bottom
Repeat process and add same volume of the other DNA samples to other wells in the gel
Clean tip of micropipette each time
Record the DNA added to each well
How to carry out electrophoresis
Put lid on box and connect leads from gel box to power supply
Pass 100V through the gel
Run gel for 30mins or until dye is 2cm from end of the gel
Remove gel tray from the gel box and tip off excess buffer solution
Stain DNA fragments by covering surface of the gel with staining solution then rinsing gel with water
Bands of the different DNA fragments will now be visible
How to measure the size of the DNA fragments
Size of DNA fragment is measured in bases
E.g. ATCC = 4 bases or base pairs
1000 bases is one kilobase (1 kb)
How do DNA fragments separate in electrophoresis
DNA fragments are negatively charged so they’ll move through the gel towards positive electrode at far end of the gel (anode)
Small DNA fragments move faster and travel further through gel
Means DNA fragments separate according to size
How are RNA fragments separated using electrophoresis
This follows the same basic method as for DNA fragments
How are proteins separated using electrophoresis
Proteins can be positively charged or negatively charged
Before they undergo electrophoresis, they’re mixed with a chemical that denatures the proteins so they all have the same charge.
Uses of electrophoresis of proteins
e.g. to identify the proteins present in urine or blood samples, which may help to diagnose disease.
What are palindromic sequences of nucleotides
These sequences consist of the same order of bases when read backwards on the opposite strand
What are restriction enzymes
Restriction enzymes are enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) the DNA at these places
They cut the DNA via hydrolysis reactions
Also called restriction endonucleases
How can you choose what type of DNA fragment are formed
Can choose between different restriction enzymes
They can cut at different specific recognition sequences, because the shape of the recognition sequence is complementary to an enzyme’s active site.
Results in different fragments forming
E.g. the restriction enzyme EcoRI cuts at GAATTC, but HindIll cuts at AAGCTT.
How to use a restriction enzyme in a lab
The DNA sample is incubated with the specific restriction enzyme
What do you need in order to completely separate a fragment from the whole chain
If recognition sequences are present at either side of the DNA fragment you want, you can use restriction enzymes to separate it from the rest of the DNA.
How are restriction enzymes used to bind different fragments together
Sometimes the cut leaves sticky ends
These are small tails of unpaired bases at each end of the fragment.
Sticky ends can be used to bind (anneal) the DNA fragment to another piece of DNA that has sticky ends with complementary sequences.
What is a DNA profile
Some of an organism’s genome consists of repetitive, non-coding base sequences which are called short tandem repeats and are within satellite DNA
These are sequences that don’t code for proteins and repeat over and over
E.g. introns, telomeres (caps and tails), centromeres
The number of repetitions of non-coding sequences differs from person to person, so the length of these sequences in nucleotides differs too.
This is their DNA profile
PCR is used to increase DNA sample size
The number of times a short tandem repeat is repeated at different, specific places (loci) in a person’s genome (and so the number of nucleotides there) can be analysed using electrophoresis.
This creates a DNA profile.
Where is DNA profiling useful
PCR and DNA profiling is performed on traces of DNA left at the crime scene
eg. blood, semen, skin cells, saliva or hair
DNA profile is compared to a sample taken from a suspect or identified using criminal data base
Can be used to prove paternity of a child
Can be used in immigration cases to prove or disprove family relationships
DNA profiling is used to identify which species an organism belongs to
Used to demonstrate evolutionary relationships between different species
Can be used to identify individuals who are at risk of developing particular diseases
Certain non-coding microsatellites, or the repeating patterns they make, have been found to be associated with an increased risk or particular disease E.g. Huntington’s disease, sickle cell anaemia
Process of DNA sequencing
DNA for sequencing is mixed with a primer, DNA polymerase, a excess of normal nucleotides (containing bases A, T, C, G) and terminator bases
Mixture undergoes PCR in thermal cycler
At 72°C DNA polymerase starts to build up new DNA strands by adding nucleotides with the complementary base to the single-strand DNA template
Each time a terminator base is incorporated instead of a normal nucleotide, the synthesis of DNA is terminated since no more bases can be added
As the chain-terminating bases are present in lower amount and are added at random, this results in many DNA fragments of different lengths depending on where the terminating bases have been added
All possible DNA chains will be produced after many cycles as the reaction is stopped at every base
Fragments separated according to their length by capillary sequencing which is like gel electrophoresis in small capillary tubes
Final base is identified on each strand by fluorescent markers
Lasers detect different colours which shows the order of the sequence
The order of bases in capillary tubes shows the sequence of the complementary strand of DNA which is used to determine the sequence of the original DNA strand
How whole genomes be studied quickly
Chain termination technique still used but the tube contains all the modified nucleotides each with a fluorescent label
The machine reads the sequence out
High throughput sequencing can read 1000 times more bases in a given time
How does high throughput pyrosequencing work
Section of DNA cut into fragments, split into single strands and a strand from each fragment is attached to a small bead
PCR used to amplify fragments on each bead
Each bead put into separate well
Free nucleotides added to the wells attach to the DNA strands via complementary base pairing
Wells also contain specific enzymes which emit light when bases are added to DNA strand
Computers analyse occurrence and intensities of the light emitted in the different wells and process the information to interpret the DNA sequence
Technique can sequence about 400 million bases in a 10 hour period
Overview of process of how chain termination method is used to study whole genomes
Genome fragmented
Fragments inserted into BACs
DNA extracted from colony
DNA sequenced using chain termination method
Fragments put back in order
First step of preparing genome to be sequenced: BACs
Chain termination technique can only sequence fragments up to 750bp long
Genome is cut into smaller fragments (about 100 000 bp) using restriction enzymes.
Fragments are inserted into bacterial artificial chromosomes (BACS)
The BACs are then inserted into bacteria
Each bacterium contains a BAC with a different DNA fragment
Second step of preparing genome to be sequenced: DNA extracted from bacteria
The bacteria divide to create colonies of cloned (identical) cells that all contain a specific DNA fragment.
Together the different colonies make a complete genomic DNA library.
DNA is extracted from each colony and cut up using restriction enzymes (to make fragments small enough to sequence)
Produces overlapping pieces of DNA.
Third step of sequencing whole genomes: fragments sequenced
Each piece of DNA is extracted from BAC and sequenced using the chain-termination method (including terminator base types of all nucleotides)
Fragments separated out according to size with electrophoresis, fragments differ by 1 base each
Electrophoresis sheet then put through computer where laser reads the bases (by radioactive tag of fluorescent marker) and creates base sequence
Finally the DNA fragments from all the BACs are read out by computers, to complete the entire genome.
What are bacterial artificial chromosomes
These are man-made plasmids
Fragments are inserted into these BACs which are inserted into bacteria to create colonies of different fragments
Not needed specifically to sequence genome
Just there to provide genomic library so the fragments are to hand if needed
What can you deduce from sequencing a gene
The sequence of amino acids the gene codes for
Used to find primary structure of polypeptide
What is synthetic biology
This is how biological molecules are made through predicting the structure of a protein derived from a gene that has been sequenced
Uses of synthetic biology
Building biological systems from artificially made molecules (e.g. proteins)
Can see whether they work in the way we think they do.
Redesigning biological systems to perform better and include new molecules.
Designing new biological systems and molecules that don’t exist in the natural world, but could be useful to humans,
e.g. energy products (fuels) and drug products.
Can also be used to build artificial pathways, organisms or devices
How is synthetic biology different from genetic engineering
Genetic engineering involves the direct transfer of DNA trom one organism to another
Whereas in synthetic biology DNA is created from scratch
How is artemisinin a product of synthetic biology
Artemisinin is an antimalarial drug
Used to be obtained by extracting it from a plant.
Using synthetic biology, scientists have created all the genes responsible for producing a precursor to artemisinin.
Successfully inserted these genes into yeast cells
We can now use yeast to help produce artemisinin
How are gene sequences and whole genome sequences compared and what can they be compared between
Can be compared between organisms of different species and organisms of the same species
Involves computational biology and bioinformatics
What is computational biology
Using computers to study biology and analyse large amounts of biodata
Can build theoretical models of biological systems to predict what will happen in different circumstances
Can work out 3D structures of proteins and understanding processes such as gene regulation
e.g. to create computer simulations and mathematical models
What is bioinformatics
Developing and using computer software that can analyse, organise and store biological data in very large quantities
What areas of biology involves the comparison of gene sequences and genomes
To study genotype-phenotype relationships
Epidemiological studies
To help understand evolutionary relationships
How does studying genotype-phenotype relationships use comparison of gene sequences and genomes
Used to predict a phenotype by analysing the genotype
Using DNA sequencing we now know that there are many more unique proteins than coding genes in human DNA
This suggests that the sequence of amino acids (phenotype) is not always what would be predicted from the genome sequence alone
Example of using the comparison of gene sequences and genomes to study genotype-phenotype relationships: Marfan syndrome
Marfan syndrome is a genetic disorder caused by a mutation of the FBN1 gene
Position and nature of mutation on gene affects the symptoms the person has (vision, cardiovascular system or muscles)
Sequencing FBN1 gene of sufferers and documented them along with phenotypes
Bioinformatics allowed scientists to compare all the data and identify genotype-phenotype correlations
Can help with treatment as gene sequencing can help predict what problems they may face
How do epidemiological studies use comparison of gene sequences and genomes
Epidemiological is the study of health and disease within a population
Considers distribution, causes and effects of disease
Gene mutations have been linked to a greater risk of disease
Computerised comparisons between genomes of people with a disease and those without can be used to detect particular mutations that could be responsible for the increased risk of disease
However, the risk of developing diseases has been found to be a product of the environment and our genes
How can the comparison of gene sequences and genomes be used to understand evolutionary relationships
Whole genomes of different species can be sequenced
They are then analysed using computer software to see how closely related different species are
All organisms evolved from shared common ancestors
Closely related species evolved away from eachother more recently so share more common DNA
How can the comparison of gene sequences and genomes be used to understand human migratory patterns
When groups of humans separated and moved to different parts of the world their genomes changed in slightly different ways
Can use computers to compare the genomes of people from different parts of the world
Can be used to build picture of early human migration
4 steps or genetic engineering
- Isolate the gene
- Package the gene (in a vector)
- Transfer the vector to the recipient cell
- Recipient cell expresses the gene
Processes of isolating the gene in genetic engineering
Use DNA probe to identify the gene then use restriction enzymes to cut the gene out
The restriction enzymes used are specific to DNA strands which have a certain base sequence
DNA fragments are left with sticky ends
Harvest mRNA from cells where it is being expressed
Use reverse transcriptase to convert mRNA to cDNA (complementary DNA)
Add primers and polymerase to build double stranded DNA
Processes packaging gene into a vector in genetic engineering
Gene can be sealed into bacterial plasmids
This uses same restriction enzymes to leave the complementary sticky ends on bacterial DNA
Once fragment sticky ends are lined up with bacterial sticky ends, DNA ligase forms phosphodiester bonds between the sugar and the phosphate groups on the two strands of DNA (joins up sugar phosphate backbones), this is called ligation
Attenuated viruses or yeast chromosomes also used
Recombinant DNA is now formed
Processes transferring the vector to the recipient cell in genetic engineering
Multiple methods of transformation
Plasmids taken up by host cell
Can use electroporation where a high voltage pulse to disrupt the membrane and allow plasmids to move into cells
Can also culture bacterial cells and plasmids in a calcium rich solution and increase temperature which causes bacterial membrane to become permeable and plasmids can enter
Bacteriophage vector infects the bacterium by injecting its DNA into it
The phage DNA with desired gene in it then integrates into the bacterial DNA
What do vectors do in genetic engineering and what is commonly used
Most commonly used are bacterial plasmids
Plasmids can recombine with host DNA to form recombinant DNA
How do scientists identify if transformation has taken place in genetic engineering
Plasmids that are used as vectors usually have a marker gene
May have a gene for antibiotic resistance which enables scientists to determine that the bacteria have taken up the plasmid by growing the bacteria in a media containing antibiotic
After recombinant DNA is formed the vector plasmids are given second marker gene to show plasmid contains recombinant gene
If DNA fragment is inserted successfully the marker gene will not function indicating process has worked
Marker genes can be for antibiotic resistant, fluorescence or enzyme which causes colour change
Current uses for genetically modified microorganisms
Microorganisms can be genetically modified to produce substances that are useful to people such as insulin or vaccines which can be produced in large quantities this way
GM microorganisms are used ti store living record of DNA of another organism in DNA libraries
Future uses of genetically modified microorganisms
GM pathogens can be used in research for cancer treatment
Scientists found tumour cells have receptors on their membranes for the poliovirus
So the poliovirus will recognise and attack them
By genetically engineering polioviruses to inactivate genes that cause poliomyelitis, scientists can use it to kill cancer cells without causing disease
This can lead a treatment for cancer
Positive ethics for use of genetically modified microorganisms
This means that previously untreatable diseases can now be treated which reduces the suffering they cause
Negative ethics for use of genetically modified microorganisms
Scientists researching pathogens could become infected with the live pathogen and cause an outbreak of disease
Genetically modified version of pathogen could revert back to its original form and cause an outbreak of disease
Knowledge of how to genetically engineer dangerous pathogens could be used to create agents for biowarfare
Researchers have to follow strict protocols to prevent these
Current uses for genetically modified plants
Scientists inserted a gene originally found in bacteria which codes for the Bt protein
The Bt protein is toxic to some of the insects that feed on soybeans
Soy beans can also be engineered to be resistant to a common weed killer and to contain Bt protein
Means that farmers can spray to get rid of weeds and reduce the use of pesticides
Leads to much higher yield of soya beans
Positive ethics of use of genetically modified plants
Can reduce amount of chemical pesticides farmers use on crops which harm the environment
Increases availability of food for famine prone countries
Negative ethics of use of genetically modified plants
GM soybean plants can encourage monoculture which decreases biodiversity and could leave the whole crop vulnerable to disease because all plants are genetically identical
Growing genetically modified crops can mean that the normal crop is not grown anymore, if a new pest is introduced that kills all the modified variants, a possible gene that could make them resistant to that pest may be present in the normal crop which has been lost
Transferred genes may spread to wild populations to produce super weeds
Future uses of genetically modified plants
Can be used to grow crops which are resistant to pesticides
Fruits can be genetically modified to contain various vaccines which can stop the spread of disease for a fraction of the normal price
Crops can be modified to grow in hostile environments such as salt water
Applications of genetically modified animals
Swine fever resistant pigs
Faster growing salmon
What is pharming
Genetically engineering animals to create human medicines or pharmaceuticals
Types of uses of pharming
Creating animal models for certain diseases so they can act as models for new therapies
Creating human proteins by introducing human gene coding for a medically required proteins
Example of pharming
Hereditary anti thrombin deficiency is a disorder that makes blood clots more likely to form in the body
The risk of forming blood clots can be reduced with infusions of the protein antithrombin
Scientists found a way to produce high yields of this protein using goats
DNA fragments that code for human antithrombin are injected into goat embryo
Embryo implanted into female goat
Offspring is tested for presence of protein when it’s born
If it does it is selectively bred to produce a herd of goats that produce antithrombin in their milk
Protein is extracted from milk and used to produce the drug ATryn that can help treat people with the deficiency
Positives of pharming
Drugs can be made in large quantities to make them more available to people
Negatives of pharming
Manipulating animals genes can cause harmful side effects for the animal
It enforces the idea that animals are merely assets that can be treated however we choose
How can genetically engineered organisms be owned by companies
Scientists from different institutions collaborate knowledge to improve techniques of genetic engineering
A group of scientists or the company they work for may want to obtain a patent to control, by law, who uses the product and how for a set period of time
Positive ethical issues of getting a patent for a GM organism
Owner of patent will get money generated from selling the product
Encourages scientists to compete to be the first to come up with a new beneficial genetic engineering idea
Means we get products faster
Negative ethical issues of getting a patent for a GM organism
Farmers in poorer countries can’t afford patented genetically modified seeds
If they can afford it for one year, the patent means they can’t use it for more than one year without paying again
What happens in gene therapy
Involves altering alleles inside cells to cure genetic disorders
Why may there be two ways to cure a genetic disorder using gene therapy
What approach is used depends on whether the disorder is caused by a dominant or recessive allele
How to carry out gene therapy to cure a disorder caused by two recessive alleles
You can add a working dominant allele to make up for them
How to carry out gene therapy to cure a disorder caused by a dominant allele
You can ‘silence’ the dominant allele
e.g. by sticking a section of DNA in the middle of the allele so it doesn’t work any more
How to get new allele inside of cell in gene therapy
Allele inserted into cells using vectors
These can be: altered viruses, plasmids or liposomes (spheres made of lipid)
Types of gene therapy
Somatic therapy
Germ line therapy
What is somatic gene therapy
Involves altering the alleles in body cells
Such as cells that are most affected by the disorder have the mutant allele replaced by healthy allele
E.g. cystic fibrosis (CF) is a genetic disorder that’s damaging to respiratory system
Somatic therapy for CF targets the epithelial cells lining the lungs.
Somatic therapy doesn’t affect sex cells so any offspring could inherit the disease
Only temporary solution as healthy cells are replaced by stem cells which have faulty allele through mitosis
What is germ line therapy
Involves altering the alleles in the sex cells.
Means that every cell of any offspring produced from these cells will be affected by gene therapy and they won’t inherit the disease.
Germ line therapy in humans is currently illegal though.
Positive ethical issues of gene therapy
Prolong lives of people with genetic disorders
Improve quality of life for people with genetic disorders
Carriers of genetic disorders can conceive a baby without disorder or risk of cancer (only germ line)
Decrease number of people suffering from genetic disorders (germ line)
Negative ethical issues of gene therapy
Technology can be used in ways other than medical treatment like treating effects of aging
Potential of doing more harm than good by using technology
E.g. risk of overexpression of genes
Gene therapy is expensive and health services should spend resources on treatments which have passed clinical trials instead
Disadvantages of gene therapy
Effects may be short lived (somatic only)
Patient may have to undergo multiple treatments (somatic only)
May be difficult to get allele into specific body cells
Body could identify vectors as foreign bodies and start immune response against them
An allele could be inserted into the wrong place in DNA, possibly causing cancer
An inserted allele can be overexpressed leading to too much of the missing protein
How is DNA profiling used in forensic science
Samples of DNA from crime scene is taken and isolated
E.g. semen, skin cells, saliva, hair
PCR used to amplify areas containing short tandem repeats (repeats in DNA unique to individuals)
Primers used to bind to either side of repeats so whole repeat amplified
PCR products run on electrophoresis gel and DNA profiles produced are compared to see if any match samples collected from suspects
Match by having same pattern on bands on gel
If samples match it links a person to a crime scene
How can DNA profiling be used in medical diagnosis
Can be used to analyse risks of genetic disorders
Useful when the specific mutation isn’t known
Useful when several mutations could have caused disorder as it identifies a broader, altered gene pattern
Example of how DNA profiling is used in medical diagnosis
Preimplantation genetic haplotyping (PGH) screens embryos created by IVF for genetic disorders before they’re implanted into the uterus.
The faulty regions of the parents’ DNA are used to produce DNA profiles
These are compared to the DNA profile of the embryo.
If the profiles match, the embryo has inherited the disorder.
Can be used to screen for cystic fibrosis or Huntington’s disease etc
How to collect results from gel electrophoresis
Hybridisation:
DNA probes are added to label the fragments
These radioactive probes attach to specific fragments
Development:
Membrane with radioactively labelled DNA fragments is placed onto an X-ray film
Development of the X-ray film reveals dark bands where the radioactive or fluorescent DNA probes have attached
Process of producing a DNA sample
Extracting DNA using reverse transcriptase on mRNA or DNA probe (small pieces amplified by PCR)
Digesting sample using restriction endonucleases
Separation using electrophoresis to produce clear recognisable pattern
Hybridisation using radioactive or fluorescent probes (p
Analysing evidence
Why would a genome have to be fragmented before sequencing
Takes less time
Smaller fragments to sequence improves accuracy
You can divide the job over different labs to save time
Difference between RNA polymerase and DNA polymerase
RNA polymerase:
Makes mRNA, tRNA or rRNA by transcription
Uses one strand of DNA and forms one strand
DNA polymerase:
Uses in DNA replication
Semi conservative replication where both strands are used and 2 strands are formed
Used before cell division
