CMB2000 Essential Biomedical Research Skills Flashcards
What is PCR
Polymerase Chain Reaction
What does DNA replication require?
-Template DNA
-40 or more proteins
-Helicase
-Primase
-Polymerases
-Nuclease
-Ligase
-ssDNA binding proteins
-Sliding clamps
What are the advantages of PCR?
-Sensitive (can amplify as little as one molecule)
-Specific (cam amplify a unique target sequence)
-Cheap
-Rapid
-Robust (DNA is very stable)
What’s required for PCR?
-Template dsDNA
-2 primers (small ssDNA)
-Polymerase (copies the template)
-dNTPs
-Magnesium (cofactor for DNA polymerase enzyme)
-Buffer (maintains pH and provide necessary salt)
What is the function of the sliding clamp in DNA replication?
The clamp protein binds DNA polymerase and prevents this enzyme from dissociating from the template DNA strand.
Describe the primers used in PCR?
-One for template and for complementary strand
-Single stranded DNA
-Length of 18-24 bp
-40-60%G/C content
-Start and end with G/C pairs
-Melting temp of 50-60C
What must primer pairs not have to prevent primer dimers?
Complementary regions to each other
Why is Magnesium required in PCR?
Magnesium acts to enhance the enzymatic activity (specifically of DNA polymerase) thereby supporting DNA application
What makes up the buffer found in PCR?
-pH of 8-9.5
-Tris HCl
-Potassium ions (KCl) promotes annealing
What are the 3 stages of PCR?
-Denaturation
-Annealing
-Elongation
What is Nancy520 an example of?
An Intercalating agent, which aids in detection of DNA.
How may we detect PCR products?
Run products on agarose gel, using intercalating dye to stain DNA to determine size and yield.
Give some key steps involved in cloning
-Bioinformatics searching
-Design primers
-PCR
-Choose plasmid and insert
-Transform into competent E Coli
-Select correct colonies
Give some uses of PCR in biotechnology
-Cloning
-Manipulating DNA
-Knock out genes
-Fuse host proteins
What must our template DNA for PCR be?
-Clean and pure
-Contaminent free
-High concentration
Describe reverse transcriptase PCR
-Convert RNA to cDNA, using reverse transcriptase (a retroviral enzyme) that converts RNA to DNA
-Amplify DNA by PCR (including qPCR)
How does qPCR/Real time PCR work?
-Using a fluorescent report in the PCR reaction
-Couples amplification of the sequence with quantification of the concentration of DNA
How does qPCR using SYBR green work?
Fluorescent that binds to groove of dsDNA
How does qPCR using TAQman work?
Fluorescent that uses probes with fluorescent reporter and quencher
In qPCR, if there is a time to reach the cycle threshold, what does this signify?
more cDNA in the sample
What may we use as reference genes in qPCR?
-House keeping genes
-As these have a constant level of expression, which is not affected by experimental factors
Why do we use reference genes in qPCR?
-Essential to support validity of qPCR
-Confirms RNA extraction was good and efficient
Give some common uses of PCR in diagnosis.
-Genotyping the patient
-Genotyping the pathogen
-Phenotyping the disease
Why may we use PCR to genotype a patient?
-Diagnose genetic traits
-Detection of carriers of genetic traits
-Tissue matching (HLA typing)
-Pharmacogenetics
Why may we use PCR to genotype a pathogen?
Diagnose a strain of infecting pathogen
Why may we use PCR to phenotype a disease?
-Measuring disease progression
-Measuring disease severity
What types of PCR are used when genotyping a patient?
PCR-RFLP or ARMS-PCR
What is PCR-RFLP?
PCR-Restriction Fragment Length Polymorphism
What is ARMS-PCR?
Amplification Refractory Mutation System-PCR
How does PCR-RFLP work?
-Works when diseased alleles carry a site for a specific restriction endonuclease, but healthy does not
-When looking at bands, healthy and diseased can be differentiated
How may you identify diseased or healthy in PCR-RFLP banding?
-If healthy, there is only 1 band
-If homozygous for the diseased allele there are 2 bands
-If heterozygous with the diseased allele there are 3 bands
Give some disadvantages of PCR-RFLP
-Only possible if the site contains a known RE site
-Some RE are expensive
-Only possible if a single nucleotide variation
-Can be time consuming
Give some advantages of PCR-RFLP
-Cheap
-Easy design
-Applied to microindels and SNPs
-Simple resources with commonly used techniques
How does ARMS PCR work?
-One PCR comprises allele specific primer at 5’ end and a common primer at 3’ end
-If the presence of an amplified mutant is detected by agarose gel electrophoresis, it suggests that the target sequence contains the mutant allele
Give some advantages of using PCR over other methods of microbial diagnosis.
-Sensitive (can detect single copy of genome)
-Specific (Can identify strain)
-Quick, taking only a few hours
-No need for culture
-Detects DNA/RNA so not reliant on immune response
What may we use to phenotype a disease using PCR?
-Quantitative PCR
-Measures abundance of DNA or RNA in a sample
Why should we make competent cells instead of buying them?
-Saves money
-Ensures we don’t run out
What OD600nm should we grow competent cells to?
0.3, which is mid exponential growth
When forming competent cells, what may we plate them on?
Agar containing Xgal and ampicillin
Why do we use “double selection” ie plating competent cells on Amp+Xgal
-Ampicillin selects for bacteria that have taken up the plasmid (need resistance to survive)
-Xgal shows white colonies for the bacteria that have an insert as the lacZ gene no longer works and the lacZ makes the colonies blue
What is Xgal?
-When cleaved by βgalacotosidase creates an intensely blue product which is insoluble
-So when LacZ is functional blue product is formed.
Why do we use calcium chloride when forming competent cells?
-Disrupts cell membrane to allow plasmid uptake
-Aids binding of DNA to the surface of the cell, which can then enter the cell after a short heat shock
How can we ensure the proper orientation of the insert in PCR?
Using 2 different restriction enzymes
What is the percentage of agarose used in agarose gel electrophoresis?
1.5%
In Agarose gel electrophoresis, what is agarose dissolved in?
TAE
What is a clinical study?
Involves research using human volunteers (also called participants) that is intended to add to medical knowledge. These can be interventional and observational
Give some examples of types of experimental studies
-Randomised control trials
-Non randomised control trials
Give some examples of types of observational studies
-Cohort studies
-Case control studies
-Cross sectional studies
-Ecological studies
Describe the characteristics of a randomised control trial.
-Units are randomised to one of two or more groups
-Experimental group has the intervention being tested
-Comparison group has an alternative intervention
OR
-Control group has a placebo
Describe the characteristics of a cohort study.
-A defined group is followed over time
-Cohort includes people with similar characteristics
What is the difference between a prospective and a retrospective cohort study?
A ‘prospective’ cohort study recruits participants before any intervention and follows them into the future. A ‘retrospective’ cohort study identifies subjects from past records describing the interventions received and follows them from the time of those records.
What are case-control studies?
-An observational study to find out the possible cause of a disease or condition
-Done by comparing a group of patients who have the disease or condition with a group of controls.
What are cross-sectional studies?
-A snapshot observation of a set of people at 1 time
-Aimed to describe a variable
Describe ecological studies
Ecological studies are used to understand the relationship between outcome and exposure at a population level, where ‘population’ represents a group of individuals with a shared characteristic
Give the phases of a clinical trial
-Laboratory studies
-Phase I
-Phase II
-Phase III
-Phase IV
Give the goals of preclinical (laboratory) phase of research?
-Model the desired biological effect of a drug
-Predict treatment outcome in patients (EFFICACY)
-Identify and characterise toxicities associated with a drug (SAFETY)
Give the goals of Phase I of research?
Study the drug to learn about safety and side effects
Give the goals of Phase III of research?
-Gathering information about safety and effectiveness
-Focusing on determination of dosage
Give the goals of Phase II of research?
-Focus of effectiveness
-Obtain data on efficacy in those with a certain condition or disease
Give the goals of Phase IV of research?
-Post marketing monitoring stage
-Monitering polypharmacy
What things should you consider when setting up and running a clinical trial?
-Consent
-Controls
-Randomisation
-Blinding
-Sample size
-Statistics
-Ethics
What is ethics?
Moral principles that govern a person’s behaviour or the conducting of an activity
What is bias?
Systematic errors that encourage one outcome over others.
What is randomisation?
Randomisation its the process by which treatments are assigned to participants by chance rather than by choice
Give some types of randomisation
-Simple randomisation
-Block randomisation
-Stratified randomisation
What is simple randomisation?
-Randomisation based on a single sequence of random assignments
-Maintaining complete randomness of a subject to a particular group.
What is block randomisation?
-Designed to randomise subjects into groups that result in equal sample sizes.
-Once block size is determined, all possible balanced combinations of assignment must be calculated.
-Blocks are then randomly chosen to determine assignment.
What is stratified randomisation?
-Ensures key characteristics are evenly distributed between different groups
-Divide into groups based on key characteristics (eg group A male group B female)
-Randomise within each strata to different treatment groups
-This ensures each treatment group has a similar mix of participants
What is blinding?
Studies are designed to prevent members of the research team and study participants from influencing the results.
What is the difference between single and double blinded studies?
Single - Only the researcher knows the allocation
Double - Neither the participants nor researcher knows the allocation
Why do double blinding?
Prevents the research team from
-Influencing patient management
-Influencing withdrawing patient from trial
-Decide to adjust drug dose or therapy intensity
What is the placebo effect?
The placebo effect is when a person’s physical or mental health appears to improve after taking a placebo treatment
What is a hypothesis?
A proposed assumption for a phenomenon that may or may not be true
What is the issue with having a sample with too few participants?
-Real effect may be missed as it will be indistinguishable from chance variation
-If the study does not yield useful results, time and money may have been wasted
What is the issue with having a sample with too many participants?
-Smaller trials could reach firm conclusions so no need for participants beyond this
-Cost implications
What are the different types of end points in a study?
-True endpoint
-Surrogate endpoint
What is the true endpoint of a study?
A clinical meaningful endpoint that directly measure patients eg how a patient feels, functions or survives
What is the surrogate endpoint of a study?
A measurement of a specific outcome used in place of another as a predictor to tell if a treatment works. Usually occurs before a true endpoint and yields conclusions about the effect of treatment.
What is the dissemination of results?
Getting the findings of your research to the people who can make use of them, to maximise the benefit of the research.
What are some of the principles that make up the Nuremberg code for ethical research?
-Voluntary consent
-Research is for the good of society
-Based on previous knowledge to justify the experiment
-Avoids unnecessary physical and mental suffering
-Conducted by qualified persons
-Subjects can withdraw
What does the Helsinki declaration do?
Guide doctors operating in the dual role of researcher and clinician.
What is a human participant in research?
-A living human being
-Human beings who have recently died (including body parts)
-Foetus
-Embryo
How can Human participants be involved in research?
-Directly through physical presence
-Indirectly through stored human tissue/body fluids or use of human data
What is informed consent?
The act of providing information to a potential research participant to enable them to
-Understand their involvement in a study
-Understand the researcher’s responsibilities
How would you describe the multi stepped process that is gaining informed consent?
1 - Giving of information
2 - Discussion, clarification and review
3 - Obtaining written/verbal consent
4 - Ongoing revalidation of consent
What does the common law dictate a child as?
Below the age of 18 years old
How do we gain informed consent from children?
Under 16 years, it is often best practice to gain the child’s assent and legal guardian provides informed consent.
What laws dictate the ability to gather informed consent from vulnerable adults?
-Mental Capacity Act 2007
-Medicines for Human Use (clinical trials) Regulations 2004
-Common Law
Describe the Human Tissue Act 2004
Framework for the regulation of storage and use of human tissue from the living, and the removal, storage and use of tissue and organs from the deceased for specified purposes.
What is inducement in gaining consent?
The provision of an incentive to entice a person to carry out an action
What is GDPR?
-General Data Protection Regulation
-Governs the protection and control of personal data
What qualifies as personal data under GDPR?
Information that relates to an identified or identifiable individual
What are the principles of GDPR?
-Lawfulness fairness and transparency
-Purpose limitation
-Data minimisation
-Accuracy
-Storage limitation
-Integrity and confidentiality
-Accountability principle
What sort of data may be personal?
-Identifiable data
-Confidential information
-Sensitive information
-Coded data
-Linked anonymised
-Unlinked anonymised
-Data that relates to an individual
What are the three divisions of medicines in the Medicines Act 1968?
-Prescription only medicines
-Pharmacy medication
-General medication
What is the role of the UK research ethics committee?
-Ensure the dignity, rights, safety and well being of individuals are protected
-Reducing the risks associated with research
How are participants allocated to UK Research Ethics Committee or University Ethics Committee?
Patient participants -> UK Research Ethics Committee
Non-Patient participants -> University Ethics Committee
What do the UK Research Ethics Committee consider?
-Has the proposal been reviewed for scientific merit?
-Will the health of the research subject be affected
-Is what is planned justified, reasonable and appropriate?
-Are there hazards and if so remedies?
Name some meta ethical theories
-Moral absolutism/dogmatism
-Moral relativism
-Pyrrhonian moral scepticism
Simplify moral absolutism/dogmatism
-Ethics from the pulpit
-“I know that X is right or wrong”
-Anyone who disagrees is wrong
Simplify moral relativism
-Ethics from the restaurant
-The statement that X is right or wrong is synonymous with the statement that someone approves or disapproves of X
Simplify pyrrhonian moral scepticism
I believe that X is right or wrong
What is legalism?
-Excessive conformity to the law
-It is always important to reflect on the law
What must we use when looking at the ethics of a situation
-Use logic
-Use analogies
-Avoiding slippery slope arguments
Describe the subjective aspect to the need for ethics
-People need to justify their behaviour
-People need to explain why their behaviour is (un)acceptable
Describe the objective aspect to the need for ethics
Many things deserve moral consideration
What is an ethical theory?
An account of which principles should be followed, and of how to balance them against each other
What are formal ethical theories?
Formal theories about the sorts of abstract principles and values that should matter (eg happiness, pleasure, health)
What are material ethical theories?
Theories about the concrete things/entities that should matter
Name some formal ethical theories
-Principlism
-Consequentialism
-Deontology
-Virtue ethics
Describe principlism in medicine
4 principles approach, focusing on
-Autonomy
-Beneficence
-Non maleficence
-Justice
What is connected to how we value things?
Our ontologies
What are the two values that contribute to relative moral significance?
-Intrinsic value (value for oneself)
-Instrumental value (value for others)
What are the two ontologies that dominate western philosophy?
-Mechanistic materialism
-Dualism
Describe mechanistic materialism
-Reality is composed of separate entities that act in a machine like fashion
-Mental phenomena (feelings) should be explained entirely in terms of the components that constitute them (neurones)
-So there is no consciousness or free will
Describe dualism
Reality is composed of two fundamentally distinct things - Things with minds and things that lack minds
Describe panexperientalism
Experience is fundamental and ubiquitous throughout nature, and that mentality is not essential to it
What is speciesism?
The practice of treating members of one species as morally more important than members of other species
What are the issues surrounding research involving animals?
-Ethical concerns
-Scientific concerns
-Institutional/professional requirements
-Legal requirements
-Application to medical research
What is the law on using nonhuman animals for biomedical science?
-Animals (scientific procedures) act 1986 (ASPA) demands that some forms of protection should be applied to certain animals
-Applies to scientific procedures which are defined as procedures that cause harm, suffering, pain or distress where this is more than what is caused by the insertion of a needle
What organisms does ASPA 1986 cover?
-Any living vertebrate other than man and any living cephalopod
-In the case of a mammal, bird, or reptile when the gestation or incubation period for the relevant species has elapsed
-In any other case, it becomes capable or independent feeding
What are the three Rs in animal welfare ethics?
-Replacement (substitution of conscious living higher animals for insentient material)
-Reduction (reduce number of animals used)
-Refinement (decrease the incidence of severity of inhumane procedures)
What does ASPA demand of researchers?
-Researchers obtain a personal licence
-For specific projects a licence is obtained
-The place where the research is conducted has a certificate of designation which requires
-A certificate holder
-A named veterinary surgeon
-A named animal welfare and care officer
What must a project licence required by ASPA need?
-Must include a condition requiring the holder to ensure that the specified programme of work
-does not involve the application of any regulated procedure
-to which there is a scientifically satisfactory alternative method or testing strategy
-not entailing the use of a protected animal
What are AWERBs?
Animal welfare and ethical review body
Name some alternatives to nonhuman animal research?
-Microdosing
-Observational studies
-Randomised controlled studies
-Use of human tissues, cells and genes
-Computer testing
What is the purpose and methods of biomedical research?
Purpose - Researching the causes of health and illness of biological organisms
Methods - Using biological organisms to identify the causes of health and illness
What are the legal requirements in relation to consent?
The person must
-Have capacity/competency
-Is informed adequately
-Not be coerced
Is it ethical to involve those who lack capacity in clinical research?
-At times, eg with diseases surrounding children or those with alzheimers
-Can be circumvented using assent
Describe the freedom of information act?
-Aim is to promote openness by public authorities
-Members of the public have a statutory right of access to information held by public authorities
-Some anonymised information must be disclosed if requested under this act
What are the 6 data protection principles in the data protection act?
1 - Processing must be lawful, fair and transparent
2 - Purposes of processing be specified, explicit and legitimate
3 - Personal data must be adequate, relevant and not excessive
4 - Personal data must be accurate and kept up to date
5 - Personal data is kept for no longer than is necessary
6 - Personal data is processed in a secure manner
Name some examples of scientific misconduct
-Falsification
-Fabrication
-Plagiarism
-Self plagiarism
-Gift authorship
What does the view of absolute dominion lead to?
Speciesism
Why believe in absolute dominion over animals?
Validity - Animals make good models for humans, current alternatives are insufficient
Necessity - Fundamental part of medical research, as it is a legal requirement before drugs are released, whole organisms are needed to model complex organisation
What are the levels of animal use in drug development?
-In silico (computational)
-In vitro (molecular, cellular, tissue and organ)
-In vivo (whole animal)
What does drug safety testing involve?
-A limited range of in vitro and in vivo screens
-A relatively small number of animals (100s)
Why believe in abolition of using animals in research?
-Animal use is unnecessary medical advances have been achieved using
-Human based research
-Public health measures
-Population studies
-Computer models and analysis
-Non animal tests
-Alternative medicine
-Money is better spent on prevention not cure
Name some drug experiments that would be misleading if animals were used
-Penicillin (lethal in guinea pigs)
-Paracetamol (lethal in cats)
-Thalidomide (tested in the incorrect species to model to humans)
Which experimental procedures on animals are regulated?
If it causes
-Death, disease and injury
-Physiological and psychological stress
-Discomfort and disturbance to normal health
-Anaesthetic or analgesic administration
When is an animal considered to be living?
Until circulation ceases/brain is destroyed
What procedures does ASPA not cover?
-Causes less pain, suffering, distress or lasting harm than that of the insertion of a hypodermic needle
-Recognised veterinary practice
-Identification method
-Killing of an animal using approved methods
how does a cost benefit analysis work in theory?
-Assign a value to the costs (eg animals interests)
-Assign a value to the benefits (eg research value)
-Which is greater decides whether it will be completed
What are the pillars of academic integrity?
-Honesty
-Fairness
-Accountability
-Transparency
-Responsibility
What is plagiarism?
Copying and passing another person/source’s work off as your own
Give some examples of types of plagiarism
-Text (Copying text from a book, paper, etc)
-Diagrams
-Idea
-Auto (copying from yourself)
How do you avoid plagiarism?
-Read and take from multiple sources of information
-Make notes in your own words
-DO NOT COPY AND PASTE
What is an essay mill?
Getting someone to write an essay on your behalf
What are some methods of detecting scientific fraud?
-Algorithms
-Social media
-Awareness
-Public domain
-Standards
Name some consequences to scientific fraud
-Distrust from public
-Practice changing
-Implication on health and fatalities
-Controversies in the field
-Long lasting effects
Name some reasons for DNA isolation
-PCR
-DNA analysis
-Genetic testing
-Forensics
-Ecological
-Archaeological
Name the steps of DNA isolation
1 Cell lysis
2 DNA purification from cell extract
3 DNA concentration
4 Measurement of DNA purity and concentration
What do we not want in a sample of DNA when isolating it?
-Proteins
-Ribosomes
-mtDNA
-Lipid
-Plasmids
Name some methods of cell lysis
-Biological methods
-Physical methods
-Mechanical methods
Describe biological methods to lyse cells
Using enzymes to disrupt cell membranes, different enzymes are required for different cells, eg
-For plants, cellulase is used
-For bacteria, lysozyme is used
-For eukaryotic cells, Sappanin is used
Give examples of physical methods to lyse cells
-Using osmotic pressure, ie excess water moves into the cell when cells are placed in a hypotonic solution, bursting it
-Using freeze-thaw, ie repeated cycles of freezing and thawing ruptures cell membranes through ice crystal formation
Describe mechanical methods to lyse cells
Grinding, ie
-Pestle and mortar is used to disrupt plant cells and hard tissue
-Bead mill is used to beat and grind tough samples
-Vortex is used with beads or by itself for small cell numbers
Describe DNA purification by phenol-chloroform extraction
-Lysed cells or tissue are mixed with equal volumes of a phenol-chloroform mixture
-These are then centrifuged forming two distinct phases as water does not mix with P-C mixture
-DNA is concentrated with 0.3M sodium acetate and 2.5 volumes ethanol, forming a DNA precipitate
Describe DNA purification using commercial kits
-Column contains a silica membrane that binds DNA in the presence of a high concentration of salt
-Impurities such as salts are washed away and then a low salt buffer is used to release the DNA from the membrane and collect it
-Results in purer DNA than phenol-chloroform extraction
Give some methods to measure quantity and quality of DNA
-UV absorbance
-Fluorescence dyes
-Agarose gel electrophoresis
-Capillary electrophoresis
-Diphenylamine
Why is DNA isolation and purification important?
-Efficient extraction leads to efficient science
-Without a good starting point there isn’t a good output
-Genomic testing wouldn’t be possible
-PCR/cloning wouldn’t work
-Forensic science would be unreliable
What are restriction endonucleases?
-Enzymes produced by bacteria to protect against viral DNA infection
-Act on specific DNA sequences (restriction sites)
-Cleaving the phosphodiester bond within a polynucleotide
How do restriction enzymes cut DNA?
-make one cut in each of the sugar phosphate backbones of the double helix (breaks bond between O and P) at their recognition site in the presence of Mg2+
What is Star activity of restriction endonuclease?
-Relaxation or alteration of the specificity
-When reaction conditions differ significantly from the optimum for the enzyme
-
Describe the principle of agarose gel electrophoresis
-Polymerised agarose is porous, allowing for the movement of DNA
-It acts as a molecular sieve
-Smaller, more charged molecules travel further
-Glycerol ensures DNA is heavy enough to stay in gel
What do we use to visualise agarose gel electrophoresis
Intercalating dyes
How is the size of DNA fragments in agarose gel electrophoresis determined?
-Size of product is compared to DNA ladder
-Look by eye
-Plot a Log10 graph and mm band migrated down the gel
-Using the MW ladder to create a standard curve
What is genome editing?
A type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using nucleases -
-Enabling specific targeting of sequences within the genome without impacting the rest of the sequence
-Potential to cure genetic diseases in a patient specific manner
What does CRISPR stand for?
Clustered Regulatory Interspaced Short Palindromic Repeats
What is Cas9
CRISPR Associated Proteins
What are the three components that make up the CRISPR complex?
-Cas9 (protein component)
-crRNA (RNA component)
-tracrRNA (RNA component)
Describe how CRISPR acts as an adaptive immune regulator in prokaryotes?
-Invading DNA recognised is cut by Cat protein complexes into fragments termed protospacers
-Protospacers integrated into CRISPR locus located in the bacterial genome
-Upon viral reinfection, transcription of the protospacers to RNA is activated which bind to Cas9
-Cas9/RNA duplex is recruited to complementary sequence on the invading strand of DNA
-Cas9 cuts DNA strand creating a double strand break to prevent reinfection
Give some key components of the CRISPR locus
-Transactivating RNA
-Operon of cas genes encoding cas protein components (eg cas1, cas2, cas9)
-Identical repeat arrays
-Spacers of invading DNA
Describe what guide RNA consists of in CRISPR
The complex formed between transactivating RNA and the protospacer/CRISPR RNA (crRNA) which enables selective binding of cas9 to invading DNA sequences
What enables Cas9 mediated DNA cleavage?
Protospacer Adjacent Motifs (PAM), Cas9 will not cut invading DNA without a PAM site irrespective of Cas/gRNA binding
How is CRISPR/Cas9 engineered for biomedical studies?
-The crRNA and tracrRNA is linked using a linker loop
-Deposition of the Cas9/gRNA complex at a desired locus of the genome will enable site-specific cleavage through nuclease activity
-The repair of the DNA break by endogenous DNA repair pathways enables specific genomic edits to be introduced
What is required following CRISPR-mediated DNA cutting?
Cellular DNA repair pathways such as
-Homology directed repair (HDR)
-Non homologous end joining (NHEJ)
Describe Non-homologous end joining (NHEJ)
Error prone as it
-Introduces insertions or deletions (indels) into DNA, generating frameshifts
-Impacts gene function
-Key to knockout studies
Describe Homology Directed Repair (HDR)
-DNA is precisely repaired using sister chromatid during S phase (In CRISPR a template is introduced that will be used)
-PAM sites are removed from HR template to prevent retargeting of region
-Key to CRISPR knock in studies
Describe Ex vivo methods to deliver CRISPR in the clinic
-Remove cells from the patient
-Edit genome
-Screen cell populations
-Engraft cells back into patient
Describe In vivo methods to deliver CRISPR in the clinic
-Package CRISPR/Cas in a delivery vehicle
-Deliver to patient
Describe how CRISPR has been used to knock out the Androgen receptor gene as a treatment for prostate cancer
1 Generate prostate cancer cell line expressing Cas9
2 AR gene locus was targeted by CRISPR to validate activity of Cas9, using NHEJ, introducing stop codon into AR gene
3 Prostate cell growth is reduced
Describe how CRISPR has been used to knock in variants of the Androgen receptor gene as a treatment for prostate cancer
1 gRNA designed to exon 5 (ligand binding domain)
2 HDR template with point mutation encodes a stop codon, stopping formation of full length androgen receptor
3 Absolute loss of full length AR expression in CRISPR edited cells
What should we consider with CRISPR cell therapies
-Efficacy of delivery
-Regulatory guidelines
-Mosaicism
-Specificity (off target effects)
-Immunogenicity
-Germline vs Somatic
Describe how CRISPR is used to treat HIV?
-CCR5Δ32 is introduced into those with HIV
-This disrupts CCR5 coreceptor, which allows for a reconstitution of a functional immune system
When did the Human genome project run?
From 1990 til 2003
When was the first complete sequence of a bacterial completed?
1995, when H influenza was sequenced
Why sequence the human genome?
-It is the blueprint of life
-Decipher what is coding and non coding
-Regulatory sequences
-Higher order structure
-Chromosome maintenance
-Comparative searches
How is the entire human genome sequence obtained?
-Obtain the organisms genomic DNA
-Break the DNA into small fragments
-Search for overlaps to “reconstruct” the genome sequence
Describe using model organisms in sequencing
-Small genome so “value for money”
-Easy organisms to manipulate
-Provide info on fundamental processes
-Useful in comparative genomics
What were the outcomes of the human genome project?
-Provided a detailed map of the human genome
-Spurred the development of high throughput sequencing technologies, bioinformatics tools and data storage solutions, reducing cost
-Identified genetic variants associated with various diseases
-Fostered international collaboration among scientists
What are some major issues of identifying genes within genomes?
-Identifying open reading frames
-Identification of RNA splice sites (as sequence changes when mRNA)
Give some limitations of genome sequencing
-Incomplete coverage
-Does not capture the full extent of genetic variation
-Does not completely teach us function
How has the human genome project aided research and medicine?
-Foundation for precision medicine (pharmacogenomics)
-Gene therapy
-Genetic screening
-Several open databases being accessible
-Comparative genomics (widely popular)
Give the benefits of studies in model organisms
-Gives insight into functional characterisation of mutant proteins
-Understanding human genetic variation
-Crucial for developing personalised medicine
-Understanding the effects of mutation
Describe using computational analyses of a genome to predict protein localisation
-Programmes predict the sub cellular location of a protein by statistically analysing patterns in its amino acid sequence
-eg PSORTII can determine if a protein is nuclear, cytoplasmic, cytoskeletal, vacuolar, etc
What can computational analyses of sequence be used to do?
-Predict function
-Predict protein localisation
-Predict protein domains
-Identify regulatory sequences
-Characterisation of protein families
What do functional genomics experiments describe?
Gene functions and interactions
Give some types of functional genomics studies
-Protein/DNA interactions
-DNA methylation
-Gene expression
-Protein protein interactions
-Loss of function
What do microarrays measure?
Hybridisation
Describe different types of microarray experiments and what they sample?
Expression - cDNA
ChIP - Immunoprecipitation
SNP - Whole genome
Methylation - Whole genome
CGH - Whole genome
Describe the method to microarrays measuring RNA expression
-Extract RNA and reverse transcript
-In vitro transcription with biotin labelled cRNA
-Fragment and hybridise with to GeneChip
-Wash away non specific binders and stain
-Scan array with lasers detect fluorescence with CCD and read image to computer
Name what has replaced microarrays
High-throughput Sequencing
Describe illumina sequencing
1 - DNA is fragmented and specific adapters are ligated to both ends of fragments
2 - This library is loaded onto a flow cell, where fragments undergo bridge amplification
3 - The flow cell is flooded with fluorescently labeled nucleotides, with each base having a different fluorophore
4 - Images are processed to identify the emitted fluorescence from each cluster, determining the sequence
Describe RNA sequencing
-RNA is converted to cDNA
-cDNA used in high throughput sequencing technologies
-Used for sequencing library generation
-Allowing quantification, profiling and discovery of RNA
Describe the steps of RNA sequencing
-Sample is PolyA enriched or rRNA depleted to focus on coding RNA, fragmented and adapters are ligated
-These fragments are reverse-transcribed and amplified
-These are then probed and read
How is RNA PolyA enriched?
Done using a beat containing a sequence of Ts to which the Poly A tail will bind
How is ribosomal RNA removed from a sample of RNA?
Done using a probe that bind to ribosomal RNA, and then dsRNAase enzymes are used to cut these
What should we consider when using RNA sequencing?
-Big data sets require expert processing
-Expression data can be noisy
-Easy for confounding factors to dominate
Describe ChIP sequencing
-Cells are treated with formaldehyde to crosslink proteins to DNA and preserve interactions
-Chromatin is fragmented
-DNA is enriched using immunoprecipitation
-Crosslinks are reversed through heating, and this DNA is sequenced
-Sequence data is analysed to identify binding sites of the protein across the genome
Describe ATAC sequencing
-Nuclei are isolated
-Treated with transposes enzyme, inserting sequencing adapters into accessible chromatin
-DNA fragments are purified to remove unincorporated transposase
-Amplification
-Sequencing, and regions of open chromatin are identified, which correlate to regulatory elements like enhancers, promoters, etc
Describe Bisulfite sequencing
-Genomic DNA is treated with sodium bisulphite, which converts unmethylated cytosine to uracil, while methylated cytosines remain unchanged
-Bisulphite treated DNA is then amplified, where uracil is converted to thymine, resulting in a sequence where methylated cytosines are retained, while unmethylated appear as thymines
-These are then high throughput se
Describe reduced representation bisulphite sequencing
-Genomic DNA is first digested with restriction enzymes that cut near CpG, capturing regions of interest
-DNA fragments are size selected to enrich for fragments focusing on promoters and enhancer regions
-Selected fragments undergo bisulphite sequencing
-This allows for the study of key regulatory regions in epigenetic studies, allowing for a more cost effective way to study.
Give the standard transgenic approach in mice?
-DNA is microinjected into the pronucleus of a fertilised mouse oocyte
-Injected oocytes are transferred to a pseudo-pregnant recipient mouse
-All offspring are screened for expression of the trans gene by DNA analysis
Name some model organisms
-Mouse (Mus musculus)
-Clawed frog (Xenopus sp.)
-Zebrafish (Danio rerio)
-Fruit fly (Drosophila melanogaster)
-Nematode worm (C elegans)
Give the gene targeted transgenic approach in mice?
-An isogenic trasngene with a drug selection gene is introduced into embryonic stem cells
-Drug selection is used and surviving cells are screened for the correct integration of the trans gene
-Correctly targeted cells are micro injected in mouse blastocysts
-Blastocysts are transferred to pseudo pregnant recipient mouse
-Chimaeric offspring are identified and mated to test for gremlin transmission of trans gene
Give some strengths and weaknesses of transgenic mouse models
+Cheap and cheerful
+Multiple founders are generated
-Cannot control site of integration into genome
-Wild type gene product is still present and may interfere with phenotype
Describe gene trapping (knockout mouse models)
-Gene trap vector is constructed containing a reporter gene and a selectable marker
-This is introduced into mouse embryonic stem cells using electroporation
-ES cells are selected using the selectable marker, only cells with the gene trap vector will survive
-Cells that survive selection are screened for the reporter gene
-Selected ES cells are injected into mouse blastocysts, which are then implanted into pseudopregnant female mice, producing chimeric mice
-These are then bred, to measure germline transmission
Give some strengths and weaknesses of knock out mouse models
+Gene traps available for virtually all genes
-may not accurately model a known human disease
+Can make conditional knockout using cre recombinase
+Can stress or cross onto a different background
Describe multiple sequence alignment
-Used to align 2 or more biological sequences
-To identify similarities, differences and conserved regions amongst them
-Characterises aligned bases into either Match, mismatch (substitution) or Gap (indel)
Give some uses of multiple sequence alignment
-Phylogenetic analysis (inferring evolutionary relationships)
-Functional annotation (inferring important sites in proteins or genes)
-Comparative genomics (highlight genetic variations)
-Protein structure prediction (help identify conserved regions that may correspond to structural motifs)
What is global alignment in pairwise alignment?
-Aligns whole sequences end to end
-eg Needle alignment tool
What is local alignment in pairwise alignment?
-Focuses on best matching parts of sequences
-eg Water alignment tool
Describe the progressive method to multiple sequence alignment
-Progressive alignment builds up the final alignment step-by-step.
-It starts by aligning the two most similar sequences (usually based on a guide tree), then progressively adds other sequences based on their similarity to the existing alignment.
Describe the iterative method to multiple sequence alignment
-Iterative alignment also starts with an initial alignment, often using a progressive approach
-But then iteratively refines the alignment to improve accuracy.
-This refinement process involves realigning sequences multiple times based on a scoring function or other criteria.
Give some software examples of progressive multiple sequence alignment
-ClustalW
-T Coffee
Give some software examples of iterative multiple sequence alignment
-MAFFT
-MUSCLE
Describe the simple steps for multiple sequence alignment using CLUSTAL
-Compare sequences to obtain a similarity matrix
-Using this, make a guide tree that relates all the sequences
-Perform progressive alignments, adding new sequences according to the guide tree
Give some methods to predict secondary protein structure and a brief summary
-Statistical methods (uses probabilities based on residue patterns from protein data bank)
-Machine learning (Uses neural networks/algorithms eg PSIPRED trained on sequence data)
-Deep learning approaches (models eg AlphaFold use transformers and attention mechanisms)
Describe template based modelling to predict tertiary structure
-Find a template protein that has a similar sequence using BLAST
-Sequences are aligned using tools like Cluster or MUSCLE
-Model is built and refined
What are the two main approaches to predicting tertiary protein structure
-Template based modelling
-Ab initio modeling
What competition is held to systematically asses protein prediction quality?
Critical Assessment of Structure Prediction (CASP)
What can be used to assess protein prediction quality?
Global Distance test, which compares the predicted 3D structure to a known experimental reference structure, giving a score
-GDT calculates how many residues in the predicted structure are within a certain distance of their corresponding residues
Describe AlphaFold technology
-Developed by DeepMind, AlphaFold is an AI system used to predict a protein’s 3D structure directly from its primary structure
-Removing the need for Xray crystallography or cryo-electron microscopy
Describe Mass spectrometry
-Analytical technique used to identify and quantify molecules by measuring their mass-to-charge ratio (m/z)
-Used to analyse proteins, drugs and chemicals
-Used to study molecular structures and concentrations of molecules in a sample
Name some protein databases
-UniProt
-PDB
-AlphaFold
Describe Peptide Mass fingerprinting
-Method used to identify proteins by breaking them down into smaller pieces, called peptides, and measuring the masses of these peptides
-Each protein has a unique set of peptide masses after digestion, so PMF allows for protein identification
Describe High dimensional flow cytometry
-A variation of flow cytometry that measures many different characteristics on each cell
-Teaching us about the cell’s function, type or health
Describe cytometry
-Technique for quantitatively measuring markers on cells
-Labelled antibody binds to marker and is detected
-Used in medical diagnostics, research and immunology
Describe mass cytometry
-A combination of flow cytometry and mass spectrometry
-Using heavy metal tagging with antibodies
-More useful than using fluorescence as there is no overlap in metal masses
Give some issues with high throughput data generation
-Noisy (which can drown out the signal)
-Prone to experimental effects (increasing noise)
-Expensive (tendency to reduce sample size)
Describe fMRI
-Functional MRI (fMRI) is used to gather large volumes of brain activity data efficiently, allowing researchers to study brain functions across many individuals or under multiple experimental conditions
-Tracks brain activity by tracking oxygen levels in the blood (indicating active cells)
In statistics, what is a random variable?
An unknown quantity that we can measure
In statistics, what is an observation?
The actual value of a random variable that we see
In statistics, what is data?
Observations collected together
What is statistics, and what does it enable?
-Methods of collecting, analysing and interpreting data
-Enables us to distinguish between random variation and real differences
In statistics, what is a population?
(N), a collection of individuals or all the possible observations that they represent, recognised by parameters
In statistics, what is a sample?
(n), a collection of individuals that we observe. Sample statistics are analogues of population parameters
What is the limiting set of relative frequencies in a sample?
The distribution
When each value within a certain range is equally likely, what form of distribution do we find?
Uniform distribution
As sample size increases, what happens?
The true shape of the population can be visualised
Describe the different types of quantitative data
Continuous - Measured on a continuous scale (eg height)
Discrete - Measured by counts (eg number of years at Newcastle University)
Describe the different types of qualitative data
Nominal - Categories with no logical order
Ordinal - Categories with a logical order
What determines the methods of statistical analysis?
Data type (eg continuous quantitative)
Give some methods to display quantitative data
-Histogram
-Stem and leaf plot
-Box plot
Give some methods to display qualitative data
-Contingency table
-Bar chart
-Pie chart
Describe a histogram
-Width of each bar relates to a range of values for the variable (eg 170<x<180)
-Height of bar is proportional to the frequency in that range
Describe a stem and leaf chart
-A vertical stem consists of the first few digits of the values, arranged in order
-Protruding from this stem are the final digits of each of the ordered values written horizontally
What may we focus on when analysing graphical displays?
-Location (size) ie the mean, mode and median
-Dispersion (spread) ie the IQR, Variance and SD
-Distribution (Shape)
-Outliers
-Sample size
How do we calculate the mean?
Sum of values / Number of values
How do we calculate the median?
-For odd amounts = 0.5x(n+1)th term
-For even amounts = Average of middle two observations
When analysing graphical displays, what should we use with symmetric distributions?
Mean and Standard deviation
When analysing graphical displays, what should we use with asymmetric distributions?
Median and Interquartile Range
When analysing graphical displays, what should we use with categorical information?
Mode
What is the focus of genomics?
Genes
What is the focus of transcriptomics?
mRNA
What is the focus of proteinomics?
Protein
What is the focus of metabolomics?
Metabolites
How may we identify proteins in proteomics?
We measure the weight of their components using mass spectrometry
How do we process proteins into the mass spectrometer?
We use porcine made trypsin that cuts at arginine and proline, so that we work with shorter peptides instead
What are each axis in a mass spectrometry graph?
X axis is mass/charge ratio
Y axis is the abundance of ions
In mass spectrometry, what may cause peaks with very similar mass/charge ratios?
Same peptide but with different isotopic combinations
How do we break peptides before using them in mass spectrometry?
Using helium bullets
What do we do from information gathered from mass spectrometry?
-We get a huge database with many peptides, simulate the spectra that we would obtain from them, and check which is the most similar to the experimental one
-We measure the distances between peaks, and check which amino acids would fit in between, reconstructing the peptide sequence
-We check the experimental spectra from previous analyses, and see what was identified when those were sequenced
What is an experiment?
Activity where we do not know what will happen but we will observe what does
What is an outcome?
One of the possible things that can occur in an experiment
What is a sample space?
The set of all the possible outcomes
What is an event?
A set of outcomes
What is it called when two events cannot occur at the same time?
They are mutually exclusive
What is it called when two events do not affect each other?
Independent events
When is the classical interpretation of probability used?
When all possible outcomes are equally likely
Give the formula to work out probability in the classical interpretation?
Probability = Total number of outcomes in which the event occurs / Total number of possible outcomes
When is the frequentist interpretation of probability used?
When the outcomes of an experiment are not equally likely
Give the formula to work out probability in the frequentist interpretation?
Probability = Number of times an event occurs / Total number of times experiment is performed
When is the subjective interpretation of probability used?
When the experiment can’t be repeated (eg bases in DNA)
Give the formula to work out probability in the subjective interpretation?
Relative frequency of A = Number of occurrences of A / Total number of letters
What does the addition rule describe in probability?
The probability of any of two or more events occurring
Give the formula for the addition rule (using events A and B)
P(A or B) = P(A)+P(B) - P(A and B)
When is the multiplication rule used in probability?
During Conditional probability (ie Given … what’s the probability of…)
Give the formula for the multiplication rule (using events A and B)
P(A and B) = P(A) x P(B given A)
Describe how you find the allele frequency of P (recessive) from a given data set
-Given Aa = 2, aa=3, AA=4,
-You work out total number of recessive alleles (2 + (3x2)) = 8 alleles
-Then divide this by total alleles ((2x2)+(2x3)+(2x4)) = 18
-To give allele frequency (8/18)
What is the limiting set of relative frequencies called?
Distribution
What are the mean, variance and standard deviation symbols in a sample?
X bar, S squared, and S
What are the mean, variance and standard deviation symbols in a population?
µ, σ squared, σ
What parameters do we need to determine the parameters of a normal distribution?
Mean and standard deviation
What are critical values?
Numbers we multiply the standard deviation by so that it contains variant populations
How may we assess if normal distribution is suitable?
-Plotting a histogram (and checking if normal and bell shaped)
-Plotting a probability plot (If P>0.05 we assume the distribution is normal)
How may we convert data to look normally distributed?
-Apply the function to the measured values
-Create normal probability plot
-Check data is now approximately normally distributed
-Analyse transformed data with normal distribution theory
-Convert transform results to original scale
What is the standard deviation of sample mean?
Standard error, and will be small when the standard deviation is small or the sample size is large
If you want to calculate where 95% of data will be, what must you do?
Multiply Standard deviation by 2 before adding and subtracting from sample mean
If you want to construct a 95% confidence interval for the population mean, what must you do?
Multiple the standard error by 2 before adding and subtracting from the sample mean.
