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Year 2 Semester 1 > CMB2000 Essential Biomedical Research Skills > Flashcards

CMB2000 Essential Biomedical Research Skills Flashcards

1
Q

What is PCR

A

Polymerase Chain Reaction

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2
Q

What does DNA replication require?

A

-Template DNA
-40 or more proteins
-Helicase
-Primase
-Polymerases
-Nuclease
-Ligase
-ssDNA binding proteins
-Sliding clamps

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2
Q

What are the advantages of PCR?

A

-Sensitive (can amplify as little as one molecule)
-Specific (cam amplify a unique target sequence)
-Cheap
-Rapid
-Robust (DNA is very stable)

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3
Q

What’s required for PCR?

A

-Template dsDNA
-2 primers (small ssDNA)
-Polymerase (copies the template)
-dNTPs
-Magnesium (cofactor for DNA polymerase enzyme)
-Buffer (maintains pH and provide necessary salt)

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4
Q

What is the function of the sliding clamp in DNA replication?

A

The clamp protein binds DNA polymerase and prevents this enzyme from dissociating from the template DNA strand.

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5
Q

Describe the primers used in PCR?

A

-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

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5
Q

What must primer pairs not have to prevent primer dimers?

A

Complementary regions to each other

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6
Q

Why is Magnesium required in PCR?

A

Magnesium acts to enhance the enzymatic activity (specifically of DNA polymerase) thereby supporting DNA application

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7
Q

What makes up the buffer found in PCR?

A

-pH of 8-9.5
-Tris HCl
-Potassium ions (KCl) promotes annealing

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8
Q

What are the 3 stages of PCR?

A

-Denaturation
-Annealing
-Elongation

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9
Q

What is Nancy520 an example of?

A

An Intercalating agent, which aids in detection of DNA.

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10
Q

How may we detect PCR products?

A

Run products on agarose gel, using intercalating dye to stain DNA to determine size and yield.

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11
Q

Give some key steps involved in cloning

A

-Bioinformatics searching
-Design primers
-PCR
-Choose plasmid and insert
-Transform into competent E Coli
-Select correct colonies

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12
Q

Give some uses of PCR in biotechnology

A

-Cloning
-Manipulating DNA
-Knock out genes
-Fuse host proteins

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13
Q

What must our template DNA for PCR be?

A

-Clean and pure
-Contaminent free
-High concentration

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14
Q

Describe reverse transcriptase PCR

A

-Convert RNA to cDNA, using reverse transcriptase (a retroviral enzyme) that converts RNA to DNA
-Amplify DNA by PCR (including qPCR)

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15
Q

How does qPCR/Real time PCR work?

A

-Using a fluorescent report in the PCR reaction
-Couples amplification of the sequence with quantification of the concentration of DNA

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16
Q

How does qPCR using SYBR green work?

A

Fluorescent that binds to groove of dsDNA

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17
Q

How does qPCR using TAQman work?

A

Fluorescent that uses probes with fluorescent reporter and quencher

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18
Q

In qPCR, if there is a time to reach the cycle threshold, what does this signify?

A

more cDNA in the sample

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19
Q

What may we use as reference genes in qPCR?

A

-House keeping genes
-As these have a constant level of expression, which is not affected by experimental factors

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20
Q

Why do we use reference genes in qPCR?

A

-Essential to support validity of qPCR
-Confirms RNA extraction was good and efficient

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21
Q

Give some common uses of PCR in diagnosis.

A

-Genotyping the patient
-Genotyping the pathogen
-Phenotyping the disease

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22
Q

Why may we use PCR to genotype a patient?

A

-Diagnose genetic traits
-Detection of carriers of genetic traits
-Tissue matching (HLA typing)
-Pharmacogenetics

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23
Q

Why may we use PCR to genotype a pathogen?

A

Diagnose a strain of infecting pathogen

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24
Q

Why may we use PCR to phenotype a disease?

A

-Measuring disease progression
-Measuring disease severity

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25
Q

What types of PCR are used when genotyping a patient?

A

PCR-RFLP or ARMS-PCR

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26
Q

What is PCR-RFLP?

A

PCR-Restriction Fragment Length Polymorphism

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27
Q

What is ARMS-PCR?

A

Amplification Refractory Mutation System-PCR

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28
Q

How does PCR-RFLP work?

A

-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

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29
Q

How may you identify diseased or healthy in PCR-RFLP banding?

A

-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

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30
Q

Give some disadvantages of PCR-RFLP

A

-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

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30
Q

Give some advantages of PCR-RFLP

A

-Cheap
-Easy design
-Applied to microindels and SNPs
-Simple resources with commonly used techniques

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31
Q

How does ARMS PCR work?

A

-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

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32
Q

Give some advantages of using PCR over other methods of microbial diagnosis.

A

-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

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33
Q

What may we use to phenotype a disease using PCR?

A

-Quantitative PCR
-Measures abundance of DNA or RNA in a sample

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34
Q

Why should we make competent cells instead of buying them?

A

-Saves money
-Ensures we don’t run out

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35
Q

What OD600nm should we grow competent cells to?

A

0.3, which is mid exponential growth

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36
Q

When forming competent cells, what may we plate them on?

A

Agar containing Xgal and ampicillin

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37
Q

Why do we use “double selection” ie plating competent cells on Amp+Xgal

A

-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

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38
Q

What is Xgal?

A

-When cleaved by βgalacotosidase creates an intensely blue product which is insoluble
-So when LacZ is functional blue product is formed.

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39
Q

Why do we use calcium chloride when forming competent cells?

A

-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

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40
Q

How can we ensure the proper orientation of the insert in PCR?

A

Using 2 different restriction enzymes

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41
Q

What is the percentage of agarose used in agarose gel electrophoresis?

A

1.5%

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42
Q

In Agarose gel electrophoresis, what is agarose dissolved in?

A

TAE

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43
Q

What is a clinical study?

A

Involves research using human volunteers (also called participants) that is intended to add to medical knowledge. These can be interventional and observational

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44
Q

Give some examples of types of experimental studies

A

-Randomised control trials
-Non randomised control trials

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45
Q

Give some examples of types of observational studies

A

-Cohort studies
-Case control studies
-Cross sectional studies
-Ecological studies

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46
Q

Describe the characteristics of a randomised control trial.

A

-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

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47
Q

Describe the characteristics of a cohort study.

A

-A defined group is followed over time
-Cohort includes people with similar characteristics

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48
Q

What is the difference between a prospective and a retrospective cohort study?

A

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.

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49
Q

What are case-control studies?

A

-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.

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50
Q

What are cross-sectional studies?

A

-A snapshot observation of a set of people at 1 time
-Aimed to describe a variable

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51
Q

Describe ecological studies

A

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

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52
Q

Give the phases of a clinical trial

A

-Laboratory studies
-Phase I
-Phase II
-Phase III
-Phase IV

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53
Q

Give the goals of preclinical (laboratory) phase of research?

A

-Model the desired biological effect of a drug
-Predict treatment outcome in patients (EFFICACY)
-Identify and characterise toxicities associated with a drug (SAFETY)

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54
Q

Give the goals of Phase I of research?

A

Study the drug to learn about safety and side effects

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55
Q

Give the goals of Phase III of research?

A

-Gathering information about safety and effectiveness
-Focusing on determination of dosage

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55
Q

Give the goals of Phase II of research?

A

-Focus of effectiveness
-Obtain data on efficacy in those with a certain condition or disease

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56
Q

Give the goals of Phase IV of research?

A

-Post marketing monitoring stage
-Monitering polypharmacy

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57
Q

What things should you consider when setting up and running a clinical trial?

A

-Consent
-Controls
-Randomisation
-Blinding
-Sample size
-Statistics
-Ethics

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58
Q

What is ethics?

A

Moral principles that govern a person’s behaviour or the conducting of an activity

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59
Q

What is bias?

A

Systematic errors that encourage one outcome over others.

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60
Q

What is randomisation?

A

Randomisation its the process by which treatments are assigned to participants by chance rather than by choice

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61
Q

Give some types of randomisation

A

-Simple randomisation
-Block randomisation
-Stratified randomisation

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62
Q

What is simple randomisation?

A

-Randomisation based on a single sequence of random assignments
-Maintaining complete randomness of a subject to a particular group.

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63
Q

What is block randomisation?

A

-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.

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64
Q

What is stratified randomisation?

A

-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

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65
Q

What is blinding?

A

Studies are designed to prevent members of the research team and study participants from influencing the results.

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65
Q

What is the difference between single and double blinded studies?

A

Single - Only the researcher knows the allocation
Double - Neither the participants nor researcher knows the allocation

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66
Q

Why do double blinding?

A

Prevents the research team from
-Influencing patient management
-Influencing withdrawing patient from trial
-Decide to adjust drug dose or therapy intensity

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67
Q

What is the placebo effect?

A

The placebo effect is when a person’s physical or mental health appears to improve after taking a placebo treatment

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68
Q

What is a hypothesis?

A

A proposed assumption for a phenomenon that may or may not be true

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69
Q

What is the issue with having a sample with too few participants?

A

-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

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70
Q

What is the issue with having a sample with too many participants?

A

-Smaller trials could reach firm conclusions so no need for participants beyond this
-Cost implications

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71
Q

What are the different types of end points in a study?

A

-True endpoint
-Surrogate endpoint

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72
Q

What is the true endpoint of a study?

A

A clinical meaningful endpoint that directly measure patients eg how a patient feels, functions or survives

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73
Q

What is the surrogate endpoint of a study?

A

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.

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74
Q

What is the dissemination of results?

A

Getting the findings of your research to the people who can make use of them, to maximise the benefit of the research.

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75
Q

What are some of the principles that make up the Nuremberg code for ethical research?

A

-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

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76
Q

What does the Helsinki declaration do?

A

Guide doctors operating in the dual role of researcher and clinician.

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77
Q

What is a human participant in research?

A

-A living human being
-Human beings who have recently died (including body parts)
-Foetus
-Embryo

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78
Q

How can Human participants be involved in research?

A

-Directly through physical presence
-Indirectly through stored human tissue/body fluids or use of human data

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79
Q

What is informed consent?

A

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

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80
Q

How would you describe the multi stepped process that is gaining informed consent?

A

1 - Giving of information
2 - Discussion, clarification and review
3 - Obtaining written/verbal consent
4 - Ongoing revalidation of consent

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81
Q

What does the common law dictate a child as?

A

Below the age of 18 years old

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82
Q

How do we gain informed consent from children?

A

Under 16 years, it is often best practice to gain the child’s assent and legal guardian provides informed consent.

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83
Q

What laws dictate the ability to gather informed consent from vulnerable adults?

A

-Mental Capacity Act 2007
-Medicines for Human Use (clinical trials) Regulations 2004
-Common Law

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84
Q

Describe the Human Tissue Act 2004

A

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.

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85
Q

What is inducement in gaining consent?

A

The provision of an incentive to entice a person to carry out an action

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86
Q

What is GDPR?

A

-General Data Protection Regulation
-Governs the protection and control of personal data

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87
Q

What qualifies as personal data under GDPR?

A

Information that relates to an identified or identifiable individual

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88
Q

What are the principles of GDPR?

A

-Lawfulness fairness and transparency
-Purpose limitation
-Data minimisation
-Accuracy
-Storage limitation
-Integrity and confidentiality
-Accountability principle

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89
Q

What sort of data may be personal?

A

-Identifiable data
-Confidential information
-Sensitive information
-Coded data
-Linked anonymised
-Unlinked anonymised
-Data that relates to an individual

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90
Q

What are the three divisions of medicines in the Medicines Act 1968?

A

-Prescription only medicines
-Pharmacy medication
-General medication

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90
Q

What is the role of the UK research ethics committee?

A

-Ensure the dignity, rights, safety and well being of individuals are protected
-Reducing the risks associated with research

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91
Q

How are participants allocated to UK Research Ethics Committee or University Ethics Committee?

A

Patient participants -> UK Research Ethics Committee
Non-Patient participants -> University Ethics Committee

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92
Q

What do the UK Research Ethics Committee consider?

A

-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?

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93
Q

Name some meta ethical theories

A

-Moral absolutism/dogmatism
-Moral relativism
-Pyrrhonian moral scepticism

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94
Q

Simplify moral absolutism/dogmatism

A

-Ethics from the pulpit
-“I know that X is right or wrong”
-Anyone who disagrees is wrong

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95
Q

Simplify moral relativism

A

-Ethics from the restaurant
-The statement that X is right or wrong is synonymous with the statement that someone approves or disapproves of X

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96
Q

Simplify pyrrhonian moral scepticism

A

I believe that X is right or wrong

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97
Q

What is legalism?

A

-Excessive conformity to the law
-It is always important to reflect on the law

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98
Q

What must we use when looking at the ethics of a situation

A

-Use logic
-Use analogies
-Avoiding slippery slope arguments

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99
Q

Describe the subjective aspect to the need for ethics

A

-People need to justify their behaviour
-People need to explain why their behaviour is (un)acceptable

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100
Q

Describe the objective aspect to the need for ethics

A

Many things deserve moral consideration

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101
Q

What is an ethical theory?

A

An account of which principles should be followed, and of how to balance them against each other

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102
Q

What are formal ethical theories?

A

Formal theories about the sorts of abstract principles and values that should matter (eg happiness, pleasure, health)

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103
Q

What are material ethical theories?

A

Theories about the concrete things/entities that should matter

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104
Q

Name some formal ethical theories

A

-Principlism
-Consequentialism
-Deontology
-Virtue ethics

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105
Q

Describe principlism in medicine

A

4 principles approach, focusing on
-Autonomy
-Beneficence
-Non maleficence
-Justice

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106
Q

What is connected to how we value things?

A

Our ontologies

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107
Q

What are the two values that contribute to relative moral significance?

A

-Intrinsic value (value for oneself)
-Instrumental value (value for others)

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108
Q

What are the two ontologies that dominate western philosophy?

A

-Mechanistic materialism
-Dualism

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109
Q

Describe mechanistic materialism

A

-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

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110
Q

Describe dualism

A

Reality is composed of two fundamentally distinct things - Things with minds and things that lack minds

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111
Q

Describe panexperientalism

A

Experience is fundamental and ubiquitous throughout nature, and that mentality is not essential to it

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111
Q

What is speciesism?

A

The practice of treating members of one species as morally more important than members of other species

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112
Q

What are the issues surrounding research involving animals?

A

-Ethical concerns
-Scientific concerns
-Institutional/professional requirements
-Legal requirements
-Application to medical research

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113
Q

What is the law on using nonhuman animals for biomedical science?

A

-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

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114
Q

What organisms does ASPA 1986 cover?

A

-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

115
Q

What are the three Rs in animal welfare ethics?

A

-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)

116
Q

What does ASPA demand of researchers?

A

-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

117
Q

What must a project licence required by ASPA need?

A

-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

118
Q

What are AWERBs?

A

Animal welfare and ethical review body

119
Q

Name some alternatives to nonhuman animal research?

A

-Microdosing
-Observational studies
-Randomised controlled studies
-Use of human tissues, cells and genes
-Computer testing

120
Q

What is the purpose and methods of biomedical research?

A

Purpose - Researching the causes of health and illness of biological organisms
Methods - Using biological organisms to identify the causes of health and illness

121
Q

What are the legal requirements in relation to consent?

A

The person must
-Have capacity/competency
-Is informed adequately
-Not be coerced

122
Q

Is it ethical to involve those who lack capacity in clinical research?

A

-At times, eg with diseases surrounding children or those with alzheimers
-Can be circumvented using assent

123
Q

Describe the freedom of information act?

A

-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

124
Q

What are the 6 data protection principles in the data protection act?

A

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

125
Q

Name some examples of scientific misconduct

A

-Falsification
-Fabrication
-Plagiarism
-Self plagiarism
-Gift authorship

126
Q

What does the view of absolute dominion lead to?

A

Speciesism

127
Q

Why believe in absolute dominion over animals?

A

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

128
Q

What are the levels of animal use in drug development?

A

-In silico (computational)
-In vitro (molecular, cellular, tissue and organ)
-In vivo (whole animal)

129
Q

What does drug safety testing involve?

A

-A limited range of in vitro and in vivo screens
-A relatively small number of animals (100s)

130
Q

Why believe in abolition of using animals in research?

A

-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

131
Q

Name some drug experiments that would be misleading if animals were used

A

-Penicillin (lethal in guinea pigs)
-Paracetamol (lethal in cats)
-Thalidomide (tested in the incorrect species to model to humans)

132
Q

Which experimental procedures on animals are regulated?

A

If it causes
-Death, disease and injury
-Physiological and psychological stress
-Discomfort and disturbance to normal health
-Anaesthetic or analgesic administration

133
Q

When is an animal considered to be living?

A

Until circulation ceases/brain is destroyed

134
Q

What procedures does ASPA not cover?

A

-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

135
Q

how does a cost benefit analysis work in theory?

A

-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

136
Q

What are the pillars of academic integrity?

A

-Honesty
-Fairness
-Accountability
-Transparency
-Responsibility

137
Q

What is plagiarism?

A

Copying and passing another person/source’s work off as your own

138
Q

Give some examples of types of plagiarism

A

-Text (Copying text from a book, paper, etc)
-Diagrams
-Idea
-Auto (copying from yourself)

139
Q

How do you avoid plagiarism?

A

-Read and take from multiple sources of information
-Make notes in your own words
-DO NOT COPY AND PASTE

140
Q

What is an essay mill?

A

Getting someone to write an essay on your behalf

141
Q

What are some methods of detecting scientific fraud?

A

-Algorithms
-Social media
-Awareness
-Public domain
-Standards

142
Q

Name some consequences to scientific fraud

A

-Distrust from public
-Practice changing
-Implication on health and fatalities
-Controversies in the field
-Long lasting effects

143
Q

Name some reasons for DNA isolation

A

-PCR
-DNA analysis
-Genetic testing
-Forensics
-Ecological
-Archaeological

144
Q

Name the steps of DNA isolation

A

1 Cell lysis
2 DNA purification from cell extract
3 DNA concentration
4 Measurement of DNA purity and concentration

145
Q

What do we not want in a sample of DNA when isolating it?

A

-Proteins
-Ribosomes
-mtDNA
-Lipid
-Plasmids

146
Q

Name some methods of cell lysis

A

-Biological methods
-Physical methods
-Mechanical methods

147
Q

Describe biological methods to lyse cells

A

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

148
Q

Give examples of physical methods to lyse cells

A

-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

149
Q

Describe mechanical methods to lyse cells

A

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

150
Q

Describe DNA purification by phenol-chloroform extraction

A

-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

151
Q

Describe DNA purification using commercial kits

A

-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

152
Q

Give some methods to measure quantity and quality of DNA

A

-UV absorbance
-Fluorescence dyes
-Agarose gel electrophoresis
-Capillary electrophoresis
-Diphenylamine

153
Q

Why is DNA isolation and purification important?

A

-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

154
Q

What are restriction endonucleases?

A

-Enzymes produced by bacteria to protect against viral DNA infection
-Act on specific DNA sequences (restriction sites)
-Cleaving the phosphodiester bond within a polynucleotide

155
Q

How do restriction enzymes cut DNA?

A

-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+

156
Q

What is Star activity of restriction endonuclease?

A

-Relaxation or alteration of the specificity
-When reaction conditions differ significantly from the optimum for the enzyme
-

157
Q

Describe the principle of agarose gel electrophoresis

A

-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

158
Q

What do we use to visualise agarose gel electrophoresis

A

Intercalating dyes

159
Q

How is the size of DNA fragments in agarose gel electrophoresis determined?

A

-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

160
Q

What is genome editing?

A

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

161
Q

What does CRISPR stand for?

A

Clustered Regulatory Interspaced Short Palindromic Repeats

162
Q

What is Cas9

A

CRISPR Associated Proteins

163
Q

What are the three components that make up the CRISPR complex?

A

-Cas9 (protein component)
-crRNA (RNA component)
-tracrRNA (RNA component)

164
Q

Describe how CRISPR acts as an adaptive immune regulator in prokaryotes?

A

-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

165
Q

Give some key components of the CRISPR locus

A

-Transactivating RNA
-Operon of cas genes encoding cas protein components (eg cas1, cas2, cas9)
-Identical repeat arrays
-Spacers of invading DNA

166
Q

Describe what guide RNA consists of in CRISPR

A

The complex formed between transactivating RNA and the protospacer/CRISPR RNA (crRNA) which enables selective binding of cas9 to invading DNA sequences

167
Q

What enables Cas9 mediated DNA cleavage?

A

Protospacer Adjacent Motifs (PAM), Cas9 will not cut invading DNA without a PAM site irrespective of Cas/gRNA binding

168
Q

How is CRISPR/Cas9 engineered for biomedical studies?

A

-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

169
Q

What is required following CRISPR-mediated DNA cutting?

A

Cellular DNA repair pathways such as
-Homology directed repair (HDR)
-Non homologous end joining (NHEJ)

170
Q

Describe Non-homologous end joining (NHEJ)

A

Error prone as it
-Introduces insertions or deletions (indels) into DNA, generating frameshifts
-Impacts gene function
-Key to knockout studies

171
Q

Describe Homology Directed Repair (HDR)

A

-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

172
Q

Describe Ex vivo methods to deliver CRISPR in the clinic

A

-Remove cells from the patient
-Edit genome
-Screen cell populations
-Engraft cells back into patient

173
Q

Describe In vivo methods to deliver CRISPR in the clinic

A

-Package CRISPR/Cas in a delivery vehicle
-Deliver to patient

174
Q

Describe how CRISPR has been used to knock out the Androgen receptor gene as a treatment for prostate cancer

A

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

175
Q

Describe how CRISPR has been used to knock in variants of the Androgen receptor gene as a treatment for prostate cancer

A

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

176
Q

What should we consider with CRISPR cell therapies

A

-Efficacy of delivery
-Regulatory guidelines
-Mosaicism
-Specificity (off target effects)
-Immunogenicity
-Germline vs Somatic

177
Q

Describe how CRISPR is used to treat HIV?

A

-CCR5Δ32 is introduced into those with HIV
-This disrupts CCR5 coreceptor, which allows for a reconstitution of a functional immune system

178
Q

When did the Human genome project run?

A

From 1990 til 2003

178
Q

When was the first complete sequence of a bacterial completed?

A

1995, when H influenza was sequenced

179
Q

Why sequence the human genome?

A

-It is the blueprint of life
-Decipher what is coding and non coding
-Regulatory sequences
-Higher order structure
-Chromosome maintenance
-Comparative searches

180
Q

How is the entire human genome sequence obtained?

A

-Obtain the organisms genomic DNA
-Break the DNA into small fragments
-Search for overlaps to “reconstruct” the genome sequence

181
Q

Describe using model organisms in sequencing

A

-Small genome so “value for money”
-Easy organisms to manipulate
-Provide info on fundamental processes
-Useful in comparative genomics

182
Q

What were the outcomes of the human genome project?

A

-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

183
Q

What are some major issues of identifying genes within genomes?

A

-Identifying open reading frames
-Identification of RNA splice sites (as sequence changes when mRNA)

184
Q

Give some limitations of genome sequencing

A

-Incomplete coverage
-Does not capture the full extent of genetic variation
-Does not completely teach us function

185
Q

How has the human genome project aided research and medicine?

A

-Foundation for precision medicine (pharmacogenomics)
-Gene therapy
-Genetic screening
-Several open databases being accessible
-Comparative genomics (widely popular)

186
Q

Give the benefits of studies in model organisms

A

-Gives insight into functional characterisation of mutant proteins
-Understanding human genetic variation
-Crucial for developing personalised medicine
-Understanding the effects of mutation

187
Q

Describe using computational analyses of a genome to predict protein localisation

A

-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

187
Q

What can computational analyses of sequence be used to do?

A

-Predict function
-Predict protein localisation
-Predict protein domains
-Identify regulatory sequences
-Characterisation of protein families

188
Q

What do functional genomics experiments describe?

A

Gene functions and interactions

189
Q

Give some types of functional genomics studies

A

-Protein/DNA interactions
-DNA methylation
-Gene expression
-Protein protein interactions
-Loss of function

190
Q

What do microarrays measure?

A

Hybridisation

191
Q

Describe different types of microarray experiments and what they sample?

A

Expression - cDNA
ChIP - Immunoprecipitation
SNP - Whole genome
Methylation - Whole genome
CGH - Whole genome

192
Q

Describe the method to microarrays measuring RNA expression

A

-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

193
Q

Name what has replaced microarrays

A

High-throughput Sequencing

194
Q

Describe illumina sequencing

A

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

195
Q

Describe RNA sequencing

A

-RNA is converted to cDNA
-cDNA used in high throughput sequencing technologies
-Used for sequencing library generation
-Allowing quantification, profiling and discovery of RNA

196
Q

Describe the steps of RNA sequencing

A

-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

197
Q

How is RNA PolyA enriched?

A

Done using a beat containing a sequence of Ts to which the Poly A tail will bind

198
Q

How is ribosomal RNA removed from a sample of RNA?

A

Done using a probe that bind to ribosomal RNA, and then dsRNAase enzymes are used to cut these

199
Q

What should we consider when using RNA sequencing?

A

-Big data sets require expert processing
-Expression data can be noisy
-Easy for confounding factors to dominate

200
Q

Describe ChIP sequencing

A

-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

200
Q

Describe ATAC sequencing

A

-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

201
Q

Describe Bisulfite sequencing

A

-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

202
Q

Describe reduced representation bisulphite sequencing

A

-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.

203
Q

Give the standard transgenic approach in mice?

A

-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

203
Q

Name some model organisms

A

-Mouse (Mus musculus)
-Clawed frog (Xenopus sp.)
-Zebrafish (Danio rerio)
-Fruit fly (Drosophila melanogaster)
-Nematode worm (C elegans)

204
Q

Give the gene targeted transgenic approach in mice?

A

-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

205
Q

Give some strengths and weaknesses of transgenic mouse models

A

+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

206
Q

Describe gene trapping (knockout mouse models)

A

-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

207
Q

Give some strengths and weaknesses of knock out mouse models

A

+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

208
Q

Describe multiple sequence alignment

A

-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)

209
Q

Give some uses of multiple sequence alignment

A

-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)

210
Q

What is global alignment in pairwise alignment?

A

-Aligns whole sequences end to end
-eg Needle alignment tool

211
Q

What is local alignment in pairwise alignment?

A

-Focuses on best matching parts of sequences
-eg Water alignment tool

212
Q

Describe the progressive method to multiple sequence alignment

A

-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.

213
Q

Describe the iterative method to multiple sequence alignment

A

-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.

214
Q

Give some software examples of progressive multiple sequence alignment

A

-ClustalW
-T Coffee

215
Q

Give some software examples of iterative multiple sequence alignment

A

-MAFFT
-MUSCLE

216
Q

Describe the simple steps for multiple sequence alignment using CLUSTAL

A

-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

217
Q

Give some methods to predict secondary protein structure and a brief summary

A

-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)

218
Q

Describe template based modelling to predict tertiary structure

A

-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

218
Q

What are the two main approaches to predicting tertiary protein structure

A

-Template based modelling
-Ab initio modeling

219
Q

What competition is held to systematically asses protein prediction quality?

A

Critical Assessment of Structure Prediction (CASP)

220
Q

What can be used to assess protein prediction quality?

A

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

221
Q

Describe AlphaFold technology

A

-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

222
Q

Describe Mass spectrometry

A

-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

222
Q

Name some protein databases

A

-UniProt
-PDB
-AlphaFold

223
Q

Describe Peptide Mass fingerprinting

A

-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

224
Q

Describe High dimensional flow cytometry

A

-A variation of flow cytometry that measures many different characteristics on each cell
-Teaching us about the cell’s function, type or health

224
Q

Describe cytometry

A

-Technique for quantitatively measuring markers on cells
-Labelled antibody binds to marker and is detected
-Used in medical diagnostics, research and immunology

225
Q

Describe mass cytometry

A

-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

226
Q

Give some issues with high throughput data generation

A

-Noisy (which can drown out the signal)
-Prone to experimental effects (increasing noise)
-Expensive (tendency to reduce sample size)

227
Q

Describe fMRI

A

-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)

228
Q

In statistics, what is a random variable?

A

An unknown quantity that we can measure

229
Q

In statistics, what is an observation?

A

The actual value of a random variable that we see

230
Q

In statistics, what is data?

A

Observations collected together

231
Q

What is statistics, and what does it enable?

A

-Methods of collecting, analysing and interpreting data
-Enables us to distinguish between random variation and real differences

232
Q

In statistics, what is a population?

A

(N), a collection of individuals or all the possible observations that they represent, recognised by parameters

233
Q

In statistics, what is a sample?

A

(n), a collection of individuals that we observe. Sample statistics are analogues of population parameters

234
Q

What is the limiting set of relative frequencies in a sample?

A

The distribution

235
Q

When each value within a certain range is equally likely, what form of distribution do we find?

A

Uniform distribution

236
Q

As sample size increases, what happens?

A

The true shape of the population can be visualised

237
Q

Describe the different types of quantitative data

A

Continuous - Measured on a continuous scale (eg height)
Discrete - Measured by counts (eg number of years at Newcastle University)

238
Q

Describe the different types of qualitative data

A

Nominal - Categories with no logical order
Ordinal - Categories with a logical order

239
Q

What determines the methods of statistical analysis?

A

Data type (eg continuous quantitative)

240
Q

Give some methods to display quantitative data

A

-Histogram
-Stem and leaf plot
-Box plot

241
Q

Give some methods to display qualitative data

A

-Contingency table
-Bar chart
-Pie chart

242
Q

Describe a histogram

A

-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

243
Q

Describe a stem and leaf chart

A

-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

244
Q

What may we focus on when analysing graphical displays?

A

-Location (size) ie the mean, mode and median
-Dispersion (spread) ie the IQR, Variance and SD
-Distribution (Shape)
-Outliers
-Sample size

245
Q

How do we calculate the mean?

A

Sum of values / Number of values

246
Q

How do we calculate the median?

A

-For odd amounts = 0.5x(n+1)th term
-For even amounts = Average of middle two observations

247
Q

When analysing graphical displays, what should we use with symmetric distributions?

A

Mean and Standard deviation

248
Q

When analysing graphical displays, what should we use with asymmetric distributions?

A

Median and Interquartile Range

249
Q

When analysing graphical displays, what should we use with categorical information?

A

Mode

250
Q

What is the focus of genomics?

A

Genes

251
Q

What is the focus of transcriptomics?

A

mRNA

252
Q

What is the focus of proteinomics?

A

Protein

253
Q

What is the focus of metabolomics?

A

Metabolites

254
Q

How may we identify proteins in proteomics?

A

We measure the weight of their components using mass spectrometry

255
Q

How do we process proteins into the mass spectrometer?

A

We use porcine made trypsin that cuts at arginine and proline, so that we work with shorter peptides instead

256
Q

What are each axis in a mass spectrometry graph?

A

X axis is mass/charge ratio
Y axis is the abundance of ions

257
Q

In mass spectrometry, what may cause peaks with very similar mass/charge ratios?

A

Same peptide but with different isotopic combinations

258
Q

How do we break peptides before using them in mass spectrometry?

A

Using helium bullets

259
Q

What do we do from information gathered from mass spectrometry?

A

-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

260
Q

What is an experiment?

A

Activity where we do not know what will happen but we will observe what does

261
Q

What is an outcome?

A

One of the possible things that can occur in an experiment

262
Q

What is a sample space?

A

The set of all the possible outcomes

263
Q

What is an event?

A

A set of outcomes

264
Q

What is it called when two events cannot occur at the same time?

A

They are mutually exclusive

265
Q

What is it called when two events do not affect each other?

A

Independent events

266
Q

When is the classical interpretation of probability used?

A

When all possible outcomes are equally likely

267
Q

Give the formula to work out probability in the classical interpretation?

A

Probability = Total number of outcomes in which the event occurs / Total number of possible outcomes

268
Q

When is the frequentist interpretation of probability used?

A

When the outcomes of an experiment are not equally likely

269
Q

Give the formula to work out probability in the frequentist interpretation?

A

Probability = Number of times an event occurs / Total number of times experiment is performed

270
Q

When is the subjective interpretation of probability used?

A

When the experiment can’t be repeated (eg bases in DNA)

271
Q

Give the formula to work out probability in the subjective interpretation?

A

Relative frequency of A = Number of occurrences of A / Total number of letters

272
Q

What does the addition rule describe in probability?

A

The probability of any of two or more events occurring

273
Q

Give the formula for the addition rule (using events A and B)

A

P(A or B) = P(A)+P(B) - P(A and B)

274
Q

When is the multiplication rule used in probability?

A

During Conditional probability (ie Given … what’s the probability of…)

275
Q

Give the formula for the multiplication rule (using events A and B)

A

P(A and B) = P(A) x P(B given A)

276
Q

Describe how you find the allele frequency of P (recessive) from a given data set

A

-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)

277
Q

What is the limiting set of relative frequencies called?

A

Distribution

278
Q

What are the mean, variance and standard deviation symbols in a sample?

A

X bar, S squared, and S

279
Q

What are the mean, variance and standard deviation symbols in a population?

A

µ, σ squared, σ

280
Q

What parameters do we need to determine the parameters of a normal distribution?

A

Mean and standard deviation

281
Q

What are critical values?

A

Numbers we multiply the standard deviation by so that it contains variant populations

282
Q

How may we assess if normal distribution is suitable?

A

-Plotting a histogram (and checking if normal and bell shaped)
-Plotting a probability plot (If P>0.05 we assume the distribution is normal)

283
Q

How may we convert data to look normally distributed?

A

-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

284
Q

What is the standard deviation of sample mean?

A

Standard error, and will be small when the standard deviation is small or the sample size is large

285
Q

If you want to calculate where 95% of data will be, what must you do?

A

Multiply Standard deviation by 2 before adding and subtracting from sample mean

286
Q

If you want to construct a 95% confidence interval for the population mean, what must you do?

A

Multiple the standard error by 2 before adding and subtracting from the sample mean.

Year 2 Semester 1 (5 decks)

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