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Introduction to Biomedicine > Lab Techniques - Year 1 > Flashcards

Lab Techniques - Year 1 Flashcards

1
Q

Aim of Northern Blotting

A

quantities how much candidate mRNA transcript is present in a sample; SDS-page

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2
Q

Aim of Southern Blotting

A

detects specific DNA sequences in a sample; agarose gel

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3
Q

Aim of PCR

A

synthesises several copies of a DNA or RNA segment

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4
Q

Aim of RT-PCR

A

synthesise several copies of a DNA or RNA segment

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5
Q

Aim of qRT-PCR

A

quantifies how much mRNA transcript is present in a sample

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6
Q

Aim of DNA microarrays

A

analyse gene expression at whole genome level

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7
Q

Aim of RNA-seq

A

analyse gene expression at whole genome level

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8
Q

Aim of transcriptomics

A

analyse gene expression at whole genome level

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9
Q

Aim of Western Blotting

A

quantify how much protein is expressed; agarose gel

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10
Q

Aim of Immunoprecipitation

A

identify protein-protein interactions

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11
Q

Aim of Proteomics

A

analyse protein expression at whole genome level

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12
Q

Aim of Immunofluorescence

A

visualise protein location

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13
Q

Aim of Metabolimics

A

analyse metabolites and biochemical pathways

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14
Q

Aim of Electrophysiology

A

analyse ion channel activity and function

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15
Q

Aim of RNA interference

A

knocks down mRNA/protein expression to investigate function

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16
Q

Name 3 places that fund biomedical research in academia

A

e.g. industry, Medical Research Council (MRC), BBSRC, Wellcome Trust, charities

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17
Q

Define: Epidemiology

A

the quantities study of the distribution, aetiology, and prevention of disease in populations

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18
Q

Name a type of epidemiology study

A

e.g. case-control, cohort, randomised control studies

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19
Q

What is the Bradford-Hill criteria?

A

evidence to support causal association: plausibility, consistency, temporality

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20
Q

Define: Aetiology

A

study of causes of a disease or condition

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21
Q

What is needed before a human tissue sample can be taken? (4 things)

A

ethics approval, NHS R&D approval, consent, volunteers

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22
Q

Give three examples of where human tissue samples can be obtained from

A

e.g. surgery, biopsies, blood, body fluids, endoscopy, the Human Tissue Bank

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23
Q

What are the steps involved in PCR?

A

denaturation, annealing, extending

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24
Q

Describe the PCR process

A
  1. double-stranded DNA is heated to 95 degrees to break the hydrogen bonds between bases
  2. temperature is lowered to 50-65 degrees enabling DNA primers to attach to template DNA by hydrogen bonding
  3. temperature is raised to 72 degrees and the new strand of DNA is built by Taw polymerase enzyme in the 5’ to 3’ direction. It attaches to the primer then adds DNA bases to the template strand
25
Q

Define: allele-specific PCR

A

a diagnostic or cloning technique based on single nucleotide or single base differences

26
Q

What does allele-specific PCR require?

A

understanding of the DNA sequences and difference between alleles, and primers which encompass the variation

27
Q

How do PCR and reverse transcriptase PCR differ?

A

reverse transcriptase PCR starts with mRNA rather than the DNA and this is used to generate cDNA

28
Q

Describe the process of real-time PCR

A

uses two specific primers and an oligonucleotide (probe)

probe has a fluorescent reporter group and quencher group and the proximity of the quencher to the reporter prevents fluorescence before and during binding to the target sequences

probe is hydrolysed during polymerisation, releasing the fluorescent reporter, generating a signal proportional to the amount of the target amplified

29
Q

Define: mutant allele-specific PCR

A

PCR amplification of wild type alleles is blocked by a PNA probe while the mutant allele is PCR amplified

30
Q

Describe the process of genetic screening using a microarray

A
  1. DNA extracted from the patient’s blood sample is amplified for a specific gene by PCR
  2. DNA is spotted onto the microarray on a glass slide
  3. fluorescent labelled oligonucleotide proves corresponding to the wild type gene and the mutant versions are hybridised onto the microarray
  4. the resulting hybridisation is revealed by the pattern and colour of the spots on the microarray; these are scanned by a laser and analysed by software
31
Q

What is in situ hybridisation (ISH)?

A

uses a labelled complementary DNA or RNA strand as a probe to localise a specific DNA or RNA sequence in a section of a tissue

32
Q

Define: transfection

A

the process of introducing nucleus acids into cells, especially for non-viral methods in eukaryotic cells

33
Q

Define: transduction

A

virus-mediated DNA transfer

34
Q

Describe the process of non-viral transfection in animal cells

A

involves opening temporary pores in the cell membrane, allowing uptake of material

can be done using calcium phosphate, by electroporation in vitro, or by mixing with cationic lipids - sometimes time produce liposomes which fuse with cell membrane and deposits the nucleic acid inside

35
Q

Why are viral methods used less often then non-viral methods for transfection, despite being more efficient?

A

They tend to trigger an immune response

36
Q

Define: Gene editing

A

the removal of defective dominant genes, the correction of defective recessive genes, and the addition of protective mutations

37
Q

Give three examples of technologies for targeted gene modification

A

e.g. Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), CRISPR

38
Q

Define: Zinc Finger Nucleases (ZFNs)

A

engineered DNA-binding proteins that enable targeted editing of the genome by creating double-stranded breaks in DNA at used-specified locations

39
Q

Define: TALENs

A

restriction enzymes that can be engineered to cut specific sequences of DNA

40
Q

Describe the process of generating antibodies

A
  1. the purified natural or recombinant antigen protein is injected into an animal- often with an adjuvant to increase immune response
  2. animal’s immune system produces antibodies for the protein
  3. the antibodies are collected through bleeding then may be purified
41
Q

Describe the process of western blotting

A
  1. SDS-page
  2. transfer proteins to membrane: usually nitrocellulose or PVDF. the bound antibody is detected by colour or light
  3. detection of specific protein: antibodies are used to stain the protein of interest
42
Q

What is ELISA?

A

Enzyme-Linked Immuno-Sandwich Assay

a quantitive measure of concentration of a specific protein

43
Q

Give 4 uses of proteomics

A

e.g. sequencing proteins, identifying proteins in complex mixtures, peptide mass fingerprinting follows identify unknown proteins, characterisation of changes to the composition of complex mixture

44
Q

What are the steps in mass spectrometry?

A
  1. ion source
  2. mass analyser
  3. detector
  4. data system
45
Q

What is MALDI used for?

A

matrix assisted laser desorption ionisation
used for the study of polymers, proteins, and peptides
a soft ionisation technique used in mass spectrometry

46
Q

Describe the process of MALDI

A
  1. sample is pre-mixed with an absorbing matrix compound
  2. laser flash ionises matrix molecules
  3. Sample molecules are ionised by proton transfer
47
Q

What is Electro-Spray Ionisation (ESI) and how does it work?

A

a soft ionisation technique used in mass spectrometry
the sample is dissolved in a polar, volatile solvent and pumped through a narrow, stainless steel capillary and voltage is applied

48
Q

Give 5 types of mass analyser techniques used for mass spectrometry

A

time of flight, quadrupole, ion trap, magnetic sector, FTMS

49
Q

How does time of flight (TOF) work?

A

measures the time taken for the ion to teach the detector after leaving the ion source

small ions reach the detector before large ones

50
Q

How does quadrupole analyse ions?

A

it has four metal rods and allows one ion to pass through at a time

it can scan through all masses or sit at one fixed mass

51
Q

What are the criteria for mass spectrometry?

A

mass accuracy

resolution: how well separated the peaks are from each other
sensitivity: the smallest amount that can be analysed

52
Q

How does peptide mass fingerprinting work?

A

proteins are degraded to peptides using trypsin
mass of peptides determined
proteins identified

53
Q

When are mass spectrometry and 2D-gel electrophoresis used in combination?

A

when all the proteins in a cell or tissue need to be identified

54
Q

Describe Tandem Mass Spectrometry (MS-MS)

A

uses two mass analyses to select and ion from a mixture
generates fragments to give structural information

  1. protein is degraded to peptides with trypsin
  2. mass spectrometry separates peptides into molecular ions
  3. individual molecular ion (the precursor ion) selected
  4. precursor ion fragmented in 2nd MS and fragmentation occurs at peptide bonds
  5. masses of product ions measured
  6. m/z data analysed to derive peptide sequence
55
Q

Nuclei with ___ sounds are most useful for NMR

A

1/2

56
Q

What are the problems with using spin as a nuclear measure of behaviour?

A

spin is not aligned

there are very small differences in energy levels of two states i.e. +1/2 and -1/2

57
Q

What does 13C NMR show?

A

the number of carbons in chemically different environments

the relative number of carbons in each environment

the environments of the carbons

58
Q

What are the advantages of 1H-NMR (proton NMR)?

A
  • high abundance of proton nuclei
  • quicker than 13C NMR
  • more sensitive than 13C NMR
  • present in all biological molecules
59
Q

Disadvantages of 1H-NMR (proton NMR)

A
  • also present in water
  • doesn’t detect all protons
  • spectra can get complicated by coupling effect

Introduction to Biomedicine (3 decks)

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