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BT2001: Biotechnology > Omics Technologies > Flashcards

Omics Technologies Flashcards

1
Q

What is Genomics?

A

The study of all of an organisms genes (the genome)

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2
Q

What is Epigenomics?

A

The study of the reversible chemical modifications to DNA

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3
Q

What is Transcriptomics?

A

The study of the complete set of RNA transcripts from DNA

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4
Q

What are the TWO dominant classes of measurement technologies for the transciptome?

A

Microarrays & RNA Sequencing

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5
Q

What is Proteomics?

A

The study of a set of proteins produced in an organism

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6
Q

What is Metabolomics?

A

The study of small molecules (metabolites) within cells

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7
Q

What are the major techniques used in Metabolomics?

A

NMR & Mass Spectroscopy

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8
Q

What is Translatomics?

A

The study of all mRNAs being actively translated in a cell

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9
Q

Who solved the 3D structure of DNA?

A

Watson & Crick
(1953)

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10
Q

Who won The Nobel Prize in Chemistry for their work on the structure of Insulin?

A

Frederick Sanger
(1958)

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11
Q

What enzymes are able to cut RNA chains at specific sites?

A

RNase enzymes

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12
Q

What was the limitation in the early efforts of nucleic acid sequencing?

A

Only able to measure nucleotide composition, and not order

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13
Q

What was the first whole nucleic acid sequence produced by Robert Holley in 1956?

A

Alanine tRNA from Saccharomyces cerevisiae

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14
Q

What was Walter Fiers’ laboratory able to produce in 1972?

A

First complete protein-coding gene

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15
Q

What technique was developed in 1977 that altered the progress of DNA sequencing technology?

A

Sanger’s ‘Chain-Termination’

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16
Q

What are the TWO techniques used in First-Generation DNA Sequencing?

A
  1. Sanger sequencing
  2. Maxam-Gilbert sequencing
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17
Q

Who won the 1980 Nobel Prize for their work into nucleic acids?

A

Berg, Gilbert, & Sanger

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18
Q

What was radiolabelling in Sanger sequencing replaced with?

A

Fluorometric based detection

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19
Q

How was detection in Sanger sequencing improved?

A

Through capillary based electrophoresis

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20
Q

What did the improvements in Sanger sequencing lead to?

A

Automated DNA sequencing machines

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21
Q

What is ‘Shotgun Sequencing’?

A

Where overlapping DNA fragments are cloned and sequenced separately, and then assembled into one long contiguous sequence

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22
Q

What automated DNA sequencing technique did Kary Mullis develop?

A

Polymerase Chain Reaction (PCR)
1983

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23
Q

What are TWO improvments in automated DNA sequencing?

A
  1. Identification of new polymerases
  2. Increasingly modified dNTPs
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24
Q

What project used the newer dideoxy sequencers?

A

Human Genome Project
(1990-2003)

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25
Q

What techique was developed by Pal Nyren in 1993?

A

Pyrosequencing

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26
Q

How does Pyrosequencing work?

A

Relies on light detection based on a chain reaction when Pyrophosphate is released

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27
Q

What THREE enzymes are used in Pyrosequencing?

A
  1. DNA polymerase
  2. ATP sulfurylase
  3. Firefly luciferase
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28
Q

What additional enzyme was introduced into Pyrosequencing to remove nucleotides that are not incorporated by the DNA polymerase?

A

Apyrase

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29
Q

What is a major difficulty with Pyrosequencing?

A

Finding out how many of the same nucleotide there are in a row at a given position

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30
Q

What biotechnology company was Pyrosequencing licensed to?

A

454 Life Sciences

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31
Q

What did the sequencing machines produced by 454 allow?

A

The mass parallelisation of sequencing reactions

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32
Q

What process is used in Illumina sequencing?

A

Bridge amplification

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33
Q

How is sequencing achieved in Illumina sequencing?

A

Using fluorescent ‘reversible-teminator’ dNTPs

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34
Q

What are TWO Third-Generation DNA sequencing techniques?

A
  1. PacBio sequencing
  2. Nanopore sequencing
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35
Q

What is PacBio sequencing?

A

Widely used third-generation technology providing a single molecule real time (SMRT) platform

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36
Q

Where does DNA polymerisation occur in PacBio sequencing?

A

In arrays of microfabricated nanostructures called zero-mode waveguides (ZMWs) which are essentially tiny holes in a metallic film covering a chip

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37
Q

What was α-Hemolysin, a membrane channel protein from Staphylococcus aureus the first nanopore to show?

A

Detect recognisable ionic current blockades by both RNA and DNA homopolymers

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38
Q

What does Nanopore sequencing allow?

A

Enables direct, real-time analysis of long DNA or RNA fragments

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39
Q

How does Nanapore sequencing work?

A

Monitoring changes to an electrical current as nucleic acids are passed through a protein nanopore

40
Q

What is RNA sequencing used for?

A

The analysis of differential gene expression (DGE)

41
Q

What are the core steps of RNA sequencing?

A
  1. RNA extraction
  2. cDNA synthesis
  3. Adapter ligation
  4. PCR amplification
  5. Sequencing and Analysis
42
Q

What are some imperfections and biases associated with RNA sequencing sample preparation and computational analysis?

A
  • Correctly identifying and quantifying which of multiple isoforms are expressed from a gene
  • Differences in how ambiguous or multi-mapped reads are handled
43
Q

What percentage of transcripts in the human transcriptome are over 2,500 bp long?

A

50%

44
Q

What are the THREE main sequencing technologies for RNA sequencing?

A
  1. The Illumina workflow
  2. The Pacific Biosciences workflow
  3. The Oxford Nanopore workflow
45
Q

What is Cap analysis of gene expression (CAGE-seq)?

A

An approach to identify and monitor the activity transcription start sites (TSSs) at single base-pair resolution across the genome

46
Q

What is Cap Trapping?

A

A technique based on the biotinylation of the 7-methylguanosine cap of Pol II transcripts

47
Q

What are the PROS of Cap Trapping?

A
  • Measures RNA expression levels and maps TSS in promoter regions
  • Provides precise mapping of TSS with single-nucleotide resolution
48
Q

What are the CONS of Cap Trapping?

A
  • Only works on total mature DNA
  • Detection is biased toward TSS of long-lived transcripts
49
Q

What are some example of genome-editing tools?

A
  • Zinc-finger nucleases (ZFNs)
  • Transcription activator-like effector nucleases (TALENs)
50
Q

What was discovered by Ishino in 1987?

A

An unusual pattern of nucleotide repeats and spacers 3’ of the iap gene in E. coli

51
Q

What is CRISPR-Cas?

A

A system in an adaptive immune mechanism which defends against specific types of phages

52
Q

What does CRISPR stand for?

A

Clustered Regularly Interspaced Short Palindromic Repeats

53
Q

What structure do most CRISPR fold into?

A

Stem-Loop or Hairpin once they are transcribed into RNA

54
Q

Does Cas-9 have palindromic repeats?

A

No.
Cas-9s crRNA base-pairs with a second piece of RNA called the tracRNA

55
Q

What are Spacers in CRISPR?

A

Small segments of phage DNA and get passed down through generations of bacteria

56
Q

What are the THREE basic steps of the CRISPR-Cas defence system?

A
  1. Spacer aquisition
  2. CRISPR RNA biogenesis
  3. Interference
57
Q

How do CRISPR systems avoid harmful destruction of the bacterium’s own genome?

A

By relying on short DNA sequences called Protospacer Adjacent Motifs or PAMs

58
Q

Where are PAMs found?

A

Next to CRISPR targets in phage DNA but never in bacterial CRISPR arrays

59
Q

Which PAM does the Cas-9 protein from Streptococcus pyrogenes recognise?

A

NGG

60
Q

What are some Cas proteins?

A
  • Helicases
  • Nucleases
  • Polymerases
61
Q

What are Adaptive modules?

A

Cas proteins participating in the acquisition of immunity
e.g. Cas1 and Cas2

62
Q

What are Effector modules?

A

Cas proteins involved in the destruction of mobile genetic elements through their recognition and cleavage
e.g. Cas9

63
Q

Who won The Nobel Prize in Chemistry in 2020 for their work on CRISPR gene editing?

A

Emmanuelle Charpentier & Jennifer Doudna

64
Q

What is the role of CRISPR RNA (crRNA)?

A

Dictates what site Cas9 will cut

65
Q

What is the role of a Trans-Activating CRISPR RNA (tracrRNA)?

A

Attaches to the crRNA and acts as a handle for Cas9

66
Q

What molecule is formed when crRNA and tracrRNA are combined?

A

Single-Guide RNA (sgRNA)

67
Q

What are the steps of DNA target binding and cleavage by Cas9?

A
  1. PAM recognition
  2. Base-pairing
  3. Complete binding
  4. DNA target cleavage
68
Q

What is the process called for stitching two broken ends of DNA back together?

A

Non-Homologous Ending Joining
(NHEJ)

69
Q

What happens in a pooled CRISPR screen?

A

CRISPR proteins plus a pooled library of guide RNAs (gRNAs) are added to a large batch of cells to test thousands of genetic targets at once.

70
Q

What happens in Positive Selection in CRISPR screens?

A

Cells with selected trait survive

71
Q

What happens in Negative Selection in CRISPR screens?

A

Cells with selected trait die

72
Q

What is dCas9?

A

Catalytically dead Cas9

73
Q

What is turning gene expression DOWN with CRISPR tools known as?

A

CRISPRi

CRISPR interference/inhibition

74
Q

What is turning gene expression UP with CRISPR tools known as?

A

CRISPRa

CRISPR activation

75
Q

What happens when an organism with a CRISPR gene drive mates with another member of its species?

A

The CRISPR components of the gene drive ensures that all of the offspring carry the gene drive on both of their chromosomes

76
Q

What are some examples of RNA therapeutics?

A
  • Antisense Oligonucleotides (ASO)
  • Small Interfering RNA (siRNA)
  • Aptamers
  • mRNA Therapeutics
77
Q

Where do Antisense Oligonucleotides bind?

A

Bind to specific sequences in target RNAs via hydrogen-bonding

78
Q

In 1977, Paterson and colleagues were the first to publish research showing what?

A

Gene expression can be modified with exogenous nucleic acids

79
Q

How did Zamecnik and Stephenson improved the activity of the oligonucleotide?

A

By introducing chemical modifications at the 3’ and 5’ ends, which reduced its degradation by cellular nucleases

80
Q

What did Donis-Keller describe in 1979?

A

RNase H cleaves the RNA strand in RNA-DNA heteroduplexes

81
Q

What is the most common modification in Antisense Olginucleotides (ASO)?

A

Phosphorothioate (PS)

82
Q

What are the TWO mechanisms of gene expression modulation for Antisense Oligonucleotides (ASO)?

A
  1. Altering pre-mRNA splicing
  2. Effects on mRNA translation
83
Q

What is Viltolarsen?

A

A phosphorodiamidate morpholino antisense oligonucleotide for the treatment of Duchenne muscular dystrophy

84
Q

What is a Dicer?

A

Cleaves long dsRNA into short double-stranded fragments of approx. 21 to 23 nucleotide siRNAs

85
Q

What is Argonaute (Ago)?

A

A nuclease that binds siRNA and cuts the target RNA substrate

86
Q

What is RISC?

A

RNA Induced Silencing Complex

87
Q

Who won 2006 Nobel Prize for their discovery of RNA interference?

A

Fire & Mello

88
Q

What are the applications and advantages of mRNA therapeutics?

A
  • Protein replacement therapies
  • Ideal for vaccines
  • mRNA-based monoclonal antibodies
  • CRISPR gene editing without genome integration
89
Q

What are the challenges of mRNA therapeutics?

A
  • Delivery
  • Stability
  • Short duration of protein expression
  • Immunogenicity
90
Q

How can Cardiac Ischemia be treated with protein replacement therapy?

A

Using Vascular Endothelial Growth Factor (VEGF)

91
Q

What enzyme does self-amplifying mRNA express?

A

RNA-dependent RNA polymerase (RdRP)

92
Q

How do Aptamers bind to their target?

A

By virtue of the tertiary structure of the aptamer, rather than its sequence

93
Q

What therapeutic are Aptamers potential to replace?

A

Monoclonal antibodies

94
Q

What therapeutic are Aptamers potential to replace?

A

Monoclonal antibodies

95
Q

What are the clinical challenges of using Aptamers as a therapeutic?

A
  • Highly sensitive to nucleases
  • Readily excreted by the kidneys
96
Q

What is the function of Pegaptanib?

A

It inhibits the binding of VEGF to its receptors

97
Q

The gene for what receptor is inserted into T cells in CAR-T cell therapy?

A

Chimeric Antigen Receptor
(CAR)

BT2001: Biotechnology (10 decks)

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