1
Q
What is Genomics?
A
The study of all of an organisms genes (the genome)
2
Q
What is Epigenomics?
A
The study of the reversible chemical modifications to DNA
3
Q
What is Transcriptomics?
A
The study of the complete set of RNA transcripts from DNA
4
Q
What are the TWO dominant classes of measurement technologies for the transciptome?
A
Microarrays & RNA Sequencing
5
Q
What is Proteomics?
A
The study of a set of proteins produced in an organism
6
Q
What is Metabolomics?
A
The study of small molecules (metabolites) within cells
7
Q
What are the major techniques used in Metabolomics?
A
NMR & Mass Spectroscopy
8
Q
What is Translatomics?
A
The study of all mRNAs being actively translated in a cell
9
Q
Who solved the 3D structure of DNA?
A
Watson & Crick
(1953)
10
Q
Who won The Nobel Prize in Chemistry for their work on the structure of Insulin?
A
Frederick Sanger
(1958)
11
Q
What enzymes are able to cut RNA chains at specific sites?
A
RNase enzymes
12
Q
What was the limitation in the early efforts of nucleic acid sequencing?
A
Only able to measure nucleotide composition, and not order
13
Q
What was the first whole nucleic acid sequence produced by Robert Holley in 1956?
A
Alanine tRNA from Saccharomyces cerevisiae
14
Q
What was Walter Fiers’ laboratory able to produce in 1972?
A
First complete protein-coding gene
15
Q
What technique was developed in 1977 that altered the progress of DNA sequencing technology?
A
Sanger’s ‘Chain-Termination’
16
Q
What are the TWO techniques used in First-Generation DNA Sequencing?
A
- Sanger sequencing
- Maxam-Gilbert sequencing
17
Q
Who won the 1980 Nobel Prize for their work into nucleic acids?
A
Berg, Gilbert, & Sanger
18
Q
What was radiolabelling in Sanger sequencing replaced with?
A
Fluorometric based detection
19
Q
How was detection in Sanger sequencing improved?
A
Through capillary based electrophoresis
20
Q
What did the improvements in Sanger sequencing lead to?
A
Automated DNA sequencing machines
21
Q
What is ‘Shotgun Sequencing’?
A
Where overlapping DNA fragments are cloned and sequenced separately, and then assembled into one long contiguous sequence
22
Q
What automated DNA sequencing technique did Kary Mullis develop?
A
Polymerase Chain Reaction (PCR)
1983
23
Q
What are TWO improvments in automated DNA sequencing?
A
- Identification of new polymerases
- Increasingly modified dNTPs
24
Q
What project used the newer dideoxy sequencers?
A
Human Genome Project
(1990-2003)
25
What techique was developed by Pal Nyren in 1993?
Pyrosequencing
26
How does Pyrosequencing work?
Relies on light detection based on a chain reaction when **Pyrophosphate** is released
27
What THREE enzymes are used in Pyrosequencing?
1. DNA polymerase
2. ATP sulfurylase
3. Firefly luciferase
28
What additional enzyme was introduced into Pyrosequencing to remove nucleotides that are not incorporated by the DNA polymerase?
Apyrase
29
What is a major difficulty with Pyrosequencing?
Finding out **how many of the same nucleotide** there are in a row at a given position
30
What biotechnology company was Pyrosequencing licensed to?
454 Life Sciences
31
What did the sequencing machines produced by 454 allow?
The mass parallelisation of sequencing reactions
32
What process is used in Illumina sequencing?
Bridge amplification
33
How is sequencing achieved in Illumina sequencing?
Using fluorescent 'reversible-teminator' dNTPs
34
What are TWO Third-Generation DNA sequencing techniques?
1. PacBio sequencing
2. Nanopore sequencing
35
What is PacBio sequencing?
Widely used third-generation technology providing **a single molecule real time (SMRT) platform**
36
Where does DNA polymerisation occur in PacBio sequencing?
In arrays of microfabricated **nanostructures called zero-mode waveguides (ZMWs)** which are essentially **tiny holes in a metallic film covering a chip**
37
What was α-Hemolysin, a membrane channel protein from *Staphylococcus aureus* the first nanopore to show?
Detect recognisable ionic current blockades by both RNA and DNA homopolymers
38
What does Nanopore sequencing allow?
Enables direct, real-time analysis of long DNA or RNA fragments
39
How does Nanapore sequencing work?
Monitoring changes to an electrical current as **nucleic acids are passed through a protein nanopore**
40
What is RNA sequencing used for?
The analysis of **differential gene expression (DGE)**
41
What are the core steps of RNA sequencing?
1. RNA extraction
2. cDNA synthesis
3. Adapter ligation
4. PCR amplification
5. Sequencing and Analysis
42
What are some imperfections and biases associated with RNA sequencing sample preparation and computational analysis?
* Correctly identifying and quantifying **which of multiple isoforms are expressed from a gene**
* Differences in how **ambiguous or multi-mapped reads are handled**
43
What percentage of transcripts in the human transcriptome are over 2,500 bp long?
50%
44
What are the THREE main sequencing technologies for RNA sequencing?
1. The Illumina workflow
2. The Pacific Biosciences workflow
3. The Oxford Nanopore workflow
45
What is Cap analysis of gene expression (CAGE-seq)?
An approach to identify and monitor the activity transcription start sites (TSSs) at single base-pair resolution across the genome
46
What is Cap Trapping?
A technique based on the biotinylation of the 7-methylguanosine cap of Pol II transcripts
47
What are the PROS of Cap Trapping?
* Measures RNA expression levels and maps TSS in promoter regions
* Provides precise mapping of TSS with single-nucleotide resolution
48
What are the CONS of Cap Trapping?
* Only works on total mature DNA
* Detection is biased toward TSS of long-lived transcripts
49
What are some example of genome-editing tools?
* Zinc-finger nucleases (ZFNs)
* Transcription activator-like effector nucleases (TALENs)
50
What was discovered by Ishino in 1987?
An unusual pattern of **nucleotide repeats and spacers** 3' of the iap gene in *E. coli*
51
What is CRISPR-Cas?
A system in an **adaptive immune mechanism** which defends against specific types of phages
52
What does CRISPR stand for?
**C**lustered **R**egularly **I**nterspaced **S**hort **P**alindromic **R**epeats
53
What structure do most CRISPR fold into?
Stem-Loop or Hairpin once they are transcribed into RNA
54
Does Cas-9 have palindromic repeats?
No.
Cas-9s crRNA base-pairs with a second piece of RNA called the **tracRNA**
55
What are Spacers in CRISPR?
Small segments of **phage DNA** and get **passed down through generations of bacteria**
56
What are the THREE basic steps of the CRISPR-Cas defence system?
1. Spacer aquisition
2. CRISPR RNA biogenesis
3. Interference
57
How do CRISPR systems avoid harmful destruction of the bacterium's own genome?
By relying on short DNA sequences called **Protospacer Adjacent Motifs or PAMs**
58
Where are PAMs found?
Next to CRISPR targets in phage DNA but **never in bacterial CRISPR arrays**
59
Which PAM does the Cas-9 protein from *Streptococcus pyrogenes* recognise?
NGG
60
What are some Cas proteins?
* Helicases
* Nucleases
* Polymerases
61
What are Adaptive modules?
Cas proteins participating in the acquisition of immunity
**e.g. Cas1 and Cas2**
62
What are Effector modules?
Cas proteins involved in the destruction of mobile genetic elements through their recognition and cleavage
**e.g. Cas9**
63
Who won The Nobel Prize in Chemistry in 2020 for their work on CRISPR gene editing?
Emmanuelle Charpentier & Jennifer Doudna
64
What is the role of CRISPR RNA (crRNA)?
Dictates what site Cas9 will cut
65
What is the role of a Trans-Activating CRISPR RNA (tracrRNA)?
Attaches to the crRNA and acts as a handle for Cas9
66
What molecule is formed when crRNA and tracrRNA are combined?
Single-Guide RNA (sgRNA)
67
What are the steps of DNA target binding and cleavage by Cas9?
1. PAM recognition
2. Base-pairing
3. Complete binding
4. DNA target cleavage
68
What is the process called for stitching two broken ends of DNA back together?
Non-Homologous Ending Joining
(NHEJ)
69
What happens in a pooled CRISPR screen?
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
What happens in Positive Selection in CRISPR screens?
Cells with selected trait **survive**
71
What happens in Negative Selection in CRISPR screens?
Cells with selected trait **die**
72
What is dCas9?
Catalytically dead Cas9
73
What is turning gene expression DOWN with CRISPR tools known as?
CRISPRi
CRISPR interference/inhibition
74
What is turning gene expression UP with CRISPR tools known as?
CRISPRa
CRISPR activation
75
What happens when an organism with a CRISPR gene drive mates with another member of its species?
The CRISPR components of the gene drive ensures that **all of the offspring carry the gene drive on both of their chromosomes**
76
What are some examples of RNA therapeutics?
* Antisense Oligonucleotides (ASO)
* Small Interfering RNA (siRNA)
* Aptamers
* mRNA Therapeutics
77
Where do Antisense Oligonucleotides bind?
Bind to specific sequences in target RNAs via hydrogen-bonding
78
In 1977, Paterson and colleagues were the first to publish research showing what?
Gene expression can be modified with exogenous nucleic acids
79
How did Zamecnik and Stephenson improved the activity of the oligonucleotide?
By introducing chemical modifications at the 3' and 5' ends, which reduced its degradation by cellular nucleases
80
What did Donis-Keller describe in 1979?
RNase H cleaves the RNA strand in RNA-DNA heteroduplexes
81
What is the most common modification in Antisense Olginucleotides (ASO)?
Phosphorothioate (PS)
82
What are the TWO mechanisms of gene expression modulation for Antisense Oligonucleotides (ASO)?
1. Altering pre-mRNA splicing
2. Effects on mRNA translation
83
What is Viltolarsen?
A **phosphorodiamidate morpholino antisense oligonucleotide** for the treatment of Duchenne muscular dystrophy
84
What is a Dicer?
**Cleaves long dsRNA into short double-stranded fragments** of approx. 21 to 23 nucleotide siRNAs
85
What is Argonaute (Ago)?
A nuclease that binds siRNA and cuts the target RNA substrate
86
What is RISC?
RNA Induced Silencing Complex
87
Who won 2006 Nobel Prize for their discovery of RNA interference?
Fire & Mello
88
What are the applications and advantages of mRNA therapeutics?
* Protein replacement therapies
* Ideal for vaccines
* mRNA-based monoclonal antibodies
* CRISPR gene editing without genome integration
89
What are the challenges of mRNA therapeutics?
* Delivery
* Stability
* Short duration of protein expression
* Immunogenicity
90
How can Cardiac Ischemia be treated with protein replacement therapy?
Using Vascular Endothelial Growth Factor (VEGF)
91
What enzyme does self-amplifying mRNA express?
RNA-dependent RNA polymerase (RdRP)
92
How do Aptamers bind to their target?
By virtue of the **tertiary structure of the aptamer, rather than its sequence**
93
What therapeutic are Aptamers potential to replace?
Monoclonal antibodies
94
What therapeutic are Aptamers potential to replace?
Monoclonal antibodies
95
What are the clinical challenges of using Aptamers as a therapeutic?
* Highly sensitive to nucleases
* Readily excreted by the kidneys
96
What is the function of Pegaptanib?
It inhibits the binding of VEGF to its receptors
97
The gene for what receptor is inserted into T cells in CAR-T cell therapy?
Chimeric Antigen Receptor
(CAR)
BT2001: Biotechnology
flashcards
Decks in class (10)
# Cards
-
Traditional & Modern Biotechnology26
-
Recombinant DNA Technology32
-
Animal Biotechnology28
-
Biodegradation/Bioremediation24
-
DNA Fingerprinting & Forensic Analysis23
-
Microbial Biotechnology57
-
Vaccinations from Smallpox to SARS-CoV-235
-
Ethics & Biotechnology4
-
Plant Biotechnology34
-
Omics Technologies97
