Omics Technologies Flashcards
What is Genomics?
The study of all of an organisms genes (the genome)
What is Epigenomics?
The study of the reversible chemical modifications to DNA
What is Transcriptomics?
The study of the complete set of RNA transcripts from DNA
What are the TWO dominant classes of measurement technologies for the transciptome?
Microarrays & RNA Sequencing
What is Proteomics?
The study of a set of proteins produced in an organism
What is Metabolomics?
The study of small molecules (metabolites) within cells
What are the major techniques used in Metabolomics?
NMR & Mass Spectroscopy
What is Translatomics?
The study of all mRNAs being actively translated in a cell
Who solved the 3D structure of DNA?
Watson & Crick
(1953)
Who won The Nobel Prize in Chemistry for their work on the structure of Insulin?
Frederick Sanger
(1958)
What enzymes are able to cut RNA chains at specific sites?
RNase enzymes
What was the limitation in the early efforts of nucleic acid sequencing?
Only able to measure nucleotide composition, and not order
What was the first whole nucleic acid sequence produced by Robert Holley in 1956?
Alanine tRNA from Saccharomyces cerevisiae
What was Walter Fiers’ laboratory able to produce in 1972?
First complete protein-coding gene
What technique was developed in 1977 that altered the progress of DNA sequencing technology?
Sanger’s ‘Chain-Termination’
What are the TWO techniques used in First-Generation DNA Sequencing?
- Sanger sequencing
- Maxam-Gilbert sequencing
Who won the 1980 Nobel Prize for their work into nucleic acids?
Berg, Gilbert, & Sanger
What was radiolabelling in Sanger sequencing replaced with?
Fluorometric based detection
How was detection in Sanger sequencing improved?
Through capillary based electrophoresis
What did the improvements in Sanger sequencing lead to?
Automated DNA sequencing machines
What is ‘Shotgun Sequencing’?
Where overlapping DNA fragments are cloned and sequenced separately, and then assembled into one long contiguous sequence
What automated DNA sequencing technique did Kary Mullis develop?
Polymerase Chain Reaction (PCR)
1983
What are TWO improvments in automated DNA sequencing?
- Identification of new polymerases
- Increasingly modified dNTPs
What project used the newer dideoxy sequencers?
Human Genome Project
(1990-2003)
What techique was developed by Pal Nyren in 1993?
Pyrosequencing
How does Pyrosequencing work?
Relies on light detection based on a chain reaction when Pyrophosphate is released
What THREE enzymes are used in Pyrosequencing?
- DNA polymerase
- ATP sulfurylase
- Firefly luciferase
What additional enzyme was introduced into Pyrosequencing to remove nucleotides that are not incorporated by the DNA polymerase?
Apyrase
What is a major difficulty with Pyrosequencing?
Finding out how many of the same nucleotide there are in a row at a given position
What biotechnology company was Pyrosequencing licensed to?
454 Life Sciences
What did the sequencing machines produced by 454 allow?
The mass parallelisation of sequencing reactions
What process is used in Illumina sequencing?
Bridge amplification
How is sequencing achieved in Illumina sequencing?
Using fluorescent ‘reversible-teminator’ dNTPs
What are TWO Third-Generation DNA sequencing techniques?
- PacBio sequencing
- Nanopore sequencing
What is PacBio sequencing?
Widely used third-generation technology providing a single molecule real time (SMRT) platform
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
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
What does Nanopore sequencing allow?
Enables direct, real-time analysis of long DNA or RNA fragments
How does Nanapore sequencing work?
Monitoring changes to an electrical current as nucleic acids are passed through a protein nanopore
What is RNA sequencing used for?
The analysis of differential gene expression (DGE)
What are the core steps of RNA sequencing?
- RNA extraction
- cDNA synthesis
- Adapter ligation
- PCR amplification
- Sequencing and Analysis
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
What percentage of transcripts in the human transcriptome are over 2,500 bp long?
50%
What are the THREE main sequencing technologies for RNA sequencing?
- The Illumina workflow
- The Pacific Biosciences workflow
- The Oxford Nanopore workflow
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
What is Cap Trapping?
A technique based on the biotinylation of the 7-methylguanosine cap of Pol II transcripts
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
What are the CONS of Cap Trapping?
- Only works on total mature DNA
- Detection is biased toward TSS of long-lived transcripts
What are some example of genome-editing tools?
- Zinc-finger nucleases (ZFNs)
- Transcription activator-like effector nucleases (TALENs)
What was discovered by Ishino in 1987?
An unusual pattern of nucleotide repeats and spacers 3’ of the iap gene in E. coli
What is CRISPR-Cas?
A system in an adaptive immune mechanism which defends against specific types of phages
What does CRISPR stand for?
Clustered Regularly Interspaced Short Palindromic Repeats
What structure do most CRISPR fold into?
Stem-Loop or Hairpin once they are transcribed into RNA
Does Cas-9 have palindromic repeats?
No.
Cas-9s crRNA base-pairs with a second piece of RNA called the tracRNA
What are Spacers in CRISPR?
Small segments of phage DNA and get passed down through generations of bacteria
What are the THREE basic steps of the CRISPR-Cas defence system?
- Spacer aquisition
- CRISPR RNA biogenesis
- Interference
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
Where are PAMs found?
Next to CRISPR targets in phage DNA but never in bacterial CRISPR arrays
Which PAM does the Cas-9 protein from Streptococcus pyrogenes recognise?
NGG
What are some Cas proteins?
- Helicases
- Nucleases
- Polymerases
What are Adaptive modules?
Cas proteins participating in the acquisition of immunity
e.g. Cas1 and Cas2
What are Effector modules?
Cas proteins involved in the destruction of mobile genetic elements through their recognition and cleavage
e.g. Cas9
Who won The Nobel Prize in Chemistry in 2020 for their work on CRISPR gene editing?
Emmanuelle Charpentier & Jennifer Doudna
What is the role of CRISPR RNA (crRNA)?
Dictates what site Cas9 will cut
What is the role of a Trans-Activating CRISPR RNA (tracrRNA)?
Attaches to the crRNA and acts as a handle for Cas9
What molecule is formed when crRNA and tracrRNA are combined?
Single-Guide RNA (sgRNA)
What are the steps of DNA target binding and cleavage by Cas9?
- PAM recognition
- Base-pairing
- Complete binding
- DNA target cleavage
What is the process called for stitching two broken ends of DNA back together?
Non-Homologous Ending Joining
(NHEJ)
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.
What happens in Positive Selection in CRISPR screens?
Cells with selected trait survive
What happens in Negative Selection in CRISPR screens?
Cells with selected trait die
What is dCas9?
Catalytically dead Cas9
What is turning gene expression DOWN with CRISPR tools known as?
CRISPRi
CRISPR interference/inhibition
What is turning gene expression UP with CRISPR tools known as?
CRISPRa
CRISPR activation
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
What are some examples of RNA therapeutics?
- Antisense Oligonucleotides (ASO)
- Small Interfering RNA (siRNA)
- Aptamers
- mRNA Therapeutics
Where do Antisense Oligonucleotides bind?
Bind to specific sequences in target RNAs via hydrogen-bonding
In 1977, Paterson and colleagues were the first to publish research showing what?
Gene expression can be modified with exogenous nucleic acids
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
What did Donis-Keller describe in 1979?
RNase H cleaves the RNA strand in RNA-DNA heteroduplexes
What is the most common modification in Antisense Olginucleotides (ASO)?
Phosphorothioate (PS)
What are the TWO mechanisms of gene expression modulation for Antisense Oligonucleotides (ASO)?
- Altering pre-mRNA splicing
- Effects on mRNA translation
What is Viltolarsen?
A phosphorodiamidate morpholino antisense oligonucleotide for the treatment of Duchenne muscular dystrophy
What is a Dicer?
Cleaves long dsRNA into short double-stranded fragments of approx. 21 to 23 nucleotide siRNAs
What is Argonaute (Ago)?
A nuclease that binds siRNA and cuts the target RNA substrate
What is RISC?
RNA Induced Silencing Complex
Who won 2006 Nobel Prize for their discovery of RNA interference?
Fire & Mello
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
What are the challenges of mRNA therapeutics?
- Delivery
- Stability
- Short duration of protein expression
- Immunogenicity
How can Cardiac Ischemia be treated with protein replacement therapy?
Using Vascular Endothelial Growth Factor (VEGF)
What enzyme does self-amplifying mRNA express?
RNA-dependent RNA polymerase (RdRP)
How do Aptamers bind to their target?
By virtue of the tertiary structure of the aptamer, rather than its sequence
What therapeutic are Aptamers potential to replace?
Monoclonal antibodies
What therapeutic are Aptamers potential to replace?
Monoclonal antibodies
What are the clinical challenges of using Aptamers as a therapeutic?
- Highly sensitive to nucleases
- Readily excreted by the kidneys
What is the function of Pegaptanib?
It inhibits the binding of VEGF to its receptors
The gene for what receptor is inserted into T cells in CAR-T cell therapy?
Chimeric Antigen Receptor
(CAR)