3.3 -Genomics Flashcards
Why is Craig Venter so important?
- has a major name in DNA sequencing.
- used shotgun sequencing where random bits of DNA are sequenced.
Who is Fredrick Sanger?
- In the 1970s he developed the idea of DNA sequencing using sanger.
what is a sanger?
- Based on a DNA polymerase building a complementary strand using:
a. mostly normal dNTPs
b. rare special dNTPs that lack a 3’OH
this means they cannot be elongated further.
what is building DNA of diff lengths what are they terminated with?
- they are terminated with a labelled ddNTP.
what are the steps of sanger?
- synthesis of new DNA until the incorporation of a ddNTP
- Capillary electrophoresis of fragments and detection of fluorescent labels. (A pairs with T, so if T is yellow ten you know that A blue attaches with it)
- automated sequence reads output
what is IIlumina sequencing popular?
- represents different variations
- chips generate millions of clusters each represents a different DNA molecule being sequenced.
- we add ferminated residues ( each residue is labelled by a colour)
- reversibly terminated ( use chemistry to get rid of fluorescence and add another residue. )
- this helps identify the sequence by knowing the bases.
Reversibly temrinated dNTPs used:
- Insert one labelled residue, take an image
(A, C, G, T each a different colour of
fluorophore) - Unblock the 3’ end so you can add
another residue. Repeat. - Each round, you get an image of what
residue is at each cluster for that position
(1st nt, 2nd, 3rd…)
what is annotation?
- takes strong of letters –> managable form or action
- it is largely done by by computers but is not perfect or reliable.
- it identifies genes based on homology to known genes – many “unknown
function” or “putative XXXX” ( takes bases and finds where genes are and what they are doing) - Annotation and anaylsis lag behind sequencing (ton of sequencing and are never look at)
what is the first thing genome sequencing can tell us?
- Metabolic capabilities of an organism (what types of genes, or yes oxygen or no oxygen)
-Virulence genes, antibiotic resistance genes, etc (known antibiotic resistance, or why causing more severe disease)
- Unusual mutations that account for unusual phenotypes( what metabolic genes caused this mutation)
-Discover new genes that might be of medical/industrial interest (need genomes to study organisms)
what is the second thing genome sequencing can tell us?
-Genetics approaches (e.g. making mutations to genes)
- Transcriptomics, qPCR, etc – studies of RNA expression ( what the organism is doing)
- Proteomics – studies of proteins
- Genome-wide mutagenesis studies (looking at the effects of many
different mutations in parallel)
what can genome sequencing tell us?
- Functional & metabolic predictions for a Vampirovibrio
chlorellavorus strain based on its genome sequence. - genome you can identify what it needs to fxn and live.
- bacterial predators. –> eat others cell
- eukaryotic alge predator –> eats up the inside of eukaryote algae.
- has a chemotaxis system and flagella.
what are metagenomics?
- study mixed samples or large samples and sequence DNA of environmental sample. (ex. soil).
what do massive sequencing of DNA purified from environment provide?
- genomic information (sometimes even complete genomes) for
organisms that cannot be cultured in the lab
-tells us about how a genome is made. and identify what population will look in a sample.
- can also look at a gene level.
what is a gene level?
target looking fro non-antibiotic resistance genes.
What is Transcriptomics : RNA seq?
- RNA can be to DNA using a process called reverse
transcription, which can then be sequenced via next-generation methods - Can get a complete picture of the relative abundance of all of the
transcripts in the cell under a given set of conditions (RNA level change and tell you what goes on in the cell) - One way this is used is by comparing expressions under different conditions.
- RNA sequcne used to comapre what genes are beign expressed.
- looing at infecting patterns. (what genes go up and down or stay the same)
what is reverse transcription?
- what types of RNA and protein are in cell.
- synthesis from DNA to RNA
what is Proteomics?
- it looks are protein instead of RNA
- Often relies on knowing the genomic DNA sequence, but doesn’t use
DNA sequencing. - Instead, uses mass spectrometry to identify proteins/protein levels
- Like RNA-seq, can tell you what proteins are present under which
conditions. Can also be used in many other creative ways. E.g. which
proteins interact with a protein of interest (look for interaction of proteins, and you dont always get a uniform distribution like RNA)
what is mass spectroscopy?
- take protein and you chop it up into small bits, and separated protein chunks, this figures our peptides based on peaks.
steps of proteomics?
- protein extracted
- denaturation digestion, peptide separation
- mass spectroscopy analysis. (peaks tell you what proteins are present in sample)
- data analysis (identification, quantification and modifications)
