L7 - The Genome Revolution Flashcards
What is the purpose of DNA, mRNA and protein?
DNA: stores info
mRNA : converts info into template for protein synthesis
protein: carries out funciton
4 features of DNA
double helix (right handed), hereditary, can be passed horizontally, stores all genetic info
What makes the sugar phosphate backbone?
phosphodiester bonds
Hydrogen bonds are used to connect interacting bases, what are the diff energy levels?
CG pairs require more energy to break than AT. 1st hydrogen (CG) binds to sugar phosphate backbone, 9th (AT binds to sugar phosphate backbone
what is the difference betweeen primidines and purines? (which ones are which)
pyrimidines are single ringed (T and C) whereas purines are double ringed (A and G)
What does DNA polymerase do?
transcription, replication, regulation
More CG bonds = more or less able to dissociate
less bc stronger (more hydrogen bonds)
What is the significance of RNA evolution into DNA
RNA is not robust bc they are unstable and short-lived
What does anti parallel mean?
5’ end of one strand is paired with the 3’ end of its complementary strand
Which way is DNA synthesized
always 5’ to 3’
How are new phosphodiester bonds created and where does it get its energy
energy released from cleavage of 2 phosphate from dNTP allows creation of phosphodiester bond. - when DNTP is added to elongate DNA molecule, 2 phosphates form incoming molecule is cleaved the hydrogen is lost from sugar phosphate backbone
- the release of phoshate provides energy for new phosphodiester bond
What is dNTP
deoxyribonucleoside triphosphate
Helical turns (20 base pairs) contains a major and minor groove. where does the protein usually bind?
major groove
How is supercooling achieved? does it usually coil +ve or -ve. how is it stabilized?
topoisomerase, mostly negative, stabilized by proteins
What is the definition genome?
the total catalogue of genetic material in a cell made of DNA
What is a chromosome vs plasmid
main genetic element, circular in bacteria, supercoiled to fit in cell. plasmid = extrachromosomal
what can genomes tell us about?
- genomic content (higher CG content may suggest they they can live in higher temp)
- genome architecture (position of origin, closer to ori = transcribed more, ter, rRNA etc)
- presence of lysine in phases plasmids transposable elements, antibiotic resistance genes
- horizontal gene transfer, gene formation and evolutionary changes, specialisation (e.g. can they sporulate?)
- virulence factors (toxins, immune modulators, fimbriae, adhesions etc) -> host pathogen interactions
- metabolic potential (ability to utilize specific substrates)
- environmental potential (e.g. having the ability to survive in extreme environments - biotech potential (bioremediation, biofuel production)
Genomes tell us about life complexity: how does the smallest and largest genome differ?
- smallest gemome = only found in leaf hoppers (offers protection from gut so its lost its niche)
- adaptable to lots of diff envionments not as specialised
genomes indicate potential but they do not…
demonstrate function
what does lacking RpoS (sigma factor required for stress-response) imply?
you think it cant survive stress but it can due to alternative mechanisms
What can persister cells do?
identical cells responding differently to stress. not all cells die under antibiotics, they have same genome but just responds differently
What is a species?
a monophyletic and genomically coherent cluster of individual organisms that show a high degree of overall similarity in many independent characteristics, and is diagnosable by a discriminative phenotypic property.
what does each part of the definition mean?
- monophyletic
- genomically coherent
- overall similarity in many independent characteristics
- diagnosable by a discriminative phenotypic property?
- recent common ancestor
- similar genome sequences
- shared morphology and physiology
- biochemical tests, conserved gene sequence
How are species defined on a sequence-based definition?
- 16S rRNA sequencing
- average nucleotide identity (ANI) > 95 = related species
- DNA-DNA hybridization 70% (membrane hybridize when mixed)
within species:
CORE = genes present in all individuals
ACCESSORY = dispensable genes (strain specific)
PAN = core + accessory
What is maxam-gilbert sequencing?
- add diff chemicals and treat with heat, they will break at diff points according to the diff chemicals
- cleavage of specific bases - breakage of DNA by piperidine
- random fragments are created and gel is ran to see the combinations of fragments to get full sequence
What is sanger sequencing?
- uses E. coli DNA polymerase to synthesize DNA in vitro
- synthesis stops by incorporation of ddNTPs (dideoxynucleotides)
- PCR w fluorescent chain-terminating ddNTPs
- size separation by capillary gel electrophoresis
- laser excitation and detection by sequencing machine
PCR, when ddNTP comes in theres no O so synthesis stops
What is Ion torrent sequencing?
release proton every time nucleotide incorporated (change in pH, electrical signal)
What is nanopore sequencing
change in current every time a nucleotide passes through the pore
- in each flow cell there is a membrane which has +ve charge at the bottom
- DNA molecule that needs to be sequence gets separated
- one strand gets pulled through membrane by coltage
- as each base foes tbru membrane you can see a current generated due to diff structures of bases
What is next gen sequencing?
sequence short fragements and each bits gets adaptors, `pcr across each gragment, massively parallel sequences
DNA overlaps and joined as entire genome, find overlapping regions.
