molecular biology (exam prep) Flashcards

Question

What occurs in a reverse transcriptase reaction?

Answer 1

Reverse transcription involves the synthesis of DNA from RNA by using an RNA-dependent DNA polymerase. The DNA strand is not identical to the og

Answer 2

Linear chromosomes => telomeres *short DNA repeats (e.g. TTAGGG) *G-rich strand + C-rich strand *every round of replication loses up to 200bp Streptomyces

Answer 3

-"jumping genes" -lac operon -pcr

Answer 4

the discovery of transposable elements in maize “the jumping genes” . known as the AC/DS system, chromosome breakage occurs at the dissociation and is regulated by the activator which can also provide its own tranposition. thus replication is not always linear and the disruption caused by them on chromosome 9

Answer 5

it is a group of genes that with a single promotor that encodes genes for the transport and metabolism of lactose in bacteria (e.coli)

Answer 6

sugar production: allosterically repressed. transcription only occurs when there is lactose ready to be digested a repressor blocks the production of the the operon contains 3 types of lactose enzymes (lac C, lac Y, Lac Z) which but if IPTG promotor is added it will bind to the repressor and allow the production to occur cis elements that bind transregulators binding sites for proteins: promotor operator cbs-cap binding site a major trans regulator encoded by the operon is LACI this is the repressor that binds to stops the polyerase binding to the repressor

Answer 7

when cleaved by the β-galactosidase enzyme it produces blue product. thus denoting if a gene is expressed or not other reporter genes firefly luciferase gene in plant cells and transgenic plants

Answer 8

– 3.6 amino acyl residues per turn; 2.3 Å helix radius. *Most common helix in proteins. * Usually about 10 aa residues, but can be 4-40+. * Usually contains M, A, L, E, K aas but P and G disrupt a helix. NAD+ Acidithiobacillus thiooxidans

Answer 9

3.0 amino acyl residues per turn; 1.9 Å helix radius * . * Very strained structure. * Found in e.g. myoglobin and hemoglobin. * Usually very short - <4 aa residues. blue whale myoglobin

Answer 10

4.4 amino acyl residues per turn; 4.4 Å helix radius. * Energetically unfavourable – selected against unless functionally critical, so found near active-sites. * Usually seen as a bulge on a long alpha helix. * Usually short – 7-10 aa residues, usually. methane oxidising bacteria soluble methane monoxygenase (13)

Answer 11

right handed (one arrow) Thermal hysteresis protein YL-1 aka antifreeze protein) from mealworm inhibit the formation of large ice grains inside the cells that may damage cellular organelles or cause cell death freeze tolerance and ice adhesion first discovered 50 years ago antartic fish present in millimolar concentrations their fucntion was to stop ice crystal growth

Answer 12

Ice-binding protein (IBP) from a type of Flavobacterium very potent for a microorganism produces FH in the range of 3°C submillimolar conditions

Answer 13

Antifreeze protein isoform 501 from spruce budworm. this anti freeze protein protects organisms from freezing by adhering to ice crystals thus preventing their growth. they absorb to ice adhere to its morphology and prevent further growth. theories suggest the effectiveness of this protein is due to how well it can stop freezing at the basal plane. A study was conducted in 2008

Answer 14

hydrophobicity plots such as kyte-doolittle plot allows us to examine a primary structure regions are. graph: above 0 hydrophillic below 0 hydrophobic example of this: aquaporin (lets what in and out of a cell). 7 alpha helices per sub unit in homosapiens.

Answer 15

multiple 4-stranded beta-meanders motifs arranged in the form of blades. all joined together. active site of many enzymes in the centre of propeller. example: methanol dehydrogenases (enzyme) 8 bladed propeller common in alcohol dehydrogenases. takes electrons off of the alcohol and donates them to cytochrome c. Thus couples directly to respiration. from paracococcus bacteria.

Answer 16

beta strands with a turn, anti parallel. example: sensory protein FhaA-receptor protein from e-coli needs to be turned on for hole to open. (outside, detergent molecules show this ) 2 types: -outside hydrophobic (will disolve in a membrane) -inside hydrophobic

Answer 17

stacked pairs of alpha helices form large flexible structures found in things that need protein-protein interactions. Massive bendy surface area. example: phosphatase 2a adds or takes phospate to or from proteins

Answer 18

formed by polypeptide interactions binding of co-factors ( non-protein chemical compound or metallic ion that is required for an enzyme's role as a catalyst)

Answer 19

example in soluble methane monooxygenase.

Answer 20

soluble (not part of proteins) redox active enzymes: PQQ (quinone proteins, hydrophobic) hemes flavins porphyrin rings chlorophylls cobalamins (b12)

Answer 21

Rna and sigma factor come together- high ifinity for DNA sequence. Locate the promotor (strong association) forms closed promotor. DNA strands start opening up, transcription starts for RNA. Signals tell polymerase to stop

Answer 22

promotors: they are recongisable by 2 main sequences one at -10 & -35 upstream from start of transcription +1 start of transcription (purine normally) -10 consesus often TATAAT -35 consensus often TTGACA distance most important Top strand is coding strand other is template strand therefore the outcome RNA will be the compliment of the top strand synthesis always happens 5'- 3

Answer 23

e- 3 types of RNA polymerase 1- rRNA transcribe 2-mRNA transcribe 3-tRNA transcribe more complex promotors taata box -30-40 more sequences where pole binds and has enhancers upstream and downstream b- 1 RNA polymerase

Answer 24

3 types of rna polymerase transcription factors involved enhancer sequence which activator proteins can bind. adaptor proteins- all activate and dna folds.

Answer 25

Polyadenytion 3' poly (A) tail- to stabilise the mRNA Splicing- removing on introns capping structure added to mRNA: addition of 7-methylguaosine (binds 5'-5' phosphate at start) added to mRNA to stop degradation (DONT DESTROY) polyo virus targets this Splicing spliceisome- multi protein complex, help bind RNA around the introns recognise consenus sequence. cuts out the introns at either end and binds the 2 exons (ligase binds 2 nucleotides)

Answer 26

the same gene can produce slightly or different proteins depending on the introns or extrons used. examples: Drosphilla gene- grey always present, r/g/b only retain in certain transcripts (38) can be

Answer 27

-no histones (bacteria) -dna is circular -divided into genomic DNA, replicon DNA (plasmids, megaplasmids) -introns and exons (rare) inteins (do a similar job to introns) -HGT -prophage

Answer 28

*using random hexamers, random bit of dna *next gen sequencing, and amplify using pcr using random hexamers contigs: made up of reads *scaffolds: made up of contigs use genome of realted organism and match up the missing sequencing from the reads

Answer 29

circular plot example: thiomicrohabdus heinhorstii from fresh water in a manitee cave this region is a prophage

Answer 30

0=origin of replication in bacteria (gene) (oriC 0.24-2.5kbp) Origin of rep in archaea (oriC1,2,3,4) spaced around the genome

Answer 31

*Opening of the strands to allow replication to begin* -one region binds single stranded dna the other double stranded DUE- dna unbinding site full of a's and t's seperates from each other creating a 'bubble' and 2 single strands briefly. - (2 replication forks) - DNAa (enzyme) intitator protein, binds to the box site (double stranded) and binds to DUE (creating helix turn helix motif) binds acroos those sites and a second part of the enzyme which is an ATPase domain binds acrose DUE this winds itself up and seperates the 2 DUE strands. - attaches to single strands, zooms along seperating the 2 strands, expensive of ATP. Have seperate so enzymes can get in and a copy to need to be seperated -space between DnaA box site and DUE is important -DnaA box site it binds to needs to have correct spacing oric is a gene but doesnt make anything just facilitates

Answer 32

-same kind of size -3-4 oriC around the genome -Orbs: origin of replication boxes all found in 1-4 oric locations what happens? -initiator protein (orc1) binds to one of the oriC. It will bind well to oric number 1 and particarly to all the other oric. -in some archaea also have WHiP proteins which do the same sort of job and can also act as initiators. -many molecules of orc1 binding to oriC one will join together forming an=oligimorise this then does the job of DNAa and does the unwinding example: saccharlobus solfataricus (wu et al) oriC2 orc complexes bind to 1 and partically to another. theory to multiple if you do it in 4 times then it speeds up time.

Answer 33

-usually have one chromosome but can be a second or a third. Whatever the biggest is the primary chromosome -all other replicons are replicated in their own way not like the chromosomes. *plasmids (origin of transfer) *megaplasmids etc replions defintion: -plasmid: smallest replicon present (G/C is greater of 1% by mol) cannot contain core genes, second disk on dvds. Species-species exclusive not found in other bacteria. -megaplasmids: probably just a second chromosome -chromids: usually second insize to the chromosome proper. (G/C content is nearer to chromosome) size bigger then half million base pairs. *manmade cloning vectors: fosmids (f plasmid from bacteria can only be used in e.coli), cosmids (phage operon, can hold big inserts) phasmids (made from the f1 phage) example pUC (plasmids always start with p) -

Answer 34

its DNA from bacteriophages (incorporated into host DNA, to store until better conditions) under the right conditions the prophage will be transalted and become a mass of phage ions that kill the cell. (calcium, or magensium going up in the environment that cell is in) detecting tools; PHASTER that can scan genome sequences 1-10 prophage is typical (often mis-annotated) activated sludge in sewage works will have multiple prophage. High amount in this area, more virus particals. viral DNA cervical cancer (Hpv)- viral dna intergrating into genomes occurs often. Thats why some viruses causes cancer sin humans as the bit of DNA they intergrate into happens to be where divison and growth occurs, switiching it off leading to uncontrolled cell growth.

Answer 35

Transfer of DNA from species to species or strain to strain (sideways) being given to appear. Usually done by several sources: *phage: when it intergrates in it will bring DNA with it. *plasmids *intergrons: virus like mobile genetic elements important in antibiotic resistance, hospital situations and active sludge. genes on plasmid not essential: *virulence factors: the molecules that assist the bacterium colonize the host at the cellular level (things that make better at being a pathogen are usually on plasmids) example: - natural occuring e.coli, has a plasmid with an antibiotic resistance. Human stops taking antibiotic early, and as there is some resistance already the resistance genes can now recolonise the whole gut. all these cells now have this virulence factor they will communiate with other cells and pass it to them.

Answer 36

*transformation: one of the 2 cells has to be component (chemical, enough calcium in the cell). A piece of genomic dna from bacterium a can be trasnfered to bacteria b F-positive (sex pillas) grips f negative to it can transfer the dna. Sex pillas is needed to transfer without it bacteria can only recieve.

Answer 37

everything read in triplets. the translation genetic code is degenerate- some codons codes for more then one AA (third base wobble) ORF- open reading frame- be careful of where rna starts from genetic code frames can be different in mitocondira etc

Answer 38

tRNA: cloverfield stucture rRNA (ribosomes) 2 sub units contained in ribosomes 70's bacteria major 50's minor 30's 80s-eukarya 60s (3 different sub units) certain degree of conservation in terms of sequence and instruction. Which infers phylogenetic relationships. mods to rRNA post transcription

Answer 39

*n and c terminal (h2n to c002) trna carries new amino acid and it attaches to new chain. ribsomes are complex strutures small sub unit large sub unit: e-site, p-site, t-site.

Answer 40

123 are the current polypeptide chain that is enlongating→bound to tRNA in p site→new charged tRNA comes into the A site and has a complimentary anti codon→bond between 3-4 begins and ribosome shifts so that the og goes toward the exit site but attached to the chain as another aa and then cycle repeating (sliding of small thne large sub unit) accesory molecules that aid this: tRNA is linked to elongation factors not just floating around by itself (different in bacteria and eukarya) GTP attached to trna and elongation factor linked to an 1st initator factor bound to GTP (energy) to break down bond and new formation→Hydrolysis of GTP to GDP (release of a phosphate→ breaking high energy bond) this energy changes confirmation of ribosome to form the new bond 2nd initaitor factor- hydrolysis of GTP to GDP creates energy that for change of confirmaton (sliding of 2 subunit) formation of new bond and exit of the last one.

Answer 41

acting on different processes, so to ihibit something you need to choose the right thing example: when cloning a gene, need to insert the gene into bacteria. Need to select bacteria depending on their antibiotic resistance. Streptomycin prevents the transition from initiation complex to chain-elongating ribosome and also causes miscoding (only acts on bacteria cells) to stop certain step pick correct inhibitor

Answer 42

reminder: Polycistronic mRNAs (multiple ORFs) make lots at some time to be used in say a pathway transcripton/transaltion are coupled in space and time doesnt occur in eukarya. Less options for regulation. the start if translations is near the shine delgarno reverse compliment of whats in the ribsomes (purine-rich initiation sequence). 6-8 nucleotides before the initiating AUG.

Answer 43

Monocistronic mRNAs: info for only one type of protein unless alt splicing transcription (nucleus) and translation(cytoplasm) are seperate in space and time Transcription can occur not in cytoplasm- for mitocondria small ribosomal sub unit interacts with the elongation factors, charged tRNA, this is helped by the 5' cap this complex is at beginning of mRNA (Small subunit only)→All starts scanning to find the initating AUG→ release of elongation factor→hydrolysis of ATP→ADP this energy recruits the large sub unit = complete machinery → chain elongation begins

Answer 44

*once polypepetide chain starts growing the aa start folding they have different qualities example: hydrophobic proteins will start folding to get away from the charged cytoplasm Secondary/tericary starts to form whilst emerging from the ribsomes. protein misfolding: whilst the protein is growing there check points to ensure the protein has folded correctly→ either fixed or hydrolised (waste of material as non functional, can be dangerous/pathogens for the cell) misfolded proteins need to be eliminated, protesome: cylinder that takes in protein, protein will hydrolise in the middle of this. how cell know this? addition of a flag to the protein (Ubiquitin) ubiquitin is recycled to be used again

Answer 45

transcription not always active regulation occurs at: capping of new messenger elognation splicing polyadenation (mod) export from nucleus to cytoplasm during protein synthesis and after transcription translation protein degradation

Answer 46

*number of copies *localisation of transcripts *timing of transcripts *chromatin structure and histone mods

Answer 47

dna is wrapped around histones, they are positively charged. can have multiple levels of organisation 4 core histones: H2A,H2B, H3,H4 together make an optomer of 8 proteins DNA is wrapped around this, h1 is a linker for DNA Mods: different mods have different meanings depending on where they are- histone code hypothesis. options: methyl, acetyl, phoshate, ubiquitin

Answer 48

can change during age of organism example- at 6 weeks for embryonic globin- cytoscene has a unmethylated promotor at 6 weeks and the a methylated promotor for globin later on to shut off the production and allow adult globin to produce.

Answer 49

tells polymerase where to start, recognises what genes are needed. Regulation can occur at this level because gene transcription can be regulated by promotor equence thus sigma factors => gene regulation (up-regulation or down-regulation) Another way of regluating gene expression example: e-coli time vs optimal density in bacteria cell different sigma factors are expressing different amounts because you need different genes growth phase- sigma 70 house keeping genes (growth) stationary: σ32: Heat-shock gene transcription σ38: Stationary phase gene expression σ54: Expression of genes for nitrogen metabolism depending on what σ are present different promotors will be activated and therefore different proteins transcibed

Answer 50

rna polymerase 1 rna polymerase 2 rna polymerase 3-trna, 5s ribosome and non protein coding

Answer 51

an example of transcript factor to d-differentiate the re-differentiate cells example of a cascade: myOD- expierment from skin of a chick embyro took fibreblasts undiffertiated teh cells and activated myod making the fibroblasts muscle cells.

Answer 52

regulation due to localisation: how can a zygote differentiate in the many different cells in an organsim?- how are they differetiated postioned in the cell? through the UTR's drosophilla melanogaster example: tells the protein to be at either end of the egg. how would you test this? (utr) tatata- this sequence makes it organised. Cut the cds and attach this section to the UTR where its obviously different then the incorrect part will be localised. (in vivo)

Answer 53

3' poly tail removing of introns 5' tail examples

Answer 54

examples: *depending on avalability of charged tRNA (attached to aa) genetic code is redundant so different codons coding for *elongation factors *post translational mods- once protein produced could be phos, acetyl, gly groups could be added. these mods will be listend on uni prot

Answer 55

where are proteins produced (cytoplasm- ribosomes) an example of protein localisation *some proteins are needed in certain places intracellualr proteins: targerted to organelles (nucleus, mitocondria etc) * these proteins that are produced on free ribosomes and translocated/modified after translation Extracellular proteins: destined to plasm membranes or for excretion. Will be produced on the ER- bound ribosomes. co-translational translocation occurs in the RER lumen where further packaged into lysosomes. extracellualr protein:destined to be plasma proteins: produced on the ER

Answer 56

a sequence of the protein will have a flag that says put it in a certain place. protein is transported within the matrix and the cut signal peptidase is removed after

Answer 57

type 1 birds disease (high level of oxalate in urine) causing kidney stones theres a mutation in the signal peptide, so the oxilate goes into the mitcondria not the proxisomes thus an active enzyme in the wrong compartment

Answer 58

kinase-adds phosphertase- removes the addition or removal can activate a protein. how does the cell know what regulates the cell cycle? cyclings- proteins which change the level at variouis levels. Activated by CDK. phosoprlation at a certain levels will activate the cyclings: -formation of complex -activiation through phosphorlaytion -inhibitor is another phosphate in another position -why does the cell do this? timing its faster to have an active but prohibited cell then creating one from scratch.

Answer 59

example of when: ubiquitylation seperation of 2 chromatids before cell division cell is ready to go but they are still linked together, the phosphorlatyin of other proteins then enables this seperation. enzyme is ready but waiting

Answer 60

*transcription (will it be transcribed or not how much if so) *mRNA- processing (splicing etc) *transport in messenger RNA *where will mrna be will it be active *translational regulation (makeup protein or not) *Will the protein be used, how long will it last, hydrolysis right away? *phosphorlaytion, ubiqylation etc

Answer 61

*attach a fluorescent protein *real-time qPCR *microarray *hybridation-based assays

Answer 62

eukaryotic bigger-size- eukayrotic cells- linear bactiera-circular bacteria-polysystronic bacteria- higher gene density bacteria- presence/absence of introns HGT/LGT- bactera ori- multiple in eukarya

Answer 63

the complexity of the organism does nto correlte with the genome size drosphilla bigger then mammals although their is a relationship between genome size and protein coding genes

Answer 64

some contain the same genes *sex chromosomes the same *conserved segments example human 17 and mouse 11

Answer 65

human and fish genome are the same, in the same sequence on the same strand exons are the same but some are used as coding sequence some are cds

Answer 66

lookng at related deer species the chromosomes look very different although they appear the same could look at genes in genome.com

Answer 67

one of the milestones- as soon as the sequences are avalible they will be released to the public. *research would cost a lot more without this *24 hours post production results need to be put up whose genomes? pangenomes:

Answer 68

genes: region of the DNA which codes for an active molecule (alternative splicing causes different proteins) various RNA genes other sequences intergeneic seqeunces (low complexity repeats often found in centromere and telemeres)- most difficult parts to sequence as so repetitive mobile elemets (selfish DNA) gene transcription miRNA and gene silencing- to silence the expression of the protein it would code for. example human nuclear genome

Answer 69

cloning tools: -PCR -Plasmids

Answer 70

-obtain the gene of interest (extract dna, PCR) -cut gol and vector -seperate and isolate fragments (agarose) -ligate gol and vector insert the new recombinant moelcule to the bacteria -grow the bacteria (this creates a huge amount of the region of interest) molecular cloning is better then using PCR as it will keep the protein alive and its easy to stop it denaturing PCR: polymerase chain reaction of section between the primers heat= denatruation, anealing, polymerasiation (doubles every cycle) annealing temperature depends on the primers sequendes (G/C content)

Answer 71

*processivity (how many nucleotides will it incoroprate before it dissociates) how long *fidelity (proof reading, 3'-5' exonuclease acitivty) *how much other stuff does it amplify *thermostablity (how quickly does it degrade at the high temp) types of DNA polymerase: TAQ (no proofreading) processivity- how many nt it can imcorrperate (how good as it at duplicating) fidelity- how error free it is (proof reading 5-3 exonnuclease) specificity- how specfic is it- what other stuff does it also amplify thermostabilty- how quickly does it degrade at temp types: taq- doesnt proof read (72-75.c) doesn't correct errors issues- could make errors consistenly short half life makes sticky ends proof reading types pfu- slower then taq Platinum taq- not great proof reading Q5

Answer 72

denaturation of inhibitor (HOTSTART)-polymerase doesnt work at room temp- inhibited by anti-body but once that hat is hit then the polymerase will be activated (stop product being made before ready) Touchdown PCR Nested PCR- one after the other, One before wanted region and one after.

Answer 73

do exist in nature but ones for cloning have been editied example: pUC19 important elements: -contain genes to replicate on the chromosome -ORI -selection marker- antibiotic resistance- will kill off everything else that isn't -multiple cloning sites -way of screening blue/white cloning 260/280- absorbance level see how clean DNA is

Answer 74

transformation (bacteria the uptake of dna directly from the environment) -transduction (can happen in mammalian cells if they have been edited) *(virus mediated when it picks it up and takes it on) -conjuation (penis bit)

Answer 75

use transfection to make competent cells.

Answer 76

use overhang to ligase, using sticky ends will make the cloning a lot mor effecient use a taq etc

Answer 77

chemical transformation: chill cells in calcium chloride to permeablise membrane then heat shock to prompt dna uptake electroporation: purify cells to remove ions then shock cells with a high voltage current to create holes in the membrane both stressful methods, after the DNA is uptaken wait 2 hours to allow cell recooperation

Answer 78

culutre spread on agar they will then reproduce and create colonies on each section. but at this stage you dont know if they have uptaken the plastid. (sterille techniques) next screen the colonies

Answer 79

select bacteria- the vectors has an antibiotics resistant gene. So grow the bacteria on the agar with antibiotic then only the bacteria with the plasmid withh survive. Couple plasmid with antibiotic in the vector using white blue/colonies system lac operon (contains different genes all transcribed at a the same time) b-galacotisades lac-glc-gal also breaks down x-gal when b-gala breaks down the x-gal it creates a bright blue colony. Therefore any bacteria that has b-gala will break down the x-gal producing the colour compound and turning them blue. *if the colonies have taken up the insert they wont turn blue as the insert has gone inside and broken up the b-gala. at this point the screening of inserts is complete but need to screen for the specifc insert wanted

Answer 80

-check for the size of the insert om a restriction digest, run on agarose and check size. -colonly pcr

Answer 81

*need restirction sites in the right place *need very highly purified enzymes (can be spenny) using pcr advantages: can isolate and fuse fragments independtemly disadvantages *length constraints, G/C content too high. * difficult to join one then more region of interest

Answer 82

TA cloning Zhou and Gomez-Sanchez (2000). Universal TA Cloning. Curr Issues Mol Biol 2(1): 1. e.g. https://pcrbio.com/applications/pcr/ta-cloning/, https://www.thermofisher.com/order/catalog/product/K457502 taq polimerase is cheap and adds an overhanging a on 1 of the 2 strands. pcr amplify insert, and incubate with deoxyadinenes to create the overhanging a use plasmid with overhanging t a and t's will bind= efficient cloning topo topo-isomerase (way dna is folded) plasmid is sold with a correct overhanging t with an enzyme (topo) attached. opens the plasmid, insert anneals with and t and then ligase activity puts it back together. #dont need many restriction sites as topo is there. Has some so insert can be cut out later if needed. #conserved priming site, dont need to make new primers why are all the elements there, why are they useful?

Answer 83

attaching dna to GFP first create primers #PCR-amplify left fragment (DNA 2) PCR product has primers either end #design primers to amplify GFP (DNA 2) but foward primer needs to contain same sequence as end of first PCR product (rev) Reverse primer for GFP doesnt matter #mix 2 products stick the 2 in a tube add a 5-3 exonuclease cuts off nucleotides from this section (digests a bit of both fragments) #Products then anneal and polymerase fills the gaps ligase then acts on binding it all advantages: seamless joinign of DNA fragments cheaper no need for RE multiple fragments can be joined in one step limitations few fragments at a time need to start from a template size limit imposed by pcr

Answer 84

-modified organisms -produce a recombinant protein like insulins -recombinant animals for research -different proteins that are reacting together

Answer 85

different organisms have different perctnages of different tRNAS and codons prefernces what enginerring a construct to express a protein which codons should be used? *what is the codon usage of the organism *which codons are used the most

Answer 86

types: -plasmids can obtain large inserts. These are used in libraries for genome sequencing. -cosmids (can accept inserts of 30/40 kb) -BACS -PACS -YACS

Answer 87

#next gen pros - higher output -can do lots (parallel at same time) - requires less prep (library) -low cost per big volume of data -more data less time quicker -sequencing in different ways #next gen cons -produce more errors then sanger (assembly) in theory each read is less reliable -still under development -produce long reads -require big computers -needs specalist knowledge -data storage issues sanger- 96 max

Answer 88

how to sequence a genome (shotgun compared to libraries) Book-shred and sequence- put back together making libraries take small pieces, sequence the 'page' and put together library= a comprehensive collection of cloned DNA fragments. (Have to be cloned into a vector with an origin of replication) and the fragments need to represent the whole genome. Can be propregated in a host cell a good library has: *insert if not rearranged *even representation of source material *no vectors with multiple inserts *no empty vectors vector can be very simple but the concept of library is the same

Answer 89

take the genome, randomly fragment and look for overlap, same bases in the same sequence. Then it can be read *step on fragment *sequence each of fragment *try and put them together (align the k-mers) align fragments by looking where the same letters are *

Answer 90

Types Illumina- mysig small sequencer (bench top sequencer) How it works: output of Illumina are pictures 1)Prep the sample (library)- put a few molecules at the end (taq) one end is used to attach dna piece to a flow cell (slide) with lanes in the lanes there are small oligonucleotides attach to cell and used as an anchor for molecule you have made. *Allow your molecule to bind to the slide. in some of the sections the molecule you want, Attach molecule you want to sequence to the slide.(all are fluoresent) each of the fragment is synthesized by sequence and everytime a molecule is added its fluorscent. (parallel sequencing) *make a cluster of these molecules so they can be perceived more easily, produce a clone in each of the lanes in the slide. in each lane: there are attached olligoers, molecule of interest which have oligers attached. attaching the bits you want to sequence to the slide, every incorporation take a picture. cluster needed in each position so its easier to visualise on film. All sequenced at the same time. dideoxynucleotide wash- Sequence the complimentary wash away the og strand. Add the oxygen and rewash with a different fluorescent to see how the new positions have incorporated the added letters. producing a huge amount of data all at the same time. *library *clusters *sequencing illumina sequence short fragments including oligos, 150bp sequenced. Sequence a fragment by both ends (pair end sequencing) or single end. Issue- small fragments hard to put together. *making a mate pair for short sections. How do all the small bits get put together

Answer 91

Smrt bell template- take the fragment and add 2 hairpin linkers that has a polymerase which will make the complimentary strand put one of each molecule in small pores. Camera can see light for small pores. i Polymerase sequencing through synthesis- fragment blocked on one side- The fragment of dna (circle) can go through polymerase a number of time. t= orange etc Depending on which light is read by the camera can determine which nucleotide has gone through. Why? *Can sequence much longer fragments, *lag time between fragments-vv *can read epigentics as well but if theres mods between bases. *How bases are modded. *Lag between reading certain bases tells you if its modded or not

Answer 92

*first portable sequencer *sequencing in the field ebola pandemic- able to do in the field

Answer 93

protein coding genes rna genes NTR (promotors, mobile elements) initital gene annotation: -look for open reading frames -compare sequence to known sequence. -Compare transcriptombe from same species or similar taxa that looks similar

Answer 94

Phylogeny Functional characterisation Epignentics compare genomes detect variants population structure Rutter GA (2014). Understanding genes identified by genome-wide association studies for Type 2 diabetes. Diabetic Medicine 31(12): 1480. Example: enabled the heritable nature of type 2 diabetes to be explored. between families and genome-wide association studies. 500 genes identified

Answer 95

what we have seen so far: --inital sequencing of human genome: 2001 --human vs mouse genome --synteny (comparison between fragments of 2 humans. Same genes, but intergenetic regions are different) --carotypes of chromosomes in genomes --similarities and differences (similar species with different genomes)

Answer 96

--gene start comparison (2007) comparing fruit fly genomes- found information about the start of genes Lin et al. (2007). Revisiting the protein-coding gene catalog of Drosophila melanogaster using 12 fly genomes. Genome Research 17: 1823. found out about the start of genes: looked at protein coding in genes in different drosophilla species and found what was conserved or not. They corrected mistakes where cds was then started.

Answer 97

--alignments (workout what sections of the gene are conserved --mitochonrial genomes (comparison of the shape of mitocondrial genomes)

Answer 98

chromosome 19: --smallest --very well conserved --has a high gene density --high G/C content Harris et al. (2020). Unusual sequence characteristics of human chromosome 19 are conserved across 11 nonhuman primates. BMC Evol Biol 20: 33

Answer 99

chromotripsis- rearrangment of chromosomes that result in disease. In cancer cells, chromosomes are rearranged. certain stresses (chemical, uv) causes partial fragmentaion, then they are re-arranged wrong. shows that its not just the information that is important its how they are expressed. (regulation)

molecular biology (exam prep) Flashcards

(145 cards)