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Biological Sciences 2 > BIOL220Z Molecular Biology > Flashcards

BIOL220Z Molecular Biology Flashcards

1
Q

Make sure to recap:

A

Life
Life domains
Main similarities/differences between genomes
Macromolecules

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2
Q

What are the characteristics of life?

A
  • maintain integrity (boundaries)
  • information: store, replicate, transform it into “action”
  • perform and regulate metabolism (energy)
  • interact/signal (with environment, other cells)
  • replicate (divide)
    *etc

Life: C-based and DNA-based

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3
Q

Describe the genome in bacterial cells

A

-one single circular chromosome
-smaller
-extra chromosomal elements outside of the circular chromosomes

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4
Q

Describe the genome in eukaryotic cells

A

-linear
-bigger
-mt genome and chloroplasts

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5
Q

Describe the origin of the present-day mitochondria

A

Endosymbiosis: bacterial cell engulfed by eukaryotic cell and evolve together.

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6
Q

Name 3 types of staining

A

-binding a molecule to a specific organelle structure
-binding an antibody
-GFP staining (green fluoresent)

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7
Q

Draw the DNA nucleic acid structure

A
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8
Q

What is a nucleoside?

A

Nitrogenous base and 5 carbon sugar

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9
Q

DNA VS RNA similarities

A

-bases: A,G,C

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10
Q

DNA vs RNA differences

A

DNA:
-base T
-double-stranded
-relatively stable
-information storage
-usually one
-deoxyribose sugar

RNA:
-single-stranded
-unstable
-base U
-many functions eg transport, enzymatic etc

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11
Q

What are Okazaki fragments?

A

Okazaki fragments are the short lengths of DNA that are produced by the discontinuous replication of the lagging strand.

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12
Q

What is ori c in e.coli?

A

This is the replication origin, where DNA sequences are recognised by initiator proteins

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13
Q

What occurs in a reverse transcriptase reaction?

A

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

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14
Q

Name 3 important milestones in molecular biology

A

-“jumping genes”
-lac operon
-pcr

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15
Q

What was Barbara McClintock’s work about?

A

the discovery of transposons “the jumping genes” and the disruption caused by them on chromosome 9

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16
Q

What is the Lac Operon?

A

A classic example of an inducible operon for gene expression and control in bacteria

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17
Q

Who came up with PCR?

A

Kary Mullis et al

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18
Q

What is synthetic biology?

A

A multidisciplinary field of science that focuses on living systems and organisms, and it applies engineering principles to develop new biological parts, devices, and systems or to redesign existing systems found in nature.

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19
Q

How can lac Z be used as a cell reporter?

A

Lac Z codes for B-galactosidase and its activity serves as a marker for gene expression patterns during development eg in whole mouse embryos

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20
Q

Alpha helix info

A

3.6 amino acyl residues per turn; 2.3 Å helix radius

Perutz (1951)

Most common helix in proteins.
*
Usually about 10 aa residues

contains MALEK
Methionine, alanine, leucine, glutamate, and lysine uncharged

example of where found-myosin

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21
Q

310 helix

A

3.0 amino acyl residues per turn; 1.9 Å helix radius

Bragg et al. (1950)

Very strained structure.

Found in e.g. myoglobin and hemoglobin.

Usually very short - <4 aa residues.

example of where found - blue whale myoglobin

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22
Q

pi helix

A

4.4 amino acyl residues per turn; 4.4 Å helix radius.

Low and Baybutt (1952)
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

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23
Q

beta helix (sheet)

A

Perutz (1951)
Can be parallel or antiparallel and complex structures can form.

Each strand is usually 3-10 aa in length.

Usually contains: Valine, threonine, histidine, tyrosine and isoleucine

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24
Q

Name an example of a protein with 310 helices

A

Blue whale myoglobin

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25
Q

First genome synthesised?

A

e-coli

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26
Q

Genomics?

A

sum of chromosomal DNA

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27
Q

Transcriptomics?

A

mRNA of specfic condition/time

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28
Q

Proteomics?

A

sum of protein content

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29
Q

metagenomics

A

all chromosome DNA, all organsisms, All domains
(blend a person)

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30
Q

metatranscriptomics?

A

all mRNA

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31
Q

Panenomics?

A

all chromosomes DNA all strains vs

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32
Q

5!

A

(5x4x3x2x1)

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33
Q

Beta meander?

A

2 or more anti-parallell strands linked by hairpin loops

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34
Q

What is a Greek Key Motif?

A

Four anti-parallel strands and linking-loops.

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35
Q

What occurs at the OriC site in bacteria?

A

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) 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.
  • strands need to be seperated so copies can be made creating 2 genomes
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36
Q

Why is space between DUE and box site important?

A
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37
Q

genome structures: bacteria and archaea.

A
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38
Q

What occurs at the OriC site in archaea?

A

-Contains a series of origin of replication sites (OriC) 3-4

-2 flavours of boxes full size and mini Orbs

  • initiator protein (orc 1/ cdc6) comes and binds to an ORIc site.
    orc1 will bind well to OriC1 and slightly to all the others- this does same job as dnaA and unwinds
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39
Q

Prophage (viral sequencing)

A

a bacteriophage genome that is integrated into the circular bacterial chromosome incorporated into the host cell.

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40
Q

HGT (hor gene trans)

A

transfer of dna from species to species or gene to gene- sideways

ways this can occur:
-phage (the act of prophase intergrating,taking bits of DNA with it)

  • plasmids
  • intergrons

important in antibiotics

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41
Q

Transcription

A

Synthesis of RNA under the direction of DNA-transcript of the genes protein-building instructions (mRNA)

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42
Q

Translation

A

Synthesis of a polypeptide under the direction of mRNA.
Site of translation are the ribosomes

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43
Q

Bacteria Transcription/Transalation

A

Coupled event, as they lack nuclei.
ribosomes attach to leading strand of mRNA colecule whilst transcription is still happening

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44
Q

eukaryotic transcr/Transla

A

transcription occur in the nucleus, mRNA are sent to the cytoplasm where transalation occurs

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45
Q

Synthesis and processing of RNA

A

RNA polymerase (enzyme) pries to strands of DNA apart and hooks together the RNA nucleotides as they base-pair slong the template

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46
Q

overview of transcription

A

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

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47
Q

How does RNA polymerase recognise where to start? (bacteria)

A

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’

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48
Q

Where is start of translation? (bacteria)

A

(ATG) look downstream
usually at -6 to -8 is shine dalgamo (ribosomal binding site)

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49
Q

Differnces in transcription bacteria and eukarotic

A

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

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50
Q

Transcription in eukaryotes

A

(Other proteins invovled not just RNA polymerase)

enhancer sequence which activator proteins can bind. adaptor proteins- all activate and dna folds.

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51
Q

What does TF stand for?

A

transcription factors
part of transcriptor proteind that help activation

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52
Q

How is gene expression regulated? (after)

A

Post Transcription modifications
(Bacteria)
3’ poly (a) tail- signal degradation

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53
Q

How is gene expression regulated?
(eukaryotic)

A

(eukaryotic) post transcription

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)

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54
Q

(Bacteria) Polycystronic?

A

one mRNA makes more then one proteins, because they’re all needed at the same time

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55
Q

snRNPs?

A

help recognise nucleotide sequences from exon/introns boundaries

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56
Q

Alternative splicing

A

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 made by one gene therefore lots of different proteins

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57
Q

What are the UTR’s?

A

Untranslated region

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58
Q

Genetic code

A

the translation- CDS is read in triplets and equals one AA

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

59
Q

ORF

A

gDNA is the entire genomic DNA and therefore double stranded

60
Q

rRNA

A

ribosomes- complex molecule

Bacteria 70’s (s-value is rate of sedimentation)
2 subunit protein and different molecules rRNA (major 60, minor 30s)

bacteria and eukaryotic similarities:
terms of sequence(useful to decide different between organsims) and instruction

16s common used

mods:

61
Q

TRNA

A

Charged tRNA so bound to an AA

62
Q

Translation process

A

Proteins have a direction of reading:
n-c terminal (5’-3’)

n-c
protein synthesis happens by an addition of aa thats bound to aa tRNA and a bond between new n terminal of new nucleotide to c terminal of og- binds to growing peptide change

Messenger RNA can be tranalted by different ribsomes at the same time→ producing many copies of proteins

63
Q

Ribsome

A

not membrane enclosed organelles

3 main sites in the large subunit
e: old tRNA goes out
p: Peptidyl-tRNA-site
a:Aminoacyl-tRNA-site

64
Q

Protein synthesis

A

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)

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.

2nd initaitor factor- hydrolysis of GTP to GDP creates energy that for change of confirmaton (sliding of 2 subunit) formatin of new bond and exit of the last one.

65
Q

Inhibitors of translation (bacteria)

A

acting on different processes, so to ihibit something you need to choose the right thing

example: antibiotics
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

66
Q

Initiation of translation in bacterial cells

A

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. Less options for regulation.

67
Q

Initiation of translation in eukaryotic cells

A

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

68
Q

Once proteins are made : folding &
quality control

A

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:
check points to ensure the protein has folded correctly→ either fixed or hydrolised (can be dangerous/patogens for the cell)

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)

69
Q

Where does regulation occur in eukaryotic cell?

A

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

70
Q

how can transcription be regulated? when/ where

A

regulate how mnay times a gene is transcribed

where transcipes are kept?

timing of transcription

mods to histones and chromosome structure

71
Q

Histones

A

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.

72
Q

DNA methylation

A

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.

73
Q

Sigma factor (σ)

A

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

74
Q

regulation at transcirption (eukaryotes)

A

different polymerases
1: ribosomal
2: mRNA and RNA genes (small nuclear etc)
3 tRNA, some non coding molecules

other factors:
either improve the transcription of something or repress it

further regulation example:
different genes have different promotrs require different activator proteins
so another activator gluco that doesnt interact with dna.
Activator comes along (hormone) binds to receptor and activates the receptor so it goes to nucleus and interact with other activator protein= more gene expression

75
Q

An example of a transcript factor to de-differentiate and re-differentiate cells

A

regulation at transcription of myOD

fibroblasts taken from skin chick cells and turned into muscle cells

done by: expressing myOD (which is required for muscle cells)

76
Q

Localisation

A

Signals on UTR can effect localisation

keeping things in certain places by certain cytoplasmic deterants
If you swap over the UTRs in nanos and bicoid, the mRNAs localised in the “incorrect” region (e.g. the nanos mRNA)

77
Q

Regulation after translation

A

Occurs once mRNA is a protein
depending of avalibilty of charged tRNA

can occur at elongation factor

post translational mods (phosphorylation, acetylation, glycosylation)

localisation of the protein:
destination of newly produced protein

2 different localisation of ribosomes
post translational translocation:
proteins produced on free ribosomes are moved after production

extracellular proteins: produced on ER destined for outside nucelus

78
Q

Example of post translational translocation

A

protein destined to mitcondria
produced on ribosomes and trans out
includes a signal peptide to say where it needs to go (cut off once has arrived)

79
Q

Phosphorlation (at/ after translation)

A

phosphates added, carried out by a kinase.
(opposite phosphatase)

cell cycle: cyclase hydrolyised and used at different stages activated by cdk kinase. need phosphorlation to activate. 2 levels of regulation.

why does cell go through this?
timing, it creates the complex which takes a while, having an activated but slightly inhibited complex is easier then starting it all at one go.

80
Q

protein degrdation and example at Ubiquitylation

A

regulation by protein degradation, hydrolyising an element thats acting as an inhibitor so then the complex can perform its function

81
Q

control level reminder

A

Control at:
Transcription- is it transcribed or not, how much
Processing of mRNA- splicing
Transpot- wheres it going/whats it need to get there
Translation- make up of protein
Protein reg- how long will protein last, is it active or not (phosphorlation etc)

82
Q

How to study gene expression?

A

make protein fluoersent or attach a tag to make it visible as long as it goes in the right place and doesnt effect the final product

do a qPCR- in vitro (extract RNA) make it visible, quanify how much RNA is produced by the amount of flurosent produced

micro ray

hybridisation

83
Q

Bacteria vs eukaryotic

https://bmcbiol.biomedcentral.com/articles/10.1186/1741-7007-11-119

A

eukaryotic- bigger, more then 1 linear chromosomes, smaller genes, mulitple ori

Bacteria- circular, no associated proteins, higher gene density, gene transfer, single origin

relationships and how genomes and evolve

84
Q

c value paradox

A

number of genes per genome doesnt equate to complexity
example mammalian vs ameoba

85
Q

relationship between protein coding genes and genome size

A

small genomes- small protein coding genes
virsus- they use other proteins from what they are preying on

86
Q

chromosome packing in eukaryotic cells

A

beads on a string- dna wrapped around histones on chromatin

87
Q

karyotype

A

list of complete set of chromosomes

88
Q

autosomes

A

not sex chromosomes

89
Q

haploid vs diploid

A

complete, only 1 in germ cells
2 copies

90
Q

human genome vs mouse genome

Lander et al. (2001). Initial sequencing and analysis of the human genome. Nature 409(6822): 860.

A

similarities-
segments are similar 17 is same as 11, same gene in same form
human 20 similar to mouse 2

large regions of synteny

91
Q

Synteny

A

p- petite
fragment of human genome- few genes

3 conticts of fugal genome
genes are exactly the same when compared.
in same sequence on same strand

the sequence shows genes present are the same.
but if zoom in and look at structures are in the gene are the same too (where introns and exons are)

92
Q

first draft of human genome

https://www.nature.com/articles/s41586-023-05896-x

A

human pangenomes

93
Q

genome content
what do they contain?/elements

A

genome (is full compliment) how much codes for protein coding genes? =2%

genes region of dna that codes for an active molecule
RNA genes etc

other sequences-
intergenic sequences
low complecity repeats (centromeres, teleomeres)
mobile elements- jumping bits of DNA- behaving selfish
splicing sequences

94
Q

miRNA

A

Gene Silencing-

95
Q

minimum number of genes for an organism to survive?

A

Hutchison et al. (2016). Design and synthesis of a minimal bacterial genome. Science 351(6280): aad6253.
Glass et al. (2006). Essential genes of a minimal bacterium. PNAS 103(2): 425-430.

96
Q

recombinant technology DNA (1)

A
97
Q

What is cloning?

A

make genetically indentical clone
an organism or molecule

(steps come together which equated to- cloning)

98
Q

What are the main steps in cloning?

A

obtain region of interest

prepare region to become sticky and get it to stick to a vector (done restriction enzymetically)- creating recombinant plastid

insert new recomb into bacteria- bacteria can dublicate- genetically identical.

need single colonies of bacteria

can express proteins more or less directly

99
Q

why need molecular cloning?
instead of just pcr?

Dna farm

A

long term- stop degredation- unstable to keep, need the specfic primers to just pcr. Bacteria can be frozen(suspended animiation) and kept and they have their own determining methods that will delete mutations etc (example?)

standard polymerase in virto might not be strong enough to cut a very long sequence

100
Q

Pcr reminder

A

polymerase chain reactions- end up with an exponetial section thats between your 2 primers.

denature
annealing temperautre
lower- less specficty
higher specifity
decides strength of interaction

elongation

101
Q

Characterisitcs of Polymerases

and types

A

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

102
Q

common types of PCR

A

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.

103
Q

Restriction enzymes
blunt ends or sticky ends

A
104
Q

Where do plasmids come from?
plasmid pu19

A

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

105
Q

Horizontal gene transer

A

what?
transfer of genetic info from the same generation, not from desendants.

transformation
transduction
conjuation

Transformation- naked DNA in environment- taken up directly

transuction- virsus infect bacterium once produced picks up a little bit od DNA and put into another virus

conjuation- transfer

example?

106
Q

Recombinant Dna 2
notes:

A
107
Q

Transformation- how to make prone to take up DNA (compotent cells)

A

chemical transformation- chilling cells down (contains calcium chloride makes membrane more permiable)

then heat shock to prompt DNA uptake (30 seconds)

electroporation
purify cells to remove ions
high voltage shock make holes in membrane

after DNA uptake, cells have recovery time before selection

108
Q

after transformation
(need to grow them, grow with correct plasmid)

A

-spread bacteria culture on agar plate (single bacteria)
-dots on plate show colonease and reproduce these are the same. (steralile)

109
Q

screen colonies
select bacteria that has acquired the plasmid (vectors contain antibiotic resistance genes)
couple plasmid with antibiotic you have in screening

A

grow bacteria on an antibiotic, if the original vector has antibiotic resistance it will survive and everything else will die

110
Q

tectracycline (antibiotic)

A

blocks translation

111
Q

white/blue colonies
bacteria can transcibe messenger RNA that contains multiple genes
screening-chose the white

A

agar plate
xgal to the plate
any bacteria with b-galactoisidase will break down xgal making colonies blue

if gene in multiple cloning site, plasmid contains insert will be white.
if the b-gala will be disrupted and therefore blue (no insert)

112
Q

screen for correct insert

A

go to primers next to the insert and PCR amplfiy then run on gel

check for the size of the insert on all colonies

113
Q

culture colonies on a large scale

A
114
Q

engineering with restriction enzymes pros and cons

A

need restriction sites in the right placen

need highly purifed enzymes

advantages:
can isolate and fuse fragments independently of restriction sites

disadvantages:
length constraints
error rate
GC content (too high PCR not effecient)
difficult to join more then one gene

115
Q

how to insert more then one gene into a vector, or join multiple fragments
(molecular cloning methods)

A

differernt types of cloning
#restriction enzyme
#PCR cloning

116
Q

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

A

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

117
Q

TOPO TA cloning
(common ta cloning)

A

conserved priming site, dont need to make new primers

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.

why are all the elements there, why are they useful?

118
Q

Advantages and disadvantages of some cloning methods

A

blunt ends- less effecient then a sticky ends (non-directional cloning)

#gateway cloning
#gibson assembly

119
Q

gibson assembly

Gibson et al (2009). Enzymatic assembly of DNA molecules up to
several hundred kilobases. Nat Methods 6: 343.

A

2 different needing to be joined together.

advantages-
- all reactions happen in one tube at one temperature
different pieces of dna at one temp and end up with one molecule
- seamless joining of any DNA fragments
- dont need RE
-mulitple fragment joining in one step
-cheaper then starting from scratch

disadvantages- difficult to create primers
few fragments at a time (5/6)
always need to start from a template
Based on PCR therefore still has limitations of PCR

2 different needing to be joined together.

120
Q

Gibson assembly

A

design primers to amplify GFP (DNA 2) but foward primer needs to contain same sequence as end of first PCR product (rev)

attaching dna to GFP
first create primers
#PCR-amplify left fragment (DNA 2)
PCR product has primers either end

Reverse primer for GFP doesnt matter

add a 5-3 exonuclease
cuts off nucleotides from this section (digests a bit of both fragments)

ligase then acts on binding it all

121
Q

What to do with cloned genes?
why use molecular cloning, what are the applications?

A

produce a recombinant protein
(for localisation)

need to produce a lot of protein

study certain diseases in an organism (animal testing)

study how proteins interacting together

122
Q

codon optimisation
which codons to choose?

Figure 10-27: The genetic code.
Griffiths et al. (2000). An Introduction to Genetic Analysis. 7th Edition. New York: W. H. Freeman

A

what codon useage does the host organism use?

-genetic code is redundant-certain triplets can code for the same protein

-different organisms have different tRNAs

#which codons are more used

123
Q

types of vectors?

A

a dna molecule that you can put another molecule into.

-plasmids: size? 5-10kb
-cosmids- bacteria phage 30-40kb
(used for packing)
-BACS
-PACS
-YACS
(UP in size)

124
Q

Next gen vs sanger sequencing

A

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

-produce more errors then sanger (assembly)
-still under development
-produce long reads
-require big computers
-needs specalist knowledge
-data storage issues

sanger- 96 max

125
Q

how to sequence a genome
(shotgun compared to libraries)
Book-shred and sequence- put back together

A
126
Q

What are libraries?
what makes a good library?

A

Library- comprehensive collection of clone DNA fragments- ligated into a vector. (Includes all key elements, like ori) characteristics needed- no empty vectors.

-insert is not modded
-no empty vectors
-not multiple inserts

Figure 7-3: General procedure for cloning a DNA fragment in a plasmid vector.
Molecular Biology of the Cell. 4th Edition. New York: Garland Science
www.nature.com/nrg/journal/v2/n8/fig_tab/nrg0801_573a_F6.html

127
Q

shotgun sequencing:
-steps
-coverage

A

Cutting/fragment to start of the same size, sequence each of the fragments, look for overlaps of the same letters- align the k-mers (length of the fragment) then can read the sequence. How many times each positon has been sequenced (in the final assesmbly) coverage-how many times each bit is identified. Gaps.

coverage- the more time bits are represented the higher coverage

128
Q

sequencing types/technologies

A

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) once end is used to attach dna piece to a flow cell (slide) small oligonucleotides attach to cell and used as an anchor for molecule you have made. Allow your molecule to bind to the slide.
Attach molecule you want to sequence to the slide.(all are fluoresent)
make a cluster of these molecules so they can be perceived more easily.
dideoxynucleotide wash-
Sequence the complimentary wash away the og strand

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

129
Q

pacbio SMRT.
single molecule real time sequencing

Flusberg et al. (2010). Direct detection of DNA methylation during
single-molecule, real-time sequencing. Nature Methods 7: 461.

A

Smrt bell template- one of each molecule in small pores. Camera can see light for small pores.

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.

130
Q

oxford nanopore
(minION)

A

Advantage- sequencing in the field.

131
Q

how to annotate a genome?
genome content?

A

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

132
Q

What can you do with NGS?

A

Phylogeny
Functional characterisation
Epignentics
compare genomes
detect variants
population structure

133
Q

application of NGS example

Rutter GA (2014). Understanding genes identified by genome-wide association studies
for Type 2 diabetes. Diabetic Medicine 31(12): 1480.

A

enabled the heritable nature of type 2 diabetes to be explored. between families and genome-wide association studies.
500 genes identified

134
Q

Comparative genomics
what is it used for?

A

compare genomes if theres similarities it shows that regions are conserved, and that they are beter with then without.

conserved regions will show what needs to be targerted.

shows changes over time

135
Q

genomes sizes and number of genes (example of comparative genomics)

Figure 1-38: Genome sizes compared.
Alberts et al. (2002). Molecular Biology of the Cell. 4th Edition. New York: Garland Science

A

size of genome (number of nucleotides) in different species.

136
Q

human vs mouse genome
first example of comparative genomics

Lander et al. (2001). Initial sequencing and analysis of the human genome. Nature 409(6822): 860.

A
137
Q

drosophilla example
Schaeffer et al. (2008). Polytene chromosomal maps of 11 Drosophila species: the order of genomic
scaffolds inferred from genetic and physical maps. Genetics 179: 1601.

A
138
Q

how to study this?

A

alignments- looking at whats conserved across organisms will show the function and the similarites between organisms.
looking at entire mitocondrial genomes

139
Q

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.

A

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.

140
Q

chromosome 19 (example)

Harris et al. (2020). Unusual sequence characteristics of human chromosome
19 are conserved across 11 nonhuman primates. BMC Evol Biol 20: 33.

A

certain characteristics are conserved and the function isnt obvious but the conservation shows it is.

141
Q

conservation of genomic regions
(medicine)
Maher & Wilson (2012). Chromothripsis and human disease: piecing together the shattering process. Cell 148: 29.

A

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.

142
Q

comparative genomics in other organisms (ensembl only shows ensembl)

A
143
Q

synthetic genomes

A
144
Q
A

Biological Sciences 2 (11 decks)

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