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Biosci 202 > Final exam - LM3 > Flashcards

Final exam - LM3 Flashcards

1
Q

PCR steps

A
  • add oligonucleotide primer
  • denature
  • anneal
  • extend
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2
Q

E coli genome vs human genome

A
  • e coli = 4.7m bp
  • human = 3.2b bp
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3
Q

Sanger sequencing

A
  • DNA from PCR combined with a primer
  • DNA polymerase binds
  • dNTPs bind
  • ddNTP cause termination
  • product put into capillary sequencer to detect tagged DNA products
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4
Q

If you want to sequence something you dont know

A
  • clone unknown into a plasmid using universal sequencing primers
  • ligate unknown DNA to known DNA as known DNA can be used as primers
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5
Q

Illumina sequencing

A
  • ligate adaptors
  • clusters form on flow cell
  • bridging amplification via clonal PCR = add ddNTPs
  • sequence via synthesis at each cluster
  • scan surface for fluorescent ddNTPs
  • short reads only
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6
Q

advantages of illumina

A
  • accurate
  • high throughput
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7
Q

Pacbio

A
  • single molecule
  • real time sequencing
  • long reads
  • expensive
  • sequence by synthesis = ddNTPs
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8
Q

Nanopore

A
  • single molecule
  • very long reads
  • portable
  • fast and convenient
  • charge measured for each base that passes through pore
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9
Q

How to assemble a new genome

A
  • build a scaffold using long reads (pacbio or nanopore)
  • overlay with short reads (illumina)
  • collect reads and align for reference
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10
Q

challenges in assembling genomes

A
  • repeat sequences which look identical
  • telomere sequences are identical between chromosomes
  • pseudogenes that are similar
  • large deletions/insertions that vary between individuals
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11
Q

Mutations

A

changes made in DNA sequence

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12
Q

Variants

A

different versions of DNA sequences that differ by one or more mutations

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13
Q

Synonymous mutation

A

alter codon but same AA

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14
Q

missense mutation (conservative)

A

alter codon and changes AA that is chemically similar

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15
Q

missense mutation (nonconservative)

A

alter codon and different amino aci

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16
Q

nonsense mutation

A

premature stop codon

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17
Q

Exome sequencing

A
  • used when most variants are exomic
  • cheaper than full genome
  • cant see large deletons
  • exome is pulled out of genome after DNA shearing into ssDNA
  • DNA is sequenced and mapped to genome
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18
Q

likelihood of mutation being casual determined by

A
  • effect on coding sequence
  • knowledge of function
  • homology in other species
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19
Q

De novo mutation

A

neither parent will have the same variation as offspring

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20
Q

Why we want to clone

A
  • need more DNA
  • may want to express recombinant proteins to test function or for medicinal use
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21
Q

What happens if you reinfect same strain of E.coli

A

same titre of bacteriophage if you infect with a different strain you get a reduced titre as you have different restriction enzymes

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22
Q

Why bacteria cuts phage DNA but not its own

A

DNA is methylated VIA methylases which protects DNA

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23
Q

Restriction enzyme characteristics

A
  • usually palindromic
  • 4-8 nucleotides in length
  • cleave phosphodiester bond at recognition site
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24
Q

2 types of restriction enzymes

A
  • sticky cutter = cut at different points, higher in frequency
  • blunt cutter = cut at same point
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25
Q

Cloning steps

A
  • cleave using restriction enzyme
  • ligate into plasmid vector
  • clone
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26
Q

Ligation

A
  • T4 DNA ligase reconnects phosphodiester bonds between 3-OH and 5-PO4
  • needs ATP for energy and Mg as cofactor
  • forms recombinant DNA plasmid
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27
Q

components of plasmid vectors

A
  • polylinker = destination for DNA
  • amp resistance gene for positive selection
  • origin of replication
  • lac Z = inactivated when DNA added
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28
Q

Inserted DNA into plasmid can be directional or non-directional

A
  • directional = use 2 different cloning restriction enzymes, 5’-3’ direction known
  • non directional = single RE used or blunt ended
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29
Q

How to transform plasmid into E coli

A
  • chemically competent E.coli = treat cells with CaCl and heat shock making it porous to DNA
  • electroporation = zap cells so they take DNA up
30
Q

Implications for non directional inserts

A

additional screening required if orientation is important

31
Q

what is screening

A

restriction digest DNA to identify correct clones and orientatione

32
Q

end modifications in clone

A
  • dephosphorylation = stops vector fro recircularise
  • addition of single A = makes compatible for TA cloning
  • blunting = makes compatible for blunt cloning
  • phosphorylation = allows ligation
33
Q

Why we might want more plasmid DNA

A
  • construction of cDNA libraries
  • subcloning small fragments for RE mapping
  • to produce protein or RNA for vaccines
34
Q

Expression plasmid vector features

A
  • origin of replication
  • ampicillin resistant gene
  • promoter for highly expressed gene
  • polylinker
  • lacI gene for inducible expression
  • HIS tah
  • repressor of primer to control copy number
35
Q

Problems with e coli recombinant expression vector

A
  • proteins expressed in bacteria are often insoluble
  • post translational modifications are different from mammals
36
Q

How to optimise recombinant protein expression

A
  • strong promoter
  • inducible expression
  • optimise translation using host codons and avoiding rare codons
  • optimise stability by removing protease
  • optimise solubility through fusions making it soluble
  • post translational modifications
37
Q

Factor VII production

A
  • expressed via T7 RNA pol and T7 late promoter through a plasmid expression vector
  • T7 RNA pol is expressed via Lac promoter in recombinant E.coli chromosome
  • RNA pol activates promoter on vector to drive production
38
Q

Insulin recombinant production

A
  • insulin cleaved into A and B chains to improve solubility
  • purify chains and then refold
  • oxidise cysteines to form disulfide bonds to form active insulin
39
Q

Genomic library

A

a set of clones containing all of the genomic DNA of an organism

40
Q

cDNA library

A

a set of clones or a pool of cDNAs containing DNA copies of all mRNAs expressed in a certain cell type

41
Q

Construction of a genomic library

A
  • prepare DNA inserts with blunt cutter and gel purify large fragments
  • insert DNA into BAC vector which has been cut with blunt cut RE
  • transform and culture
42
Q

Construction of cDNA library

A
  • isolate and collect mRNA
  • synthesise cDNA from mRNA via oligoT and reverse transcriptase
  • insert cDNA into bacterial plasmid vector
  • transform plasmids into bacteria and culture
  • isolate plasmid and purify
  • sequence DNA
43
Q

What does cDNA show

A

shows tissue specific expression

44
Q

what does genomic library show

A

intron and promoter info

45
Q

problem with PCR

A

you have to know enough about your target gene as you have to design primters

46
Q

reverse transcriptase PCR (cDNA)

A
  • anneal oligo-dT primer to mRNA with poly A tail
  • make cDNA VIA reverse transcriptase
  • digest away RNA with enzyme and make a DNA copy of cDNA
  • you can add restriction sites
  • make DNA with primers and linker connecting primers 5’ end and amplify
  • DSDNA made and can clone into vector
47
Q

what happens if you dont add linkers to DNA copy of cDNA

A

you will have a DNA pool

48
Q

Why sequence RNA

A
  • characterise complexity of genes expressed in a particular tissue
  • quantify expression levels of transcripts and compare samples
  • can discover novel genes, exons and splice isoforms
  • allows assembly of coding sequence or transcriptome
49
Q

illumina with cDNA

A

make mRNA to cDNA via reverse transcriptase

50
Q

Single cell RNA sequencing

A
  • shows you the cells within a tissue
  • input a single cell which can show expression variability and heterogeneity
51
Q

advantages of single cell RNA over bulk

A
  • can reveal new cell types
  • can identify new cell types and genes involved in disease
  • can reveal cells that are lost or damaged in disease process
  • can be used to test targeted effects of therapies
52
Q

How is polymorphism assesed

A

by measuring allele frequencies at individual loci

53
Q

how are markers chosen

A

based on how polymorphic the gene is

54
Q

restriction fragment length polymorphism

A

differences in DNA sequence at restriction sites recognised by restriction enzymes

55
Q

what are restriction fragment length polymorphisms caused by

A

base changes that remove sites or add sites

56
Q

Why have microsatellites and SNPs replaced RFLPS

A

they are based on PCR so they are faster,cheaper and automatable

57
Q

Microsatellites

A
  • small DNA sequence repeats
  • interspersed frequently throughout genome
  • highly polymorphic between individuals so is a good marker
  • high frequency rate of mutation
  • measured via fluorescence with sanger
58
Q

SNPS

A
  • single nucleotide variant
  • if there is a higher number of base changes among individuals with a disease this is informative
  • high throughput
  • less polymorphic than microsatellites
59
Q

Methods for sequencing markers

A
  • affymetrix
  • illumina SNP genotyping
60
Q

Affymetrix

A
  • DNA sheared and oligonucleotides are synthesised to glass slide
  • fluorescently tag DNA
  • detect fluorescence by laser
  • a perfect match will have high amounts of fluorescence
  • genotyping via hybridisation
61
Q

illumina SNP genotyping

A
  • genotyping via oligo extension
  • isolate genomic DNA and shear
  • anneal oligos adjacent to SNP
  • fluorescently label oligonucleotide
  • increased fluorescence when it anneals to SNP
62
Q

applications of DNA markers from genome

A

used to construct fine resolution genetic maps and population and genetic diversity studies

63
Q

applications of DNA markers linked to specific trait loci

A

identification of single genes affecting traits and multigenic trait analysis

64
Q

Ways to put DNA into cells

A
  • transformation
  • projectile gun
  • injection
  • virus
65
Q

Transformation

A

genetic alteration of a cell resulting from direct uptake and incorporation of exogenous genetic material from its surroundings through cell membrane

66
Q

Transfection

A

process of deliberately introducing naked or purified nucleic acids into eukaryotic cells

67
Q

Transduction

A

process by which foreign DNA is introduced into a cell by a virus or viral vector

68
Q

Transgene

A

a gene or genetic material which has been transferred naturally or by genetic engineering from one organism to another

69
Q

Genome editing

A

genetic engineering where DNA is inserted,deleted,modified or replaced in genome of a living organism

70
Q
A

Biosci 202 (4 decks)

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