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CMB2000 Flashcards

1
Q

stages of cloning

A
  • bioinformatics searching
  • design primers to include 2 restriction sites
  • PCR
  • create clean insert with appropriate ends
  • plasmid choice - treat with same restriction enzymes
  • ligase - to join inset and plasmid in MCS
  • transfer/transform into impotent E.coli
  • grown on selective media
  • pick right colonies PCR straight from colony/culture/plasmid
  • mini-prep
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2
Q

what is PCR

A
  • polymerase chain reaction
  • amplification of DNA
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3
Q

stages of PCR

A
  • starts with a single stranded piece of DNA
  • uses taq polymerase for repeated cycles
  • with each cycle there is an exponential increase in strands
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4
Q

what 2 things are needed for replication in eukaryotic cells

A
  • template DNA
  • polymerases
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5
Q

why PCR

A
  • sensitive - can amplify as little as one molecule of DNA
  • specific - can amplify a unique target sequence stringency depends on temperature and [mg2+]
  • cheap
  • rapid - results available in a few hours
  • robust - DNA is very stable can be amplified from old and degraded samples
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6
Q

what’s in the tube

A
  • template - double stranded DNA
  • 2 primers - to prime synthesis
  • polymerases - copies the template, extending from the 3’ end of primer
  • dNTPs - deoxyribonucleotide triphosphate
  • magnesium - co-factor for DNA polymerase enzymes
  • buffer - maintain pH and provide necessary salt
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7
Q

what are the 3 regions of tax polymerase

A
  • synthesis
  • proof reading
  • primer removal
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8
Q

what are the key parts of primer design

A
  • 2 primers - one for each strand
  • length 18-24 bases
  • 40-60% G/C content
  • start and end with 1-2 G/C pairs
    -melting temperature of 50-60 degrees
  • 3’ end must be complementary to the template DNA
  • primer pairs should not have complementary region
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9
Q

importance of magnesium

A
  • co-factor
  • a non-protein component of the reaction that’s needed to enable the activity of the catalysis
  • magnesium acts to enhance the enzymatic activity thereby supporting DNA application
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10
Q

what is the buffer used

A
  • optimal pH is 8-9.5
  • tri Hcl
  • potassium ions - promote annealing (may be replaced by ammonium sulphate, which destabilises base pairing bonds
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11
Q

names of the 3 stages in PCR

A
  • denaturation
  • annealing
  • elongation
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12
Q

what is the process of DNA synthesis

A
  • 1st cycle - synthesis of a strand of DNA in test tube
  • 2nd cycle - synthesis of two strands in a test tube
  • the rest - simultaneous synthesis of both strands
  • polymerase chain reaction
  • exponential amplification of DNA polymerase chain reaction
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13
Q

how do we detect PCR production

A
  • run products on agarose gel
  • use intercalating dye to stain DNA to determine size and yields
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14
Q

what can be identified by detection of PCR

A
  • molecular weight markers
  • PCR products
  • primers
  • template
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15
Q

why is PCR so important

A
  • good when DNA is scarce
  • manipulate DNA
  • detection of pathogens
  • diagnosis of genetic disease
  • detecting genetically modified material
  • biotechnology
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16
Q

examples of use of DNA

A
  • forensic analysis of DNA samples
  • manipulate DNA - genetic modification
  • knock out genes - study gene function
  • fuse host proteins with GFP
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17
Q

what are the stages of reverse transcriptase PCR

A
  • convert RNA to cDNA. use reverse transcriptase, retroviral enzymes that converts RNA to DNA
  • amplify DNA by PCR
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18
Q

what are the sources of RNA

A
  • gene expression (mRNA) - disease vs healthy/drug effects/environment changes
  • RNA virus infection levels
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19
Q

what are the ingredients in RTPCR

A
  • template (RNA)
  • primer
  • dNTPs
  • reverse transcriptase
  • buffer
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20
Q

features of endpoint PCR

A
  • cheap
  • semi-quantitative at best - band intensity
  • sequence, genotyping, cloning
  • see results at end, plateau
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21
Q

features of real time PCR

A
  • more expensive
  • quantity of PCR is proportional to amount of template
  • quantification of gene expression, microarray verification, quality control and assay validation, SNP genotyping, copy number variation, viral quantification, siRNA/RNAi experiments
  • measures at exponential phase - more precise
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22
Q

how do we include fluorescence

A
  • SYBR gree
  • binds to groove of dsDNA –> increases fluorescence
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23
Q

why do we need reference genes

A
  • constant level of expression - not affected by experimental factors
  • essential to support validity of qPCR results
  • confirms RNA extraction was good and efficient
  • supports conclusion of expression levels
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24
Q

examples of reference genes

A
  • beta actin
  • GAPDH
  • albumin
  • 18s rRNA
  • TATA sequencing binding protein
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25
Q

what’s in a PCR reaction tube

A
  • DNA template - the sequence to be amplified
  • primer (reverse and forward) - to start the synthesis of the new DNA strand
  • nucleotides (dATP, dCTP, dGTP, dTTP) - building blocks for the new DNA strand
  • taq polymerase - to catalyse the synthesis of the new DNA strand
  • buffer - to maintain optimal pH for synthesis
  • mgcl2 - essential for tax activity. concentrations of mg2+ ion effects stringency of primer binding
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26
Q

what are the common uses of PCR

A
  • genotyping the patient
  • genotyping the pathogen
  • phenotyping the disease
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27
Q

what can genotyping the patient be used for

A
  • diagnosis of genetic traits
  • detection of carrier of genetic traits
  • tissue matching
  • predicting the response to drugs (pharmacogenetics
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28
Q

what is HLA typing

A
  • the proteins encoded by HLAs are those on the outer part of the body cells that are unique to that person
  • any cell displaying that persons HLA type belongs to that person and therefore is not an invader
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29
Q

what can genotyping the pathogen be used for

A
  • diagnosis of species and strain of infecting pathogen
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30
Q

what can phenotyping the disease be used for

A
  • measuring disease progression
  • measuring disease severity
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31
Q

what are the 2 PCR techniques used in genotyping the patient

A
  • PCR-RFLP - restriction fragment polymorphism
  • ARMS-PCR - amplification refractory mutation system
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32
Q

what is an allele

A

any of the alternative forms of a gene that may occur at a given locus

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33
Q

what is a restriction enzyme

A

an enzyme that digests DNA at a highly specific site

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34
Q

steps of PCR-RFLP

A
  • identifies allelic variants based on presence/absence of a restriction site
  • amplify
  • cut PCR product with restriction enzyme R
  • size-fractionate by gel electrophoresis
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35
Q

what can be identified by restriction site (cutting PCR product)

A
  • if the RE site in neither allele - homozygous allele 1
  • if the RE site in both alleles - homozygous allele 2
  • if RE site in one allele - heterozygous
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36
Q

what can be identified by restriction site (gel electrophoresis)

A
  • if RE site in both products - homozygous for the disease allele
  • if RE site in neither products - homozygous for healthy allele
  • if RE site in one of the products - heterozygous
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37
Q

what is sorsbys fungus dystrophy

A
  • example of genotyping the patient
  • degenerative eye disease leading to blindness
  • mutation in TIMP3 gene introduces premature stop codon
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38
Q

what are the advantages of PCR-RFLP

A
  • cheap
  • easy design
  • applied to microindels and SNPs
  • simple resources
  • commonly used technique
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39
Q

what are the disadvantages of PCR-RFLP

A
  • only possible if the site contains a known RE site
  • some RE are expensive
  • only possible if a single nucleotide variation
  • hands on and time consuming
  • not suitable for high-throughput
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40
Q

importance of ARMS-PCR

A
  • detects allelic variants using allele specific primers
  • simple method for detecting any mutation involving single base changes or small detections
  • presence or absence of a PCR product is diagnostic for the presence or absence of the target allele
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41
Q

how can ARMS-PCR be used to diagnose cystic fibrosis

A
  • mutations of the CFTR gene leads to imbalances of cl- transport across plasma membrane
  • f508 mutation is the most common cause
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42
Q

RFLP VS ARMS (RFLP)

A
  • uses locus specific primers (will amplify all variants of the chosen DNA sequence
  • relies on the presence or absence of a restriction site to distinguish between variants
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43
Q

RFLP VS ARMS (ARMS)

A
  • uses allele specific primers
  • relies on the stringency of the PCR to distinguish between alleles
  • alternative is tetra primer ARMS-PCR, which uses addition non-allele specific primers
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44
Q

what can genotyping the pathogen be used for

A
  • identifying the species and strain of an infectious pathogen by isolating a specific gene/piece of DNA
  • this information will influence: patient management and infection control measures
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45
Q

PCR vs conventional microbial diagnosis (PCR)

A
  • sensitive - can detect single copy of genome
  • specific - can identify species and strain
  • sensitivity means no need for culture
  • PCR takes a few hours
  • detects DNA/RNA therefore not dependent on immune response
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46
Q

PCR vs conventional microbial diagnosis (conventional)

A
  • requires high levels of infecting organisms
  • often difficult to distinguish different species
  • electron microscopy required to visualise virus
  • some organisms cannot be cultured
  • culture can take weeks
  • hard to be strain specific
  • pathogen may not elicit a strong antibody response
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47
Q

how can genotyping the pathogen be used to identify tuberculosis

A
  • conclusive diagnosis depends on detection of M.tuberculosis in sputum
  • previously depended on microscopy and culture
  • now can achieve same day diagnosis using PCR
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48
Q

phenotyping the disease - what is quantitative PCR

A
  • quantitative PCR measures the abundance of DNA or RNA in a clinical sample for example
  • to measure the level of infectious pathogen in a sample
  • to measure the level of expression of a gene
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49
Q

how can DNA and cDNA be accurately quantified by real time qPCR

A
  • PCR product is measured as it is produced e.g. by incorporating fluorescent marker into the product
  • the cycle number at which the fluorescence reaches a threshold value is measured
  • the lower the ct value, the greater the quantity of DNA/cDNA in the starting template
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50
Q

how can qPCR be used in HIV

A
  • measurement of the HIV viral load by quantitative RT-PCR
  • useful for monitoring progress of disease and response to drug therapy
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51
Q

why do we isolate DNA

A
  • for genetic manipulations
  • for DNA analysis e.g. scientific, medical, forensics, ecological, archeological
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52
Q

what are the steps of DNA isolation

A
  • cell lysis
  • DNA purification from the cell extract
  • concentrate DNA
  • measurement of DNA purity and concentration
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53
Q

what is cell lysis

A
  • release the DNA from the cell by breaking down the cell membrane
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54
Q

biological method of cell lysis

A
  • uses enzymes to disrupt cell membranes. difference enzymes for different cells
  • plants - cellulase
  • bacteria - lysozyme
  • eukaryotic cells - sappanin
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55
Q

physical methods of cell lysis

A
  • osmotic pressure - excess water moves into the cell when cells are placed in hypotonic solution
  • freeze-thaw - repeated cycles of freezing and thawing ruptures cell membrane through ice crystal formation
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56
Q

mechanical methods of cell lysis

A
  • grinding e.g. pestel and mortar, bead mill, vortex
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57
Q

what do we not want in our sample of DNA

A
  • protein
  • ribosomes
  • mtDNA
  • lipid
  • plasmid
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58
Q

how is DNA purified using phenol chloroform extraction

A
  • lysed cells or tissues are mixed with equal volume of phenol:chloroform mixture
  • centrifugation - 2 distinct phases as the phenol:chloroform mixture doesn’t mix with water
  • DNA concentration - 0.3M sodium acetate and 2.5 volume ethanol can be used to precipitate DNA from salt and sugar to concentrate it
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59
Q

how can DNA be purified using commercial kits

A
  • column contains a silica membrane that binds DNA in the presence of a high concentration of salt
  • impurities such as salts are washed away
  • a low salt buffer such as water or 10 mM try-cl
  • pH 8.5 is used to release DNA from the membrane and collect out
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60
Q

ads of using commercial kits

A
  • not hazardous
  • less time consuming
  • results in purer DNA then phenol:chloroform extraction
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61
Q

dis of using commercial kits

A
  • expensive
  • small volume
  • membrane can only bind a set amount of DNA
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62
Q

steps of silica binding DNA

A
  • lyse cells
  • add high salt buffer
  • wash with ethanol buffer
  • elute with very low salt
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63
Q

how can we measure the quantity and quality of DNA

A
  • UV absorbance
  • fluorescence dyes
  • agarose gel electrophoresis
  • capillary electrophoresis
  • diphenylamine method
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64
Q

why is it important to measure the quantity and quality of DNA

A
  • efficient extraction = efficient science
  • without a good starting point you will never have good output
  • genomic testing would be impossible
    -PCR/cloning wouldn’t work
  • forensic science would be unreliable
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65
Q

what are restriction endonuclease

A
  • enzymes produced by bacteria to protect against viral DNA infection
  • restriction enzymes cut the foreign DNA
  • restriction - act on specific DNA sequences
  • endonuclease - cleave the phosphodiester bond within a polypeptide
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66
Q

why use restriction endonuclease

A
  • to make recombinant DNA molecules
  • to cut DNA into defined fragments (DNA fingerprinting and mutation analysis)
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67
Q

how do restriction enzymes cut DNA

A
  • make one cut in each of the sugar phosphate backbones of the double helix at their recognition site in the presence of mg2+
  • hydrolyse the phosphate group
  • cut ends have a 5’ phosphate
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68
Q

what makes up different types of restriction endonuclease

A
  • cut at specific sequences
  • different Res - different cutting sites
  • some are blunt ends, some sticky
  • recognition sites for restriction enzymes are often palindromic
  • 5’ GAATTC 3’
  • 3’ CTTAAG 5’
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69
Q

what can restriction endonuclease be used for

A
  • to make recombinant DNA molecules
  • to cute DNA into defined fragments
70
Q

what is star activity

A
  • relaxation or alteration of specificity - chemical/drug intervention
  • when reaction conditions differ slightly from the optimum for the enzymes
71
Q

why might reaction conditions change from the optimum

A
  • low ionic strength
  • high pH
  • high glycerol concentrations
  • presence of mg2+
72
Q

what are the principles of agarose gel electrophoresis

A
  • polymerised agarose is porous, allowing the movement of DNA
  • large towards negative end, travel towards the positive end
  • samples enter the gel and migrate according to charge, size and shape
73
Q

implication of DNA being negatively charged

A
  • migrates to positive electrode
  • smaller molecules move more easily through gel than larger molecules
  • because of the sugar-phosphate backbone migrates to anode
74
Q

how can we see the movement of DNA in gel electrophoresis

A
  • visualise with intercalating dyes e.g. Nancy red
75
Q

how can we determine the size of the DNA fragments

A
  • compare size of product of interest with DNA ladder
  • plot a graph log10 and mm band migrated down the gel (use MW ladder to create a standard curve)
  • determine size of your products
  • graph of log DNA size of the markers by distance migrated creating. straight line
  • can then plot the distance the product of interest has travelled on the line and estimate the DNA size
76
Q

what is genome editing

A
  • is a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using nucleases
  • enabling specific targeting of sequences within the genome without impacting the rest of the genome sequence
  • potential to cure genetic diseases in a patient specific manner
77
Q

what is CRISPR

A
  • clustered regulatory interspaced short palindromic repeats
    -adaptive immune system of prokaryotes
  • the complex cleaves invading DNA to prevent re-infection by viruses
78
Q

what are the 3 components systems that make up CRISPR

A
  • cas9 - protein component
  • crRNA - RNA component
  • tracrRNA - RNA component
79
Q

how can CRISPR act as an adaptive immune regulators

A
  • against invading DNA/RNA
  • invading DNA recognised and cut by cas1-cas2 protein complexes into fragment - protospacer
  • protospacers integrated into CRISPR locus located in the bacterial genome
  • upon viral reinfection, transcription of the protospacers to RNA is activated which bind to cas9
  • cas9/RNA duplex is recruited to complementary sequence on the invading strand of DNA
  • cas9 cutes DNA strands creating a double strand break to prevent infection
80
Q

what is the structure of the CRISPR locus

A
  • transactivating RNA
  • operon of cas genes encoding cas protein components
  • identical repeat array
  • spacer of invading DNA
  • the complex formed between the trans activating RNA and the protospacer is the guide RNA which enables selective binding of cas9 to invading DNA sequences
81
Q

role of the cas operon

A
  • cas operon encodes cas proteins required for DNA cleavage
82
Q

role of tracrRNA:crRNA form the guide RNA

A
  • the duplex formed between the two RNA species is known as the guide RNA
83
Q

how do protospacers adjacent motifs enable cas9-mediated DNA cleavage

A
  • 2-8 base pair sequence 3-4 base pairs downstream of the cut site
  • cas9 will not cut invading DNA without a PAM site irrespective of cas/gRNA binding
  • PAM sequences are not present in the CRISPR locus
  • prevents bacterial CRIPSR locus being targeted by cas proteins
84
Q

what two components make up gRNA in bacteria

A
  • tracrRNA
  • crRNA
  • linker loop used to link the two
85
Q

how has CRISPR/cas9 been engineered for biomedical studies

A
  • deposition of the cas9 complex at a desired locus of the genome will enable site specific cleavage through nuclease activity
  • the repair of the DNA break by endogenous DNA repair pathways enables specific genomic edits to be introduced
86
Q

why is correct gRNA design essential for selective CRISPR positioning and DNA cleavage

A
  • the gRNA should contain a photo-spacer sequence upstream of the PAM site
  • gRNAs should be selective to a single genome locus to avoid off target effects
87
Q

what is cellular DNA repair needed for

A
  • the pathways are critical for desired CRISPR-mediated DNA editing
88
Q

repair mechanisms for double strand breaks

A
  • homology directed repair
  • non-homologous end joining
89
Q

how is genetic drift generated

A
  • when cas9 cleaves DNA, a double strand break is introduced
  • homology directed repair or non-homologous end joining function down stream to repair DNA
  • this will create the desired genetic drugs
90
Q

why is DNA repairs by NHEJ error prone

A
  • introduces insertions or deletions into DNA
  • impacts gene function
91
Q

how does HDR enable precise DNA repair

A
  • DNA is precisely repaired using sister chromatid during 5 phase of the cell cycle
92
Q

how does CRISPR mediate gene knockout via NHEK

A
  • target cas9:gRNA complex to gene of interest
  • DSB introduced
  • cell repairs the break via NHEJ (error prone)
  • indels introduced generating a frameshift (premature stop codons introduced)
  • normal gene product not expression
93
Q

how does CRISPR mediate gene knock in via HDR

A
  • DBS introduced by cas9:gRNA complex
  • introduce a template that the cell will use to repair the DSB through HDR
  • HDR template requires homology arms on either side of the point of mutation inset
  • PAM sites are removed from HR template to prevent re-targeting of region
94
Q

why is androgen receptor signalling a key driver of prostate cancer

A
  • prostate cancer progression is largely driven by androgen receptor signalling
  • current treatment aims to inactivate AR by blocking ligand binding
95
Q

what are the two CRISPR based studies

A
  • to generate a cas9-expressing prostate cancer cell line to knockout AR
  • to create a modified prostate cancer cell line to study function of aberrant forms of the androgen receptor - knock in strategy
96
Q

how is a cas9 expressing prostate cancer cell line developed

A
  • generate prostate cancer cell line expressing cas9
  • to knock out the AR gene
  • AR gene locus was targeted by CRSIPR to validate activity of cas9
97
Q

how are androgen receptor variants studied in prostate cancer

A
  • alternative splicing is principally involved in the generation of AR-VS in response to AR targeting agents, such as enzalutamide
  • expression of AR-VS is elevated in advanced disease
98
Q

how is enzalutamide activated

A
  • loss of the AR LBD creates constitutively active transcription factors that are refractory to enzalutamide
99
Q

how can using a CRISPR knock in strategy create an AR-V only expressing cell line

A
  • gRNA designed to exon 5 of the AR
  • template with point mutation - stop codon
  • will block synthesis of full length AR
100
Q

what should be considered in cell therapy

A
  • efficacy of delivery
  • regulatory guidelines
  • mosaicism
  • gremlin vs somatic
  • immunogenicity
  • specificity - off target affections
101
Q

how is CRISPR delivered ex-vivo

A
  • remove cells from the patients/donor
  • edit genome
  • screen/expand cell popultation
  • engraft cells back into patients
102
Q

how is CRISPR delivered in vivo

A
  • package CRISPR/cas in a delivery vehicle
  • deliver to patient
103
Q

how did successful CRISPR editing of CCR5 in vivo confer HIV-1 resistance

A
  • long term CCR5 disruption was observed
  • CCR5 disrupted HSCs were able to reconstitute a functional immune system
  • viral titre reduction and increase in CD4+ T cells demonstrated HIV resistance
104
Q

why sequence genomes

A
  • blue print to life e.g. all genes, regulatory sequences, higher order structure, chromosome maintenance, comparative searches
105
Q

what are the issues with sequence genomes

A
  • cost and scale
  • technology
106
Q

how is an organisms genomic sequence obtained

A
  • obtain the organisms genomic DNA
  • break the DNA into small fragments
  • obtain the DNA sequence from all the fragments
  • search for overlaps to identify between the DNA sequences of the different fragments to reconstruct the genome sequence
  • fill in any missing gaps in the genome sequence
107
Q

what is the importance of model organisms

A
  • small genome - value for money
  • easy organisms to manipulate
  • provide information on fundamental biological processes
  • technology development
108
Q

what are the major issues identifying genes with genomes

A
  • how big is a valid open reading frame
  • identification of RNA splice sites
  • RNA analyses can help but depends on the expression of the gene
109
Q

why problems in gene identification emphasised by genome analysis in s.cerevisae

A
  • it is smaller than the human genome
  • genes are tightly packed with very little repetitive DNA
  • complete absence of RNA splicing to complicate gene identification
  • simple genetics can be used to analyse potential gene function
110
Q

what can be predicted from compute analyses of protein sequence

A
  • prediction of function - roles for model organisms
  • prediction of protein localisation
  • prediction of protein domains/modification
111
Q

what are the benefits of studies in model organism

A
  • functional characterisation of mutant proteins
  • understanding human genome variation
112
Q

what is an example of identification of functional characterisation of mutant proteins

A
  • analysis of predicted catalytic mutant Msh2 proteins from human colon cancer was confirmed by expression the proteins in yeast
113
Q

what did analyses of other mutant Msh2 proteins reveal

A
  • defect in critical protein-protein interaction
  • reduced steady state levels of Msh2
  • mutations affected the activity of the mismatch repair complex
114
Q

example of the importance of cellular localisation

A
  • cellular localisation of the schizosaccharomyces pome cell cycle regulatory Yox1
  • analyse the protein using the PSORTII programmed
  • yox1 localises to the nucleus in all stages of the cell cycle in schizosaccharomyces bombed
115
Q

example of the importance of prediction of protein domains/modifications

A
  • regulation of the schizosaccharomyces pome cell cycle regulator yox1
  • to understand function/regulator of the protein use a range of programmes to predict potential functional domains and protein modification sites
116
Q

how can yox1 be analysed using domains

A
  • use various programmes including BLAST to identify conserved domains
117
Q

what was discovered in the domain analysis of yox1

A

homeodomain - DNA binding domains involved in the transcriptional regulatory of key eukaryotic developmental processes; may bind to DNA as monomers or as homo and/or heterodimers, in a sequence-specific manner

118
Q

what is phosphorylation site analyses of yox1

A
  • search for potential serine/threonine/tyrosine phosphorylation sites
  • for example using NetPhos programmed
119
Q

what approaches can be taken to undergo phosphorylation sites analyses of yox1

A
  • investigate protein phosphorylation in vivo and if so whether the identified threonine residue is important
  • test genetically and biochemically the potential role of the programme predicted kinase
  • mutate the threonine residue to a glutamic acid, an aspartic acid or an alanine residue to investigate role of phosphorylation
120
Q

what are the uses of genome sequence within an organism

A
  • identification of regulatory sequences
  • characterisation of protein families
121
Q

what are regulatory sequences

A
  • identify all promoters containing a transcription factor binding site
122
Q

how are protein families characterised

A
  • kinases have well characterised homology within catalytic domains
  • genome analysis allows inference of the function of uncharacterised kinases by family studies
  • genome analysis allows identification of conserved and organism-specific families of protein kinases
123
Q

what are the different types of study for genomic experiments

A
  • protein/DNA interactions
  • DNA methylation
  • gene expression
  • protein-protein interaction
  • loss of function
124
Q

what are microarrays

A
  • many functional genomics experiments depended on microarrays
  • measurement of hybridisation
  • sample to probes on array
  • range of samples and probes for different experiment types
125
Q

steps of microarray

A
  • extract RNA
  • reverse transcription
  • cDNA
  • in vitro transcription
  • biotin labelled cRNA
  • random fragmentation
  • fragmented biotin labelled cRNA
  • cRNA hybridisation to gene chip
  • wash away non-specific binders and stain with streptavidin-phycoerythrin
  • scan array with laser, detect fluorescence with CCD, read image into computer
126
Q

how did we go from microarrays to sequencing

A
  • direct sequencing can substitute for hybridisation
  • give greater resolution and accuracy
  • issues were cost and throughput
  • development in sequencing have addressed both
127
Q

what is high-throughput sequencing

A
  • refers to range of technologies
  • competition - driven down cost and increased throughput
  • illumina sequencing dominates
128
Q

what is illumina sequencing

A
  • fragments of DNA bound to solid surface
  • binding enables by special sequences ligated to fragments
  • solid-phase bridge PCR forms clonal clusters
  • sequencing proceeds in cycles
  • modified nucleotides with fluorescent group which blocks extension
  • reversible termination allows sequencing to proceed to next cycle
129
Q

what is RNA sequencing

A
  • use of high-throughput sequencing technologies to get information about a samples RNA content
  • mRNA are converted to cDNA
  • cDNA used for sequencing library generation
  • allows quantification, profiling and discovery of RNA
130
Q

how does RNA sequencing work

A
  • polyA selection
  • fragmentation
  • random printing
  • first and second strand cDNA synthesis
  • end repair, phosphorylation and A tailing
  • adapter ligation, PCR amplification and sequencing
131
Q

how does amount of RNA effect RNA sequencing

A
  • sequences in final library are derived from RNA population in sample
  • presence is proportional to original sample - more abundant RNA species will be present more frequently in library
  • random priming is attempt to remove bias
  • actual randomness debatable
132
Q

what are the consideration for RNA sequencing

A
  • big data sets require expert processing
  • expression data can be noisy
  • easy for confounding factors to dominate
  • good practise same as for any statistical approach
133
Q

what are the other functional genomics approaches

A
  • many assays of aspects of nucleic acid biochemistry based on sequencing
  • general idea is to enrich specific molecules or regions of molecules and sequence
  • then analyse to show what you’ve enriched
134
Q

what is ChIP-sequencing

A
  • cross link proteins to DNA
  • isolate DNA and shear
  • immunoprecipitate protein of interest
  • reverse cross-linking
  • purify DNA
  • sequence
135
Q

what is ATAC-sequencing

A
  • assay for transposes accessible chromatin
  • similar to older DNAse-sequences
  • relies on transposase Tn5
  • adapter ligated fragments isolated, amplified and sequenced
136
Q

what are the feature of transposase Tn5

A
  • high activity transposase
  • highly efficient cutting of exposed DNA
  • ligation of adapters to ends
137
Q

what is bisulphite sequencing

A
  • bisulphite treatment is used to determine the methylation state of DNA
  • methylated cytosine protected from deamination
  • unmethylated cytosine converted to thymine
138
Q

what is the importance of reduced representation BS-seq

A
  • RRBS utilises Mspl restriction enzyme to enrich for CpGs
  • results in fragments which begin/end with CpG
139
Q

what are the key point of functional genomics and bioinformatics

A
  • high through-put sequencing produces large amount to data
  • data can be noisy and complex
  • computational approaches needed to make most of data
140
Q

what are the main examples of model organisms

A
  • mouse - mus musculus
  • clawed frog - xenopus sp.
  • zebrafish - danio rerio
  • fruit fly - drosophila melanogaster
  • nematode worm - caenorhabditis elegans
141
Q

what are the key point of mice for model organisms

A
  • mice are an extremely well characterised model organism
  • we share most of our genes with mice
  • mice have a short lifecycle - useful for rapid breeding of new stains
  • however, they are relatively expensive to keep
142
Q

what are the 3 licenses required for animal experiments

A
  • personal license for the researcher
  • project license for the study
  • establishment licence for the place where the study is carried out
143
Q

why aren’t in vitro models commonly used

A
  • not representative of a proteins role in a biological system like a body
  • different cell types interact with each other constantly within living tissues
  • these interactions are impossible to model outside of animals
144
Q

what are the different mouse models used to study human development and disease

A
  • transgenic mice
  • knock-out mice
  • knock-in mice
145
Q

what are the step in creating a standard transgenic mouse

A
  • gene is microinjected into the pro-nucleus of a fertilised mouse oocyte
  • injected oocytes are transferred to a pseudo-pregnant recipient mouse
  • all offspring are screened for expression of the trans gene by DNA analysis
146
Q

what are the stages in creating gene-target transgenic mice

A
  • an isogenic tranigen with a drug selection gene is introduced into embryonic stem cells
  • drug selection is used and surviving cells are screened for the correct integration of transgene
  • correctly targeted cells are microinjected in mouse blastocysts
  • blastocysts are transferred to pseudo-pregnant recipient mouse
  • chimeric offspring are identified and mated to test for gremlin transmission of trans gene
147
Q

what is required for simple vector construct (transgenic approach)

A
  • gene of interest
  • relevant promoter
  • 3’ protein tag for detection
  • poly A tail
148
Q

what are the benefits of transgenic mouse models

A

cheap
easy to make
wild type gene prod is still present - can express human genes in mice

149
Q

what are the drawbacks of transgenic mouse models

A
  • multiple founders are generated - need to characterise numerous mouse lines
  • can not control site of integration into genome
  • wild type gene product is still present
150
Q

drawbacks and benefits of gene targeting approach for knock out mice

A
  • precise
  • requires a complex vector and relies on homologous recombination between vector and host genome
151
Q

what are the stages in gene targeting screening

A
  • electroporation of targeting vector into ES cells
  • establish homologous recombinant ES cell closed by 1st screening, PCR analysis, southern blot analysis
  • production of chimeric embryos by aggregation method
  • transfer to recipient mice
  • obtaining chimeric mice. contribution of ES cells is estimated by coat colour
  • crossing chimeric mice with wild-type mice to obtain F1 heterozygous mice
152
Q

what is gene trapping

A
  • a high-throughput approach that is used to introduce insertion mutations across the genome in mouse embryonic stem cells
153
Q

what are the consequences of the insertion of a gene trap vector

A
  • disrupts gene function
  • reports gene expression
  • provides a convenient tag for the identification of the insertion site
154
Q

what is the international gene trap consortium

A
  • administers all publicly available gene trap cell lines
  • researchers can search and browse the IGTC database for cell lines of interest
155
Q

what is the relevance of genetrap for mouse phenotyping consortium

A
  • to provide targeted inactivation through recombination using FRT and loxP
156
Q

what certain genes inactivate gene expression

A
  • lacz - to localise gene expression
  • neo - for antibiotic selection
157
Q

what is the importance of FRT and loxP

A
  • FRT and loxP sites mediate site specific recombination through DNA recognition sites
158
Q

what is cre recombinase

A
  • is a tyrosine recombinase enzyme derived from p1 bacteriophage and catalyses site-specific recombination between two loxP sites
159
Q

what is flippase

A
  • is a tyrosine recombinase enzymes derived from the bakers yeast - saccharomyces cerevisae - and catalyses site specific recombination between two FRT sites
160
Q

what is the importance of loxP

A
  • loxP sites allow straight forward knock-outs to be generated
161
Q

what is the cre-lox system for conditional alleles

A
  • following manipulation the mouse has a targeted but flowed allele
  • a second mouse is transgenic for core recombinase expressed under the control of a tissue specific promoter
  • the two mice are crossed together to generate a mouse line that carries both alleles: floxed and cre
162
Q

what is the result of the crd-lox system for conditional alleles

A
  • tissue specific deletion of the floxed allele only in tissues where cre recombinase is expressed
163
Q

what are the benefits of knock out mouse models

A
  • gene traps available for virtually all genes from international mouse phenotyping consortium
  • can make conditional KO using cre recombinase
164
Q

what are the drawbacks of knock out mouse models

A
  • can stress or cross onto a different background
  • may not accurately model a known human disease –> loss of function vs dominant negative
165
Q

when is knock in technology commonly used

A
  • used to introduce a human mutation into the mouse genome
166
Q

what are the benefits of knock in mouse models

A
  • genetically relevant mouse model of a human disease
  • crisp/cas 9 has lowered cost and time
167
Q

what are the drawbacks of knock-in mouse models

A
  • traditionally time consuming and expensive
  • remaining loxP site may be a problem - might interfere with mRNA stability and/or expression
168
Q

what is CRISPR-cas9

A
  • CRISPR is a form bacterial adaptive immunity
  • it utilises short RNA molecules in concert with a DNA cutting enzyme to protect against viral infection
  • it has recently been repurposed by researched to edit DNA in vitro
169
Q

what are the examples of disease modelling using a variety of mouse transgenics

A
  • the genetic skeletal diseases
170
Q

what did the mouse model of a genetic skeletal disease

A
  • short limb dwarfism phenotype
  • mutant protein retained in ER of chromosomes
  • reduced cell proliferation
  • increased spatially dysregulated apoptosis
171
Q
A

CM2001 (13 decks)

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