Genetics MCQ

Investigating Gene Expression

True/False

  • SYBR Green binds to double stranded DNA
  • TAQMAN's strand displacement involves the use of an illuminator and a quencher which is removed using a 5'exonuclease
  • Taqman is a hydrolysis probe
  • Hybridisation probes of beacon and FRET have the same stem loop structure
  • FRET Probes 5' acceptor accepts red light and donates it to 3' fluorecsin
  • Often automated by 96 plate BIORAD cyclers , the process of QPCR can be used for genotyping , quantification of infectious agents and analysis of gene expression

True/False QPCR

  • Discrete peaks during the melting phase of QPCR indicate a good duplex , lower poorer TM if not
  • Large amounts of mRNA are studied as the reagents are cheap and large amounts of tissue easy to extract by lasers
  • Primers in RT-PCR have no DNA signal
  • Primers in RT PCR cross the exon-exon boundary
  • They must be primer-dimers

Controls in QPCR

  • Negative control (no DNA) exists to check for contamination
  • Positive control not needed (check reagents and primers work)
  • No RT control is checks for contaminating DNA
  • Negative control exists to check that original sample has Watson crick pairing
  • Positive control exists to check threshold value

Threshold Value

  • Set on curved part of graph (300 fL units)
  • Set on part of graph that may not appear linear
  • Threshold values allows discrete signals to be identified in amplification experiments
  • cT value is the cycle number when the fL crosses the threshold
  • Can plot a ct x concentration exponential graph

Northern Blotting and Scaling

  • GAPDLI
  • Cycloplatin
  • Amorpectin
  • RFGLO
  • Ribosomal RNA
  • Myosin mRNA
  • MHCI mRNA
  • Different copy number in different cells
  • Large copy number to easily measurable
  • Expression doesn't change

Antibodies

  • 2 Heavy + 2 light chains linked by ionic bonds
  • Both heavy and light chains have 1 variable domain and 2 constant domains around 110 amino acids long
  • Protein of around 12 amino acids used to generate polyclonal antibodies (+adjuvant)
  • Variable exon is created De Novo in B cells
  • VDJ rearrangement in light chains
  • Mouse spleen cells fused with plasma cell tumours (myelomas) to form hybridomas
  • Antobodies used to be labelled with Helium that would give you thyroid cancer
  • It is the preferred method now to directly label antibodies with fL
  • Dot Blot used to calculate concentration of proteins
  • NARBLE and BSA used to block non-specific interaction
  • Proteins can be boiled with SDS to remove charge
  • Proteins can be immobilised onto PVDF or nitroglucose
  • Horseradish peroxidase uses luciferon as a substrate
  • Immunoprecipitation used if 3 layer western isn't enough to visualise
  • Protein A and G have different specificities for different antibodies
  • Protein A and G can be linked to magnetic , agarose or ice cold columns
  • Formaldehyde used to cross link DNA and protein
  • Analysis of chIP done by QCPR and microarrays if sequence is known
  • Immunocytochemistry in cells uses enzymes as secondary antibody markers ?
  • Pulse-chase experiments used radoactive materials to see how long a protein is in use
  • GST Pulldown experiments can be done in vitro and in vivo
  • GST uses binding proteins as bait to 'fish' for truncated forms of binded proteins to search for binding locations
  • GST is complementary to pulse-chase experiments

RNA Interference

  • The main pathway of mRNA degradation is RNAi
  • 3UTR sequences decrease rate of deadenylation
  • Short lingering mRNA leads to little expression
  • RISC (+Template dsDNA and ATP) recognises specific site on the guide strand and cleaves them
  • DICER endonuclease will cut template for RISC with 3' overhangs
  • Cytoplasmic RNAse I is RISC
  • Dicer converts exogenous RNA and miRNA into small ssDNA
  • RISC complex consists of PAZ , PIWI , MID and N
  • Temporary Knockout of gene functions allows for mapping of gene functions
  • Barcode assays on shRNA to see what genes they target
  • miRNA is exogenous
  • miRNA is processed in nucleus by Drosha/Pasha complex (also hijacked by siRNA)
  • MiRNA is processed by DICER in cytoplasm
  • miRNA is processed by RISC outside of cell
  • miRNA produced by pol II
  • miRNA folded into stem loop (shRNA)
  • RNAi drugs may have cross reactivity
  • RNAi drugs need a lot of testing
  • IC50 = 50% degradtion of mRNA by NaOH
  • Low Conc of siRNA used due to amplification
  • Toll receptors that recognise dsRNA cause inflammatory response including apoptosis
  • Any sequence can be targeted by RNAi drugs
  • DICER may malfunction due to shape of dsRNA causing apoptosis
  • Accumulation of siRNA is cells allows for total gene shutdown
  • siRNA can be delivered to cells using + protein links that can be attatched to targeting MOTIF antibodies
  • Viruses and ribosomes good carriers of siRNA using anionic lipid core

Fluorescence technologies

  • Visible Spectrum of light ranges from 350nm-700nm
  • Stokes shift involves the absorption of long wavelengths and the emission of shorter ones
  • Less conjugated parts in a flourophore allow for higher wavelength absorption
  • Xanthene , Cyanine and Benzenene ( Pyrene derivatives)
  • Phallodin dye targets organelles
  • 5 to 6 filters in a selective modulator
  • ImmunofL must have fixed and paralysed samples
  • Shinomura cloned and expressed gfp in C.elegans
  • Tyrosine to histadine in gfp makes it brighter
  • Are cerulean , cyan , venus , citrate and yellow all variants of gfp?
  • Are plum , cherry , strawberry , blood and Martha all variants of dsRED
  • dsRED dimerized makes it double active causing activation of tyrKinase which kills cells
  • Blinking (using calculated fL half lives) can be used to image singular cells
  • brightness = molecular extinction x coefficient / 1000
  • Quantam yield = amount of fL left after binding
  • Bleaching time = fL lifetime
  • EGFP's stoke shift reduced to make it brighter (shaper filters used)
  • Cauliflower aptamer in RNA binds to DHRBI region causing fL
  • TALE proteins and CRISPIR-RNA bonds can be labelled with fL

Gene regulatory sequences and their binding proteins

  • Translational control is the most important step in gene regulation
  • Different cells have different amounts of regulatory proteins leading to different levels of production
  • EVE is the gene for segmentation in flies
  • Active 2 hours after fertilisation
  • 8 stripes of activity in the fertilised egg
  • stripes correlate to DNA diffusion
  • 7.4kb of regulatory sequence upstream and 13kb downstream of the EVE promoter and ORF
  • 30 different regulatory proteins
  • Can replace EVE ORF with a reporter gene (b-gal)
  • 480nt regulatory region
  • Make deletions via a RE map directed double digest and ligate
  • Digest with RE , add exonuclease III which digest 5' temini , Blund ends with SI nuclease
  • BAL31 digests 3' termini of exposed DNA duplex
  • Quick-change mutagenesis used for widespread nucleotide changes
  • Q5 uses non-overlapping oligonucleotides
  • Self ligation prevented using VPN1
  • Supershifts , antibody hybridisation and mass spec used to identify binding protein in gel mobility shifts
  • Affinity chromatography links regulatory sequences to an immobilised column
  • Affinity chromatography can be done in vivo
  • In vivo AC involves NLS tagging the protein to the nucleus (5' end of tag) and pulling out using streptavin/avidin/biotin system
  • Hunchback (1) + , Bicoid (5) +, Kruppel (3) - , Giant (3) -
  • Hunchback (1) - , Bicoid (5) - , Kruppel (3) + , Giant (3) +
  • + = 3' to 5'
  • += 5' to 3'

Studying gene expression

  • Human body has 37.2 trillion cells
  • Human body has 180 cell types
  • 2D electrophoresis involves isoelectric focusing
  • C.Elegans used as a model for expression due to the fact all 956 cells have been characterised
  • C.Elegans are limited by their fragility
  • Zygote cells split into P1 and AB cells
  • Evidence for selective genetics include the transplant of frog epithelial nucleus and cow epithelial oviduct nucleus into egg cells generating new young
  • Genes can be amplified
  • Chorion genes in flies encode cell membrane and are up regulated in cells in gut
  • 2 Chorion genes in flies are upregulated 16 to 64 times to generate structural protein for fly egg shells
  • Ribosomal RNA found in Xenopus frogs forms circular plasmid where it is replicated over 3000 times
  • major step in gene regulation is translational control
  • RNAseq analysis used to determine abundance of all RNA's in a cell
  • Enzymatic reporters have low stability but are reliable
  • GFP has 238 AA sequence
  • GFP has 273 AA sequence
  • B-Glucanidase also uses X-Gal as a substrate
  • Luciferin and luciferase isolated from angler fish
  • Photirus Pyah (Fish) and Renilla (Pansy) were the isolate organisms

Cloning And Manipulating Genes

Genetics Background Material

  • Eukaryotic cells have simultaneous transcription and translation
  • Prokaryotic cells have no nucleus
  • Eukaryotic transcription controlled by epigenetics
  • DNA wrapped around histone octomers
  • 2xH2A , 2xH2B , 2xH3 , 2xH4
  • 2xH2 , 2xH3 , 2xH4 , 2xH5
  • 7 Packing Ratio
  • 8 packing ratio
  • Every 160bp
  • Final packing Ratio 10(2)-10(4)
  • Primase synthesises DNA primer for lagging stand
  • Clamp controls leading strand replication
  • Mg2+ needed during DNA polymerisation to ensure correct breakdown of DNTP's
  • ADP released after DNA binding
  • rRNA takes up 80% of cell
  • mRNA takes up 15%
  • tRNA takes up 15% of cell
  • mRNA takes 3-5%
  • miRNA > 1%
  • Both prokaryotic and eukaryotic mRNA has a 5'Cap and a 3' Poly A Tail
  • Poly A tail on prokaryotic mRNA acts as a clock to facilitate degradation
  • Start Codon = Aug
  • Stop Codon = AAG , AGA , GAA
  • Stop Codon= UAG , UAA , UGA
  • Kavinksi Model is the premier model for ribosomal scanning
  • Kozaks model involves facilitated binding of 40s subunit after recognition of 5' Cap , complexing upon recognition of start codon
  • Initiation Requires
  • 7 Initiation Factors (eIF1)
  • ATP
  • 40S
  • Methionine
  • 60s bins to 40s initiation complex to form 80s initiation complex releasing GDP and Pi
  • Elongation requires .....
  • Elongation factors 1A/1B (regulation)
  • Elongation factors 2A/2B (Binding)
  • Elongation factors 1A/1B (Binding)
  • Elongation factor 2 (Translocation)
  • Termination requires stop codon and a release factor (eRF1 or 3)
  • Kinase will remove a 5' Phosphate

Gene Cloning

  • Heat Inactivation and gel purification needed at almost every step of cloning
  • PCR primers need.....
  • 30 NT long sequence
  • 5' Extra NT for good cleavage
  • End in G or C
  • Add sequences at 3' end
  • 55 Melting Temp
  • (2xAT + 4xGC)
  • Avoid primer/primer complementation
  • Cosmids have cos sites for packaging into lambda
  • Phagemids have bacteriophage promoters
  • Ligase can repair double strand breaks in DNA backbone
  • PvuI ( Genus , species ,strain)
  • Cold shock as well as CaCl2 and RbCl2 can be used to disrupt cell membrane
  • DNA will move towards cathode during gel electrophoresis as phosphates make cell positive
  • Linear Migration is proportional to log10 size
  • Ethidium Bromide and Sybr green used to visualise DNA
  • Taq polymerase contains a 5' to 3' flap exonuclease
  • Kod/Pfu polymerase conatins a knelow fragment
  • Pol I has 5' to 3' repair
  • T7 and SP6 bind to very specific promoters allowing for directed activity
  • Exonucleases include Bal52 , Exonuclease II and DNA Polymerase Exonuclease
  • Exonuclease III will digest 5' end in absence of DNTp's creating ss
  • Exonuclease III used to create blunt ends in presence of DNTP's

Cloning and sequencing

  • Plasmids can range from 1kb in size to 100kb
  • Some are as large as 500,000 kb
  • Polylinker for Lac Z complementation is in lac Z region
  • pET vectors have NCO1 and HindIII sites with start codons for insertion of fusion tags
  • His Tag purification via steptavidin column
  • Insert range of plasmids ranges from 5-10kb
  • Host modifications for genetic engineering include ..
  • Additional restriction modification for more modification
  • Knocked out homologous recombination for increased recombinant genome stability (Delete HSDR genes)
  • Add genes for central metabolism so they can survive in harsh environments
  • MACS and HACS used to create genome libraries
  • YACS and BACS used in somatic gene therapy
  • Chromosome 47 used to introduce genes

Cloning and sequencing using bacteriophage

  • Lambda genome is around 49kb
  • ssDNA with cohesive ends so can form circular or concatemer molecule
  • Lysogens resist reinfection from lambda
  • Removed CL gene allows for supression of lytic cycle
  • Original genome can only back 2kb of genetic material with cos sites
  • central region responsible for lytic activity can be removed
  • Removed using a lambda based cloning vector gt10
  • Replacement vector has a 22kb upper limit for insertion
  • Human genome 30.2 billion kb
  • Any molecule with cos sites 37-50kb across can be packaged
  • High conc of DNA used in replacement vectors to prevent self ligation
  • M13 has a 6.4kb ss Genome
  • M13 has a 16.3kb Ds genome
  • Will introduce genetic material to any cell
  • Will introduce genetic material to any cell with a F Plasmid containing cell (F pilus)
  • M13 genome becomes SS upon insertion into F+ cells
  • M13 infected cells grow extra fast
  • M13 ss can be isolated from supernantat
  • M13 ds can be isolated from centrifuged pellet cells
  • M13 has excellent transformation efficiency
  • Phage display involves the insertion of protein genes into the coat protein region of the M13 genome
  • Biopanning involves washing a plate of proteins with you phage display particles to look for specific interactions
  • After elution of binded phage , cells are infected with the phage to amplify the phage . Then it's done

Sequencing and in silico cloning

  • Not one clone for each gene when cut with Sau3a
  • 2x10(6) phage needed instead of calculated 200,000 (2x10(9) divided by 15)
  • Overlapping clones cannot be identified by molecular hybridisation
  • Genetic libraries can be used for genetic complementation tests
  • Illumina sequencing (2008) uses long reads
  • Future sequencing methods such as PacBio and Ocford Nanopore use longer reads
  • 2'3'-dideoxtnucleotides have no hydroxyl at 5' ends
  • original sanger sequencing method now obsolete
  • can be used so sequence plasmid inserts , primer extension analysis , footprinting studies
  • Modern sanger uses the knelow fragment of Pol I
  • Modern standard uses dsDNA
  • detection in modern methodology uses laser directed capillary electrophoresis
  • Radioactive labels for the DNA are p32 , s35 and p34
  • Primer walking , like sanger sequencing , is an outdated and unneeded methodology
  • Increasing sequencing time will be of great benefit to medicine and is the challenge of the acheon-x-prize
  • Illumina sequencing ....
  • Sequencing by synthesis using dyes as indicators
  • 5' Phosphate is blocked
  • fL labelled in blocked state for detection
  • Labile bond removed to remove fl and restore 3'OH
  • bridging amplification occurs
  • Up to 200million samples can be analysed at once
  • Limited by size of DNA
  • Peptide mass fingerprinting (part of in silico cloning) involves MALDI-TOF and ESI-TOF and works by breaking up proteins and analysing the samples using mass spec
  • TBLASTN can sequence DNA in all 6 possible reading frames to compare with known sequences to determine mRNA code and structure

Nucleic Acid Hybridisation

  • DNA is analysed in a southern blot
  • Tm = temperature at which all DNA is fully degraded
  • DNA can be degraded using NaOH
  • AT triple bond raises TM
  • High Salt concentration stabilises hybrids (Higher TM)
  • Formahide stabilises hybrids (higher TM)
  • RNA duplex less stable than DNA duplex
  • High salt and low temperature allows for heterozygous hybridisation
  • Random Hexamer involves using random complementary 6nt OLGNT as primers , alongside labelled DNTP's and using Knelow Fragment Enzymes
  • Nick Labelling invovles use of BAL 31 , to create nicks where alpha labeled DNTP's are added
  • Label is usually 32-P
  • Can add phage promoters to probe fragment vector to create riboprobes
  • Use T4/T7 3' to 5' exonuclease activity to digest 3' ends
  • Alpha 32-P used for end labelling
  • Can label 5' ends using T4 polynucleotide kinase and alkaline phosphatase (Y-P32)
  • Simmilar process used in EMDA
  • Oligonucleotides have 5' phosphates when synthesized
  • Non Radioactive labels include biotin and digoxigen
  • Usually incorparated with dUTP (DNA) or UTP (RNA)
  • Biotin-dUTP will replace a base
  • Avidin or coccodin are reporter proteins for biotin
  • Digoxigen uses DIG antibodies for detection
  • HCL breaks phosphate backbone of DNA randomly
  • Treating ssDNA with NaOH makes it double stranded
  • Alkaline phosphatase reacts with 4-nitrophenyl phosphate to create a yellow product
  • Horseradish peroxidase reacts with luciferin to form dianimobenzidine (light)
  • In Situ hybridisation involves the preservation and immobilisation of the cell sample using Parafin and Formalin
  • Proteinase K or Pronase permalises cells for enzyme entry in in situ hydridisation

Mutagenesis

  • Excellent strategy for creating new proteins
  • Easy to change multiple residues at once in proteins
  • Most common role is to understand AA roles in proteins
  • Form heteroduplex , synthesize complementary strand , digest original
  • M13 ssDNA used for early experiments
  • M13 useful as it has no mismatch repair
  • Modern approaches use Pol 1
  • MutL and Mut S strains of E.Coli used
  • WT stand destroyes after use using DPNI (via methylation)
  • XL10 silver cells used for mutated DNA transformation
  • Denature plasmid , anneal primers , extend , denature , anneal , digest WT
  • Saturation mutagenesis used to determine if AA is optimal
  • Every single AA made into Alanine standard independently
  • Oligotrimer synthesis used to account for degenerate code
  • Deletion mutagenesis limited around 60NT
  • Insertion mutagenesis not limited
  • Deep knowledge of protein needed to chage AA's using site directed mutagenesis
  • Directed evolution involves random mutations and selecting for improvements
  • can't improve binding properties (alter KM)
  • can enhance Kcat (catalytic properties)
  • Random mutations introduced by error prone PCR and DNA shuffling
  • Only one evolution cycle is needed

Expression Systems I

  • In Vitro/prokaryotic and yeast QUALITY
  • Fungal cells can be used for most types of proteins
  • Insect and mamallian hosts chosen due to PTM and folding
  • Fusion tags are independant of host
  • Fusion tags don;t have to be added in frame of N or C terminal
  • N terminus addition requires removal of stop codon
  • N terminus addition requires removal of ATG
  • Frame tags used for affinity chromatography
  • Maltose FP : Amylose (EDTA to strip)
  • hexa-histidine : streptactin
  • GST : Glutathione
  • Strept II tag : sterptavidin
  • Protease cleavage sites in TAG's can be used to elute proteins from column
  • Enzyme's are tagged to be removed themselves
  • Ligand wash to remove non-bound proteins
  • T7 promoter systems found in DE3 cells
  • IPTG only lifts lac repressor for T7 Pol in genome
  • T7 pol binds to T7 promoter on plasmid
  • Tet-on system involves binding of (transactivator protein) rGTA to (tetracyline responsible element) TRE in the presence of tetracycline leading to transcription
  • Only 1 vector needed
  • Tet-off system is used in functional experiments to see roles of genes in disease
  • Baculovirus used to deliver cDNA into cells
  • Army worm lung cells used as standard infectants
  • Constitutive expression driven by inducible promoter
  • Constitutive expression driven by constantly active promoter
  • Inducible promoters work by binding of transactivators and allow for creation of large amounts of toxic products
  • Transient expression leads to long and hence high expression (conserved during mitosis)
  • Stable expression occurs via integration into host genome
  • eukaryitic cells grown as monolayers or in spinner flasks
  • Lipofection involves the cation lipid covering of DNA for insertion into cells
  • Complex with Calcium ?
  • Constitutive or inducible expression based on phage promoters
  • Include Sv40 ,CMV,MMTV
  • Trasnfectants can be selected via blue/white selection
  • T-Rex system is ampicillin inducable
  • Insect ecdysome is hormone inducible
  • MMTV is hormone inducible - glucacorticoid
  • CMV-Lac I repressor - metabolite inducible - IPTG
  • Allolactose from lactose metabolism induces lac operon
  • Amplification of rne genes leads to more RNAse increasing production of mRNA