1
Q
Why is PCR determination of Homo or heterozygous important?
A
Because most heterozygous individuals do not produce a unique phenotype so there is no easy way to differentiate them from homozygous individuals
2
Q
What are the target genes for our CRISPR construct project?
A
Genes related to auxin synthesis,transport,receptors,alterations in gene function triggered by auxin, and biosynthesis pathways
3
Q
Describe the steps in SLIC cloning
A
1)Additional 15bp are added to the 5’end that are identical to plasmid ends used during PCR
2)T4 DNA polymerase digests the insert and the vector along the 3’ end in the absence of DNTPs
3)DCTP stops digestion at the next G in the sequence
4)Vector and PCR product are annealed at room temperature
5)Construct is taken up by e.coli
6)E.coli fills remaining gaps in the strands and ligates them together
4
Q
What changes were made to sequencing from Sanger to NGS
A
1)Removal of the cloning in plasmid steps
2)Development of parallel data collection methods
3)Computer assembly
5
Q
Stable vs transient expression
A
Stable expression-Gene is integrated into the host chromosome and can be maintained in culture
Transient expression- Gene will eventually be degraded after the expressed phenotype is recorded
6
Q
Site specific recombination
A
Short specific sequences that are required for recombination and are the only sites which recombination occurs at
7
Q
How is huntingtons treated by genetic engineering?
A
RNAi knockdown of the HTT gene by AAV5 vector injected into the brain
8
Q
How is fluoresence used to measure the concentration of DNA during RT-PCR
A
After extension is complete our SSDNA is now DSDNA which allows SYBER to bind to the minor groove and after excitation of the SYBER green fluorescence will occur proportionally to the amount of DNA present in the reaction.
9
Q
Transcriptional vs Translational fusion
A
1)In transcriptional fusion the promoter is fused directly to the gene, is uses the native regulators and it’s own stop codon for the gene
2)In translational fusion the gene is inserted between the the ORF of the gene,using the GOI promoter, start,and stop codon
10
Q
How is gene therapy done ex-vivo
A
1)Cells are removed from patient
2)Virus is altered so it cannot reproduce and GOI is inserted into the virus
3)Virus is mixed with cells from the patient
4)Cells of patient are genetically altered
5)Altered genes are returned to patient
6)Genetically altered cells produce desired protein or hormone
11
Q
Draw an inversion of direct repeats
A
When oriented in the same direction the XY portion gets deleted from the original fragment after A-B recombination
12
Q
What are the physical components of the Retro and Lenti viral vector?
A
1)Nucleic acid for integration
2)Reverse transcriptase
3)Viral capsule
4)Viral capsule spike proteins for entry
13
Q
Benefits of site specific recombination for cloning compared to ligation methods
A
1)Higher efficacy
2)Less skilled individual can do this
3)Wide range of vectors and able to use large inserts
4)Does not require adjustments to vectors using endonucleases
14
Q
What is Adrenoleukodystropy and how is it treated via gene therapy?
A
It is caused by long fatty acid chains in the tissues which degrades the myelin,adrenal coretex and leydig cells and is caused by a defective ABCD1 Gene
Treatment:Bone marrow cells are removed from patients and !lentiviral vector! encoding the ABCD1 gene is infused into the cell and put back into the patient
15
Q
How to perform a section hybridization give an example from lecture
A
1)Embryo is fixed so probes may enter the cell
2)Embryo is then embedded in wax and sectioned
3)Sections are placed onto a microscope slide and exposed to a radio isotope labelled probe
4)Slide is dipped in emulsion in a dark room
5)Emulsion develops and slide is examined under the scope
ex.Arabodopsis flower development cross sections
16
Q
What is transthyretin amyloidosis, how is it treated by genetic engineering
A
Caused by a mutated TTR protein that accumulates in the heart and nerves
CRISPR treatment: Cutting the gene leads to an indel leading to a frame shift which prevents the TTR protein from being produced preventing damage to heart and nerves
17
Q
Binary plasmid gene composition
A
1)Origin of replication for E.coli and A.Tumerfaciens
2)High copy # genes
3)Unique endonuclease cut sites for GOI
4)Left and right boarders recognized by the Vir proteins for excision and transfer into plant chromosome
5)Selectable marker for bacteria with the plasmid
6)Selectable marker for plant cells with the plasmid located in transfer region
18
Q
What are the 4 steps of Illumina seqeuncing
A
1)Preparation of the template to attach to the flow cell
2)Generation of clusters of identical fragments on glass by solid phase PCR
3)Sequencing by synthesis
4)Analysis by computer
19
Q
In situ hybridization
A
Uses a labelled antisense strand of mRNA to hybridize to sense mRNA,allowing the visualization of gene expression. It reveals the location of where the mRNA is present but not the cells that express the gene
20
Q
Transformation vs transfection vs transduction
A
Transformation- introduction of DNA into a bacterial cells
Transfection- Introduction of DNA into eukaryotic cells
Transduction- Introduction of DNA to prokaryotic or eukaryotic cells using a viral vector
21
Q
How is the agrobacterium used in genetic engineering?
A
By replacing the T-DNA region of opine and hormones genes with GOI and antibiotic resistance allowing for selection of transformed cells
22
Q
What the possible A+B fusion primers designs
A
1)ORF A 18 nucleotide right primer with 15nt overlap and 18nt ORF left primer without overlap
2)ORF b 18nt left primer with 15nt overlaps and ORFa 18nt right primer without over lap
3)ORFB 18nt left primer with 15nt overlap and ORF a 18nt right primer with 15nt over lap creating 30nt total overlap
23
Q
How to perform a DIG probe in situ hybridization visualization
A
1)Antisense mRNA is labelled with DIG and hybridizes to a sense strand
2)Using antibody alkaline phosphates it conjugates to the DIG probe
3)DIG alkphosphate will precipitate out and form a blue product labelling the area that has the protein
eg.FTZ gene stained with DIG
24
Q
How do we add sequences to DNA in a plasmid that we want to insert into another vector?
A
By adding restriction sites flanking our fwd and reverse primers we can create a linear product that includes restriction sites compatible with our new vector
25
How do we confirm that our reporter plasmid has the correct elements for visualization?
1)Use of fragments 1-5kb upstream of start codon as for most small genome organisms this is enough to ensure the regulatory elements
2)Compare the results with in situ hybridization
26
Protein therapy
The delivery of a missing protein that would otherwise be absent in an individual with a given illness
27
Gateway cloning method
1)AttB1/2 and AttP1/2 is recombined using the BP clonase mix which swaps the ORF of AttB with the suicide gene of AttP forming AttL1/2 regions
2)AttL1/2 vector is our entry vector and we can use the LR clonase mix to insert it into our destination vector AttR1/R2 which contains a suicide gene
3)AttR1/R2 can now be cloned!
28
Type 2s restriction enzymes
Generate a cut outside of the recognition sequence with the positions written as (X/Y) where X refers to number of spots past the sequence on the top strand and Y the number of spots past position on the bottom strand
or (X,Y) when the cut happens upstream to the seqeunce
29
How does a lower or high CT value affect the graphs position
The lower CT value will shift the graph left and the higher CT value shifts the graph right
30
What are the steps in preparing the template to attach to the flow cell
1)Randomly break the DNA into short fragments
2)Run the fragments in gel and extract the ones in the 100bp range
3)Attach universal adapters with primer sites by annealing the T overhang to the fragment thats been treated with TAQ to add a complimentary A overhang
4)PCR amplify to get a few copies of each strand
5)Denature the fragments and attach them to the flow cell via the complimentary oligos on the cell
6)Excess strands are washed away
7)PCR begins and the flow cell oligo is now extended into a complimentary strand
8)Temperature is increased to denature and separate the template strand,leaving the oligo synthesized strand on the flow cell
31
How to prepare samples for gateway cloning
1)Trandtional restriction endonuclease/ligation cloning
2)Topoisomerase cloning
3)AttB sites added via PCR and BP recombination
32
Infection process of plant with agrobacterium
1)Plant wound produces acetosyringone
2)Acetosyringone activates the virulence genes on the plasmid
3)Vir proteins are synthesize the single stranded T-DNA
4)T DNA is transferred into the host cell
5)T-DNA complex is imported into the nucleus and integrated into the host genome
33
Describe the process of oxford nanopore sequencing
1)DNA is unwound by a helicase on the nanopore
2)A protein creates a pore in the membrane and holds the DNA molecule into place aligning with the pore
3)A flow or ions through the pore creates a current
4)The adapter molecules keep bases in place as they are forced through the pore and read by the electronic sensor into a squiggle
34
What are the different post transformation information we can collect using PCR
1)PCR detection of insert
2)Identifying insertion position using PCR
3)Determining if an individual is homozygous or heterzygous for insert
35
Describe cation heat shock
Use of heat to create holes in the lipid bilayer in combination with positive cation buffer that helps the DNA enter the cell by neutralizing the negative charge, DNA must be circular and carrier DNA is used to crowd the circular pieces near the pores
36
What is the CT value in RT-PCR, how is it chosen
The CT value is the # of cycles it takes to reach an arbitrarily threshold. It has to be set within the exponential phase of the reaction, if it is too low there is interference and if its too high it will begin to plateau
37
DNA viruses that cannot integrate into the genome and trigger a weak immune response due to the fact they do not typically infect mamalin cells
Adeno-Associated viruse
38
What are the methods to applying a probe to living tissues?
1)Whole mount-in situ hybridization
2)Section mount in situ hybridization
39
What is RT-PCR
RT PCR stands for real time PCR and is a technique which allows use to visualize the progress of our PCR reaction in real time after each cycle is completed
40
Principle behind illumina sequencing
Analyzing the nucleotide sequence as the sequence is being synthesized
41
What are some of the use of Viral vectors in genetic engineering?
1)Production of a target protein within a cell
2)RNA interference
3)Gene therapies
42
How does cite specific recombination occur for Lambda phage into e.coli
1)E.coli has a recombination site that is composed of B-attB-B' which we call BOB'
2)The phage has a matching sequence composed of P-attP-P' we call POP'
3)When the sites are near one another crossing over occurs and P' combines with BO forming attL and PO combines with B' forming attR and the phage DNA is inserted between these two sites
43
How can genetic engineering be used to kill unwanted cells?
WE can engineer antibodies that recognize tumour specific epitopes which can 1)act as antibodies to trigger an immune response in the patient or 2)Kill the tumour directly by conjugating the antibody to toxic molecules
44
Chimera
A genetic chimera is a single organism composed of cells of different genotypes.
45
What are the pros and cons of oxford nanpore seqeuncing
Pros:10-100kb read, Works for DNA and RNA, Fast,cheap,scalable
Cons:Certain sequences are difficult to resolve, only 92% accuracy
46
Why are viral vectors good for introduction of DNA as opposed to lipofection?
1)High specificity
2)High infection % of cells
47
What is the goal of our CRISPR construct project on rice?
Our goal is to understand vein development genes in rice to increase vein # which we believe may be a prerequisite to turning them into C4 plants
48
What are the different ways we can use PCR to build a genetic construct?
1)Addition of fragments via PCR
2)Site directed mutagenesis
3)PCR fusion independent from plasmid
49
Binary system of T-DNA transfer
Uses agrobacterium with two plasmids. 1Ti plasmid containing the virulence genes with no T-DNA and 2.Binary plasmid a smaller second plasmid containing the TDNA and selection genes working together to create our vector
50
Methods of Introducing DNA non biologically
1)Chemichal->Cation heat shock and liposomes
2)Physical methods-Injection,Electroporation and microprojectile bombardment
51
How have we modified the phage lambda system for cloning?
We have two vectors which are as follows
A: AttB1-ORF-AttB2
B:AttP1-Suicide gene-AttP2
AttB1 only pairs with attP1 and AttB2 only pairs with AttP2, this allows the switching of the genes located between these two regions
52
Describe the advantage of Next gen sequencing
1)Does not require a plasmid prep
2)Allows for more sequence to be prepared in one run
Disadvantage:Too short clusters for complete sequence of a complex genome
53
How do we ensure all cells in an organism have the modified detection genes for reporter plasmid assay
1. antibiotic selection for single
transformed cells, followed by regeneration
of complete organism (plants)
2. transformation of gametes, followed
by formation of complete organism (plant and
animal).
3. Generation of chimeric organism, carrying
untransformed cells, but also some transformed
germ-line cells. Offspring, resulting from single
gametes, are screened for transformants
(not chimeric).
54
What is agrobacterium mediated gene transfer?
Use of single stranded T-DNA to transfer DNA into the plant cell and integrate the transfer region into the host chromosome
55
Pros and cons of section hybridization
Pros:High resolution and sensitivity
Cons:Labour intensive
56
Difficulties of in situ hybridization
1)Inefficient at penetrating tissues
2)Complex and difficult to use procedure
57
How do we create out vector for viral transduction?
1)3 plasmids are given to our compentent cells i)Tranfer cectro ii)packaging vector iii)Envelope vector
2)All three plasmids are read and products are produced
3)Only our transfer vector has the Ψ nescessary for its genes to be put into the viral evelope
4)When target cell are infected the LTR allows integration into the genome
58
How do we add sequences to linear DNA fragments using PCR primers?
We can design fwd and reverse primers that include mismatched overhangs. When they are filled in by taq polymerase the sequence will be added into the fragment
59
How can reporter plasmids help us visualize gene expression?
A plasmid which contains a reporter gene ex.GFP gives us a readout of activity near a regulatory region allowing us to study expression of promoters,enhancers,and silencers. This is because genes are modular we can simply swap out components to visualize expression.
60
If I start with twice as much template for a reaction that takes 25 cycles to hit the CT value how many cycles will it take for doubled reaction to hit the CT value?
24- Each cycle doubles the amount from the previous reaction so starting with twice as much template will save you one cycle of amplification
61
What is the problem with increasing climate in relation to RUBISCO?
As temperature increases CO2 becomes less soluble in air and is therefor less concentrated and when in competition with O2 at a substrate it looses out leading to photo respiration instead of photosynthesis
62
How does CT value correspond to starting amount?
Higher CT value-Lower starting amount, takes longer to amplify
Lower CT Value-Higher starting amount takes fewer cycles to hit threshold
63
Steps in selection
1)Plasmid containing a gene that detoxifies a chemical in growing media is introduced into the cells
2)Cells are grown in media that kills nontransformed cells
3)Transformed cells are differentiated by their ability to divide within the media
ex.HPT detoxifies hygromycin and is used to select transformed plant and animal cells
64
Why do we use multiple plasmids to design our viral transduction vector?
Were we to simply knockout these genes in our transfer vector it is possible for it to regain virulence when encountering another virus. By removing them and keeping them in separate plasmids this cannot occur ensuring saftey
65
How is PCR used to confirm the presence of transgenic genes in an individual
You can use primers that amplify the trans genes, if amplification of the correct length occurs once run on a gel you know that the insert has been added
66
How to prepare a sample of oxford nanopore sequencing
1)Extract DNA
2)DNA is fragmented by transposome complex which adds the adapters,proteins required for pore interaction, and a bar-code for identification of sample
67
Draw the inverted repeats site specific recombination reaction
When the orientation on the strand is opposite to one another in the same molecule and inversion of the XY position occurs for the gene
68
Drawbacks of the agrobacterium system
1)Low copy # plasmid
2)Plasmid is extremely large making it difficult to find unique cut sites in the T-DNA region that don't already exist in the plasmid
69
What is site directed mutageneis?
By using primers with mismatches to the target sequence you can change the sequence to include a mutation at the ends of the sequence. This mismatch is tolerated by lowering the annealing temperature to decrease specificity of primer binding
70
How can we produce human growth hormone with genetic engineering
We can express the gene for human growth hormone in E.coli and purify it out, it can treat turner syndrome
71
What modifications do we need to make to the viral vector genes before creating our vector?
1)Remove gag and pol->Packaging vectors
2)Remove env->Removes envelope vector
3)Add GOI and select-able marker between LTR Ψ______LTR
4)Additional compliment vectors for packaging and envelope production are needed to make up for missing functions
72
How to perform a whole mount in situ hybridization
1)Embryo is fixed so probes can enter the cells
2)Enzyme labelled probe is applied to the whole embryo
3)Embryo is washed
4)Colour reaction is made visible and specimen is slide mounted
73
Describe the Lambda phage system
1)Lytic phase where phage DNA replicates and new phage particles are synthesized
2)Lysogenic phase-where the phage integrates itself into the host genome
To switch between the two phases it uses site specific recombination to pull itself in and out of the host genome
74
What are the benefits of gloden gate cloning
1)Seamless addition
2)Correct insertion direction due to the sticky ends
3)Vector cannot self ligate
75
What is RUBISCO
A protein that is used by plants for photosynthesis, It can use both O2 and Co2 as a substrate
76
How can we determine the position of a transgene within an organisms genome?
1)First we use restricion enzymes which target specific cut sites in our insert, this also digests part of the flanking DNA
2)The fragment with the insert +flank is then ciruclarized into a plasmid
3)Using fwd and reverse primers that compliment moving away from one another for your insert you can transcribe the entire plasmid into a linear fragment
4)The fragment can then be sequenced and compared to a BLAST database much like how DNA barcoding works
77
Illumina vs Sanger Sequencing
Illumina:6 billion samples per run with 100bp read lengths
Sanger:384 samples per run with 800bp read lengths
78
What are the genes we use for reporter plasmid assays. What is the limitation to the gene selected?
1)LacZ-histochemical kills its host-animals
2)GUS-Histochemical-kills host-plants
3)GFP-Fluoresences-does not kill-animal/plant
The gene inserted cannot already exist within the organism of study!
79
Lentivirus vector production scheme
1)Production plasmids transfect competnent cells
2)Lentiviral vectors are produced and harvested
3)Transduction of virus into the organism
80
Screening
Being able to visually differentiate detect transformed cells from untransformed cells exp.GFP
81
Describe the process of cluster generation
1)Our DNA is now bound to the flow cell by the oligo, by lowering the tempreture the strand will bend and bind to an adjacent oligo
2)PCR is performed and now we have a second complimentary strand that is also bound to the flow cell
3)Strands are denatured and separated, now each attached oligo strand can repeat this process with adjacent cells generating a cluster of the same DNA sequence
4)After amplification, either the reverse or fwd strands are washed away and a new primer is added that has a 3' blocker
82
How to introduce a point, deletion, and insertion mutation into site direct mutagenesis for internal position change of gene
1)Point-Fwd or Rev primer has a mutation that is transcribed
2)Deletion-Fwd and Rev primers lack part of the sequence that is to be deleted and when religated will no longer include that section
3)Insertion-additional nucleotides are added to the primers which will join the sequence once religated
83
Steps in screening
1)Plasmid carries gene that produces a protein which can be visualized directly of via assay
2)After transformation cells are screened for inserted marker
3)Transformed cells are collected and grown
84
Describe microprojectiles of DNA
The main principle of the microprojectile bombardment method is to deliver genetic materials directly into target cells by bombarding them with microcarrier particles at high velocity.The microcarrier particles are coated with the genetic material of interest. They are inserted into the target cells using a high-velocity stream generated by either an electric discharge or helium pulse.
The high-velocity stream shoots the microcarrier particles toward the target cells, allowing them to penetrate the cell membrane and enter the cytoplasm. Once inside the cells, the genetic material separates from the microcarrier particles and can be used by the cell’s machinery for gene expression.
85
What are the methods to genetically prepare our organism for plasmid assay
Bacteria-Chemical transformation and bacteriphage mediated transfection
Insects-Baculoviruses
Animals-Injection into embryos and retroviral vectors
Plants-Agrobacterium tumor inducing plasmids,biolisitics,viral vectors
86
What is required to run a RT-PCR?
1)PCR thermocycler with open well plates for readings
2)Optical module which detects and excites the fluorphores using the fresnal lens
3)Computer which translates the data
87
How do we ensure safety when it comes to using viral vectors for introduction of DNA into eukaryotic cells?
1)Disease triggering genes are removed
2)Ability for the virus to propagate is removed
88
Describe the process of Gibsons assembly
By using 5' primer additions you can create 20bp overhangs with the next desired sequence. Using T5 exonuclease digest you will create 3' overhangs which can be assembled into a desired sequence
89
How to ensure proper localization of translational product
1)Make several constructs with the label at several positions on the protein and observe its function, if all localize properly we can assume construct does not interfere with localization
2)Using a genetic mutant you can incorporate construct into mutants genome and if the phenotype is corrected upon adding tagged protein we know the construct does not interfere with the function
90
Describe microinjection of DNA
Injection of DNA solution into the nucleus of not yet differentiated cells (usually male pronucleus) ex.Drosophillia transformation
1)Nuclei are exposed to DNA before celluarization and establishment of the drosophillia germ line cells
2)After cellularization presence of gene leads to germ line cells containing a selectable marker
3)Offspring are produced and selected for based off said trait
91
Describe electroporation
Use of high voltage pulses to disrupt the membrane of the cell enough for DNA to be uptaken by the cell
92
How do we select fro recombinant vectors in the gateway system?
By using the ccdB "suicide gene" the gene is removed from the destination vector when recombined so non combined destination vector perishes and recombined vector will also perish. Only the uncombined entry vector to worry about. TO select against the entry vector we put ampicillin resistance in the destination and kanomyocin resistance in the entry clone. BY growing in ampicillin the original non combined vector will perish
93
Describe the steps in sequencing by synthesis
1)Now we add a primer which recognizes the adapter sequence either fwd or reverse but are separate
2)DNA begins to be synthesized and by using DNTPS with a 3' blocker and fluorophore
3)As strands are made the solution is excited and fluorescent data is collected. Then the 3'oh is cleaved and another fluorophore is added
4)This continues as the computer records all this data to assemble the sequence
94
How to use PCR to determine if an individual is homozygous transgenic,homozygous wild type or heterzygous
By using FWD and REV primers that compliment the WT gene along side a REV primer for our insert we can use amplicon size to determine if the individual is Het, HomoWT, or HomoTG. Heterozygous indiviudals produce two fragments using the FWD and REV of WT and FWD WT and REV Transgene.
95
Describe liposome/lipofection
Use of liposomes carrying DNA into miotically developing cells
1. Transfection-active lipoplexes spontaneously form from cationic lipids and negatively
charged DNA. Lipoplexes are taken up by endocytosis.
2. The DNA is released by destruction of the endosome membrane
3. The DNA enters the cell nucleus during mitosis
96
Gene therapy
Delivery of a wild type copy of a defective gene into patient to treat a disease,usually via viral particles
97
Why must you dilute your site direct mutagensis for internal position change of gene?
Dilution must occur so that when the plasmid religates it will do so with it's own ends instead of attaching to other linear products
98
Name the viral vector types for viral transduction
1)Retroviruses
2)Lentiviruses
3)Adenovirusees
4)Adeno-associated viruses
99
What are the three categories of protein therapy give one example
1)Complimentation of a deficiency ex.Insulin and human growth hormone
2)Kill unwanted cells ex.cancer treatment
3)Block overactive/harmful pathways ex.autoimmune disease
100
What are the probes used in in situ RNA hybridization
1)DIG- Antibody linked to an alkaline phosphate which will convert substrate to blue/purple in ELIZA
2)P33 or P32-isotopes which are detected via a liquid photographic emulsion and developed like film
3)Fluorescent probes-uses microscopy equipment to detect fluorescences but can photo bleach over time
101
Describe the process of generating novel monoclonal anitbodies
1)DNA fragments of random sequence are cloned into bacteriophages(phagemid) genomes as fusion genes that are capable of producing the variable regions of the antibody
2)Fragments of the variable regions of the antibodies are expressed on the outer coating of the bacteriophage coat
3)Phages will expres and display a large set of antibodies on it's surface
4)An antigen is preseted and the phages which bind are trapped on and none compatible phages are wash away
5/6)Phages retained are eluted and infect E.coli
7)Amplification of the phage and anitbodies are run through multiple rounds of selection
8)Phage clones are isolated and subjected to sequencing to determine the anitbody sequence
9)Clone sequences produce antibodies with high affinity to antigen
102
Why do we do SLIC?
1)Label a protein with a marker like GFP
2)Fusing two genes together
3)Putting a sequence into a vector
103
How is diabetes treated with genetic engineering
It is a type of protein compliment We can make proinsulin by expressing the modified human insulin gene in E.coli
104
How do you perform site directed mutatgenesis at an internal position of the gene?
1)Placing the fragment into a plasmid means that any two points can be the "ends' of your sequence
2)Primers including 5'phosphates and a desired mutation bind to the plasmid and are transcribed AWAY from each other
3)Linear product is ligated back into a circle with the desired mutation not existing between the two primer sites.
4)Plasmid is propagated via E.coli
105
What is PCR fusion?
PCR fusion is the generation of complimentary sequences via PCR primers between two strands in order to join them together
106
RNA viruses that can integrate into the genome of dividing AND non dividing cells
Lentiviruses
107
What are the pros and cons of a whole mount in situ hybridization?
Pros:Gives an immediate overview of expression fast and simple
Cons:low sensitivity and poor penetration of tissues
108
How can genetic engineering be used to block harmful pathways and immune responses?
You can use antibodies that bind to specific receptors to prevent a troublesome cell from binding to the molecule which triggers the disease ex.Transplant rejection/autoimmune disorders
109
How do we interpret the results of RT-PCR?
BY comparing out CT value to that of a Log copy# graph we can extrapolate the starting titre. The graph is created from serial dilution data of our starting DNA.
110
How do we use genetic engineering for recombinant vaccines
We can clone genes which produce surface antigens (spike/coat proteins) and that gene can be put into a microbe which creates the protein and that can be purified and injected into people to elicit an immune response
111
Challenges of gene therapy
1)Only works on recessive conditions
2)Safety of delivery
3)Correct delivery location
4)Entry of cells and nucleus
5)Unwanted effects of genome integration
112
How do we do Golden gate cloning?
1)Add primers that contain a sequence indeitcal to the cloning site and the BSAL cut site
2)PCR amplify
3)Cleave amplicons and plasmid with BSAL
4)Ligate amplicon into the plasmid
113
Describe the steps of PCR fusion
1)Primers with complimentarity along the 3' direction are added to both strands.
2)Amplification occurs so that the additions are incorporated
3)Strands are denatured and turned into SS DNA
4)Complementary sequences between strands will anneal to one another
5)PCR is run to extend the 3' ends of the strands forming one ds DNA
114
What are the important genes found on the Retro and Lenti viral vectors?
1)LTR-Long terminal repeats used for integration into the host genome
2)Ψ-Packaging signal, controls the packaging of the retrovirus into the capsid
3)gag-Coat protein for packaging
4)Pol-viral polymerase used in replication
5)Env-viral envelope proteins that determine what proteins are present on the protein coat and control the specificity of the viral infection
115
Selection
Differentiating between cells with a construct from those without by killing off cells that lack the contruct
116
DNA viruses that cannot integrate into the genome and may trigger a strong immune response
Adenovirus
117
How to B/P recombine with PCR fragments
1)Add the AttB1 sites and AttB2 sites to your PCR primers
2)Following PCR you can recombine the vector carrying AttP1 and AttP2 sequences using the BP clonase enzyme mix
3)The sequence will now carry the AttL sequences necessary to recombine with the AttR destination vector
118
What is C4 photosynthesis, why is it better than C3 at higher temperatures?
At higher temperatures more O2 is used by C3 plants which is less efficient. In C4 plants CO2 is converted into oxaloacetic acid and then bring them to sheath bundle cells allowing C4 plants to avoid performing O2 photo respiration
119
Paired end seqeuncing
allows users to sequence both ends of a fragment and generate high-quality, alignable sequence data
120
Differences between the two fusion types in information provided
Transcriptional: Contains the 5' regulatory elements
Translational:Contains the 5' regulatory elements, the 3' elements, and allows for sub cellular localization of the native proteins
121
What are some applications of RT-PCR
1)Gene expression-Using reverse transcriptase a DNA copy of RNA can be produced and amplified allowing us to quantify the expression of a gene
2)Diagnosis-allows you to determine viral titre and adjust treatment plans
3)Food testing-allows for determination of GMO %
4)Animal and plant breeding-allows you to determine gene copy #s which correlate to quality and quantity of agricultural output
122
RNA viruses that can integrate into the genome of dividing cells
Retroviruses
123
Requirements for gene expression mapping
1)WE need to be able to genetically modify the organism of interest
2)All of the cells in an organism need to have the modified detection genes
3)We know the genetic sequence of the organim
BISC 357- Genetic Engineering
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