1
Q
Cas 9
A
breaks phage DNA preventing gene function and replication
2
Q
Neoschizomers
A
Enzymes with the same sequence specificity but cut at different location on the DNA
3
Q
Plasmid
A
Small circular pieces of DNA in a bacterial cell which can self replicate and aren’t essential to the survival of the bacteria
4
Q
Plasmid which produces few copies and are usually large
A
Low Copy # plasmid
5
Q
What does the 260/230 indicate
A
Tells you if there are phenolate,thiocyanate carbohydrate and other organic compounds in your extraction
6
Q
How do we remove RNA from a DNA extraction
A
RNAses
7
Q
Explain blue white selection
A
1)Plasmids contain and antibiotic gene- when plated on ampicillian only those with plasmid survive
2) Plasmid has the code for the alpha fragment of the LacZ gene which is damaged on the main chromosome. When plated on lactose, LacZ is activated and if the gene has not been inserted the alpha fragment gene will code and allow for production of lactase which will digest the IPTG/X-gal and turn the colonies blue on the plate.
8
Q
Florescent dye sequencing
A
Modified version of a sanger sequence where instead of radioactive isotopes as part of the chain the ddNTPs are labelled with a dye. When loaded into a capillary gel they separate by weight and are read by a computer to determine the sequence
9
Q
What are some ways that improve sticky end ligation success
A
1)Lower temperature
2)Longer overlap of sticky ends to allow more hydrogen bonding
3)Higher GC content
4)Higher concentrations
10
Q
Why is it important to have a negative and positive control?
A
1)Negative- control with no template at all, if bands appear this indicates that there is contamination
2)Positive-control with a verified template, if nothing appears then something went wrong during the set up
11
Q
How to score a formaldhyde gel for RNA?
A
1)In plants look for 25s and 18s rRNA bands
2)In animals look for 28s and 18s rRNA bands
Both ratios should be around 2 and there should be little no smearing on the gel. We use this as an indicator of mRNA since it’s presence is low. If there is a good amount of rRNA then it means the extraction was successful in preserving RNA
12
Q
What are things that must be removed from a DNA extractions
A
a)Large pellet particles
b)Proteins
c)RNA
d)Metabolites
13
Q
What are the primary strucutral diffrences betweek eukaryotic and prokaryotic genes?
A
1)Eukrayoti genes contain introns and exons
2)Eukaryotic genes are usually longer than prokaryotic genes
14
Q
Shot gun sequencing
A
1)Restriction enzymes cut at random spots along the chromosome multiple times with many copies of the chromosome
2)Insert is cloned and cut and that sequence is digested cloned and cut until you achieve fragments of small enough size
3)Small pieces are then sanger sequenced and assembled to find overlapping regions and assemble the genome.
15
Q
Replicon
A
Contains the origin of replication AT rich and genes that regulate replication affecting the copy number in lab
16
Q
Terminator
A
Contains the motif to end trascription and for the cutting of the 3’ end to add the poly-A tail
17
Q
Draw the structure of ribose
A
18
Q
Type 2 endonucleases
A
Endonucleases in bacteria which cleave DNA at specific palindromic regions and cannot cleave DNA that has been methylated like genomic DNA
19
Q
Replication
A
Creating an exact copy of something ex. Cell division
20
Q
Steps in PCR
A
1)Denaturation- at high temps the DNA will separate 95C
2)Annealing- temperature is lowered so that the primers can bind to the DNA target
3)Extension- Temperature is increased to 68 or 72C to allow strands to be fully built by the polymerase
21
Q
Identify a gene and mutate it to see the phenotypical effect
A
Reverse genetics
22
Q
Vector
A
Vehicle to transfer genetic material into a target cell
23
Q
Reverse Transcriptase
A
RNA dependant polymerase that can make a DNA copy of RNA. Good in cDNA as you can synthesize the gene without any introns
24
Q
Invivo CRISPR delivery
A
Therapeutic is delivered directly into the patient via plasmid or viruses
25
Transcription
Expression of DNA into RNA
26
What does the 260/280 indicate and what is the contamination threshold
Less than 1.7 indicates high contamination from proteins
27
Describe knockdown via RNA interference
1)Introduced DNA codes for the antisense version of a target gene which when transcribed forms dsRNA with the target
2)dsRNA is recognized by DICER and is digested into siRNA
3)RISC complex will use the antisense strand of siRNA to guide it to similar strands and cleave the mRNA
4)leads to post transcriptional inactivation of a target gene function
28
How do we remove small metabolites from a DNA extraction?
Salt and alcohol DNA precipitation and the excess liquid can be poured out to remove metabolites
29
Steps in a phenol buffer solution at acidic pH extraction for RNA
1)Trizol is mixed with tissues (Guandinium salt, low pH buffer,Phenol)
2)Chloroform is mixed into solution and centrifuged
3)Solution will separate into three layers, remove the aqueous one
4)Using isopropanol precipitate out the RNA
5)Wash the pellet with ethanol and let the remainder dry out
6)Add RNAses free water and keep at low temp and away from chaotrophic salt/phenol/and chloroform
7)If concerned about remaining RNAses RNAsin can be used to degrade them
30
How to prepare a formaldehyde gel
1)Incubate RNA in presence of formaldehyde and formamide at 55-65c for a few minutes
2)Run the solution on a gel that contains fromaldhyde
SN. Bleach also works for this
31
Sticky end
Can be in the 5' or 3' direction and is when one end of the DNA strand is longer than the other
32
DNA repair mechanisims
1)NHEJ- when two strands are directly rejoined after being cut usually leading to INDEL/loss of function
2)HR-when a DNA fragment matching the cut location is integrated into the DNA
33
Describe Knockout via homologous repair
Plasmid with a mutated target gene is inserted into the host genome via homologous repaid of DNA
34
Why would we use absorption chromatography over precipitation extraction of DNA?
Used for plasmid purification and PCR as it's faster and less set up required
35
How to calculate amount of material in weight from volume and concentration
Volume (ml) x Concentration (mg/ul)= amount in mg
36
Plasmid that can only replicate in a few species
Narrow host range plasmid
37
Plasmid that can encode all the proteins necessary for replication of the plasmid
Broad host range plasmid
38
Coding strand
Strand that will be read the same as the RNA runs 5'-3'
39
Transcriptional unit
Contains the predicted code for the protein
40
What is a agrose gel used for in DNA extraction?
Used to determine if degradation has occurred and if DNA is the appropriate length. A good quality DNA will be in the 20-40KBase with no smearing
41
Primer walking
1)chromosome is isolated and digested many times using enzymes to create over lapping sequences
2)Sequences are ligated into plasmids and taken up by competent cells which form colonies
3)Colonies are selected and the insert is cut,removed, and sequenced and a probe is created
4)Probe is then used to find the next part of the sequence via overlapping ends and that colony is selected,cut,sequenced and another probe is developed
5)Process repeats until the entire sequence is assembled
42
Why is Eukaryotic mRNA useful in genetic engineering?
1)It can be used to determine if a gene is expressed
2)It can be used to make cDNA that can express eukaryotic genes in bacteria
3)mRNA sequence can be used to determine the peptide sequence of a gene
4)It can be used to make probes
43
Insert
Piece of DNA which are you are cloning into the plasmid
44
Uptake of DNA by eukaryotic cells in tissue cultures
Transfection
45
Uses the phenotype to determine the genotype
Forward genetics includes traditional mutinization and random insertion into genes
46
What are the two groups of dna polymerases
1)DNA dependant-uses DNA as a template strand for synthesis
2)RNA dependant-uses RNA as a template for strand synthesis
47
When do we use blunt end ligations?
1)When sticky ends cannot be matched
2)When working with PCR products that have blunt ends to them
48
How are endonucleases named?
First letter- Genus name
Second and third letter-First two letters of the species name
4th letter-strain identity
Roman #-Indicates order of discovery of said endonuclease
49
What are the characteristics of type 2 endonucleases?
They cleave specific palindromic regions on invading DNA and require Mg2+ as a co factor
50
What are the possible vectors and their insert sizes
Plasmid-15kb
Phage lambda-25kb
Cosmid-45kb
Bacteriophage-70 to 100kb
PACS-130-150kb
BACS-120-300kb
YACS-250-2000kb
51
Isoschizomers
Enzymes which recognize the same sequence and cut at the same spot
52
Steps in CRISPR function in bacteria
1)Acquisition of genetic material from foreign DNA
2)crRNA biogenesis- formation of CRISPR components
3)Interference-components work to cleave similar foreign DNA
53
Exon
A segment of a gene which form a part of the final RNA product used in translation
54
What elements are required for a CRISPR gene
1)Eukaryotic promoter for Cas9
2)NLS at front of Cas9 gene
3)Cas 9 gene
3)NLS at back of Cas9 gene
4)Promoter for sgRNA
5)sgRNA
55
What do we use CRISPR for?
1)Knockouts to study gene function
2)Insertion of genes or gene fragments
3)Modification or repair of a gene
56
E coli DNA polymerase
Works 5'->3' as a polymerase
Works 3'->5' as a exonuclease in proof reading
Works 5'->3' to remove RNA primers
57
How to perform a DNA precipitation extraction
1)Create a suspension with EDTA, TRIS,NaCl, SDS, and Protinease K
2)Add salt and lower temperature to precipitate protein fragments
3)Collect aqueous layer
4)Phenol and chloroform are added and solution is centrifuged
5)Add alcohol to form a DNA pellet
6)Wash with 70% ethanol and let dry
8)Dissolve pellet in TE along with RNAses containing water
58
What does this depict?
Deoxyribose, missing an O on the 2'
59
How do we remove proteins from a DNA extraction
Use of nonpolar organic solvents like phenol/chloroform, high salt, low PH, and low temp conditions
60
Reverse primer
Reads opposite to the 5'->3' strand and will bind to the 5'->3' strand
61
Plasmid conformations and their banding
1)Nicked-circular and bulky, slowest on a gel
2)Linear- Moves at a speed relative to its length
3)Supercoiled-Compact twisted circles that move fastes on a gel
62
What is the traditional approach to determining gene function?
1) Mutinization of a population
2)Screening of the F2 and F3 generation
3)Figuring out what gene causes the phenotype observed
63
What modifications do we make to CRISPR when using it for gene modification?
1)Create guide RNA that fuses crRNA and tracrRNA into sgRNA which binds Cas9 at one end and guides CAS9 to target on other end
2)Cas9 is modified to cleave 2 strands of DNA
64
Palandromic region
Region of DNA where when read 5'->3' both strands share the same sequence
65
Loss of function knockouts/knockdowns
1)Amorph- genetic mutation that causes a complete loss of function of the gene
2)Hypomorph- A genetic mutation that reduces but not eliminates gene function
66
What does a strong detergent do in a DNA extraction?
Disrupts the cell membrane and proteins including DNAses allowing for release of DNA and inhibits degradation of DNA. Ex.SDS
67
Chaotrophic salt
Salt which disrupts hydrogen bonds and removes the hydration shell so negative DNA can bind to positive silica
68
Pros and Cons of Blunt end ligation
Pros: Allows for universal annealing of any DNA
Cons:Inefficient and strands seperate quickly
69
Intron
A nucleotide sequence in a gene that is not expressed or operative in the final RNA product
70
crRNA
hybridizes to complementary sequence in foreign DNA guiding Cas9 to corresponding sequence for cleavage
71
Plasmid types and functions
1)Cloning plasmid- used to clone DNA fragments contains ORI,MCS,Antibiotic resistance
2)Expression plasmid- used for expressing a certain gene promoter,trancriptional terminator and ORI
3)Gene knockdown plasmid- reduces the expression of a gene through mRNA interference
4)Reporter plasmids-studies the function of a gene using a reporter gene like GFP to visualize activity
5)Viral plasmids- create viral particles to infect target cells
72
Translation
Expression of RNA into protein
73
Draw the structure of deoxyribose
74
Exonuclease
nuclease that can only degrade DNA from one of it's ends
75
Ideal 260/280 for DNA and RNA
DNA:1.8 RNA:2.0
76
What are the challenges of working RNA as opposed to DNA?
1)RNAses are more stable than DNAses
2)RNA itself is less stable than DNA
77
What does Tris buffer do for DNA extraction?
Creates a Ph of 8 that supports the stability of DNA
78
What is the difference between a knockout vs a knockdown? Why do we use this method for gene study?
Knockouts cause a complete loss of function while knockdowns lead only to a reduction of function. This is useful as loss/reduction of gene function can tell us what the expressed gene does via the organisms phenotype
79
Pyrimidines
Single ringed nucleic acids, Thymine, Cytosine, Uracil
80
Exvivo CRISPR delivery
Cells are removed from patient,modified in culture and replicated then inserted back into patient
81
What is the purpose of NaCl in a DNA extraction?
Solubilizes the DNA for future precipitation
82
Limitations to using blunt end ligations for plasmid constructs and how to get around it
1) Products which were made using Tag polymerase have an additional A to the 3' end meaning it is not blunt- Use a T overhang plasmid
2)For primers that use a 5'OH end they cannot be used for ligation so only one strand of the product can be used- used a primer with 5' P end
3)Plasmid can self ligate when doing blunt end ligations-include EcoRV to prevent self ligation
4)Reactions are very infrequent-increase concentrations of reagents
83
Continuous cut
The two halves of the recognition
sequence are adjacent to each other
84
Purines
Double ringed nucleic acids, Adenine and Guanine
85
What does this depict?
Ribose
86
Enhancer
Contains the binding sites for the transcriptional activators to enhance gene expression and is independent of location
87
What components do we need in a plasmid for genetic engineering?
1)Replicon-ORI
2)Antibiotic resistance to allow for selection of plasmid containing bacteria
3)MCS- Short segments of DNA which contain restriction sites for DNA one per plasmid
4)Insert-gene and it's promoter
5)Promoter region-drives transcription of target gene vital for expression of the vector
6)Selectable marker-The antibiotic resistance gene allows for selection in bacteria. However, many plasmids also have selectable markers for use in other cell types.
7)Primer binding site- used to sequence DNA inserted into MCS
88
Ribonuclease
Catalyzes the degredation of RNA to remove from solution
89
Methods for delivering CRISPR into cells
1)Cas 9 and sgRNA are added directly
2)Cas9 and sgRNA producing genes are inserted into plasmids or viruses
90
Steps in DNA purification (GENERAL)
1)Mechanical breakdown of tissue
2)Chemical disruption of cells to access DNA
3)Step wise removal of non DNA components
91
How to perform sanger sequencing
1)Two alliquots one with forward primer and one with the reverse primer
2) 4 solutions are made with each solution containing a diffrent ddNTP
3)Kenklew fragments read the sequence generating a complementary strand and add nucleotides until it hits a ddNTP which stops the transcription process
4)Samples are then loaded into individual lanes of a polyacrylmide gel
5)After drying the gel is exposed to photographic film and the dCTPs release photons and create an image of the sequence order
6)Starting from the bottom and working towards the top you can find the sequence of the compliment strand of DNA
92
What does Ligase need to complete its reaction?
In Bacteria- NAD+
In Eukaryotes and Viral DNA- ATP
93
Silencer
Contains the binding sites for repressing transcription factors, decreasing transcription and is also independent of location
94
List the nucleotides and their acronyms for DNA synthesis
1)Adenosine-dATP
2)Guanine- dGTP
3)Cytosine-dCTP
4)Thymine-dTTP
5)All-dNTPS
95
Things to consider when designing your PCR primer
1)Avoid secondary structures in your primers
2)Avoid complimentary sequences between your forward and reverse primer
3)Make sure the primer is unique to your target
4)Make sure both primers have similar annealing properties ex.GC content
96
crRNA biogenisis step of CRISPR
When bacteria is attacked again the aquired locus is transcribed and forms tracrRNA,crRNA, and Cas 9
97
Introduction of DNA via viruses
Transduction
98
What is genetic engineering
Deliberate modification of the characteristics of an organism by manipulating it's genetic material
99
tracrRNA
Binds Cas9 and crRNA together
100
How to deal with chemical contaminants in your RNA extraction?
Additional chloroform extraction and alcohol precipitation can remove extra chemicals like phenol/trizol/ and gaunidinium salts
101
What is the purpose of using a formaldehyde gel for RNA?
1)Allows RNA to remain denatured
2)Allows more accurate molecular weight to be asssesed
3)RNAses are deactivated
4)Allows you to know if there is DNA contamination in your sample
102
Construct
The insert plus vector combination we are trying to make
103
Limiting factors in PCR
Dependant on primer concentration as Primer is incorporated into the PCR products.
104
Methods of mechanical breakdown for the different tissue types
1)Blood cells and cell culture-no breakdown
2)Large tissues:Homogenization or maceration using a motor
3)Tough/hard tissue: liquid nitrogen or pound mills
105
Acquisition step in CRISPR
Foreign DNA is inserted into the CRISPR loci when a PAM (NGG) sequence is identified on foreign DNA, signalling for cleavage 3 bp upstream
106
What type of sequencing to use for specific lengths
Sanger 700bp-1000bp
Primer walking 1kb to 10kb
Shotgun sequencing Greater than 10kb
107
How to go from DNA to Protien (all steps of processing)
1)Transcription factor binds to promoter sequence
2)RNA polymerase intiates transcription of transcription initiation site
3)RNA polymerase terminates trasription at theterminator
4)Enzymes recognize Polyadenylation signal at the 3' end cutting it to add the poly A tail
5)5' cap is added
6)Enzymes remove the introns
7)AUG triggers the binding of ribosomes
8)Ribosomes translate the mRNA until it reaches the stop Code-on
9)Peptide is produced
108
Promoter
Regulates where and when a gene is expressed
109
Plasmid which produces up to a hundred copies per cell and are the preferred plasmid for genetic cloning
High Copy # plasmid
110
What does this depict?
Cytosine
111
What are the components used to create a crude lysate
1)Tris Buffer
2)EDTA
3)Strong deterent
4)NaCl
112
What are the steps for Cloning in plasmid vector
1)Endonucleases cuts gene of interest and cuts plasmid
2)Ligase joins the fragments with compatible overhands to form individual plasmids
3)Transformation of the bacterial cell with plasmid
4)Plate colonies and inoculate broth culture to quickly create millions of copies
113
What is the point of PCR?
Using short primers to target specific DNA sequences in order to create additional copies of the target
114
Discontinous cut
The two halves of the recognition
sequence are separated from each
other and Will produce different overhangs
depending on the exact sequence cut
115
What does this depict?
Thymine
116
What are the different primers? When do we use them?
1)Oligio- hybridizes with the Poly-A0Tail in mRNA to gernate cDNA
2)Random oligio sequences- Used for DNA synsthis of RNA's lacking a poly A tail
3)Defined sequence Oliglio- Synthesis cDNA of a specific gene only
117
Different nanodrop contaminants for RNA and their warning signals
Trizol and Phenol- 270nm peak, absorbs at 230, low 260/280
Guandinium HCL- high absorption peak at 210-230nm
Guanidinium isocynate- Peak at 250nm
118
Interference step of CRISPR
TracrRNA,crRNA and Cas9 make a complex to target phage DNA and cleave it along similar points to prevent its function
119
How do we prevent RNA degradation during an RNA extraction
1)Keep the material frozen so that RNAses are inactive
2)Treat with strong denaturants like guanidinium salts
3) Quickly remove proteins (RNAses)
120
What does EDTA do in a DNA extraction?
Cleaves cations like Mg2+ that act as cofactors for DNAses, therefor preventing degradation of DNA
121
Regulator components
Elements next to the gene that direct the expression of a gene in a tissue/cell/stage
122
Product of the second round of PCR amplification
1)8 strands
2) 2 template, 4 linear, and 2 product strands
#Product=2^n where n= cycle number
123
What are the characteristics of the T4 Ligase
Most commonly used ligase in ligation reactions as it can be used for sticky and blunt end ligations. Requires ATP as a cofactor
124
Explain how CRISPR can be delivered via viruses or plasmids
1)Plasmid enters nucleus and using eukaryotic promoters is transcribed
2)Cas9 RNA and sgRNA is produced
3)Cas9 is translated outside of the nucleus but brought back inside the nucleus with Nuclear locailzation signals
4)sgRNA binds to Cas9 and it attacks target DNA
125
What are the two RNA extraction methods? When do we use them?
1)Selective precipitation of RNA using Lithium Chloride, used for RNA BP length 100>
2)Extraction with phenol buffered at an acidic PH used when BP are short and when excess nucleotides need to be removed
126
Describe plasmid insertion knockout
Agrobacterium plasmid is modified so that TDNA can be inserted into spots along the chromosome and knockout a genes function
127
pUC ori
Origin of replication for the plasmid, pUC ori has a mutation to allow for high copy #
128
Basic steps in protein making
1)Coding region is read
2)Exons and introns are both transcribed
3)Processing removes introns
4)Processed mRNA is translated into a protien
129
Taq Polymerase
Polymerase that is used in high heat enviroments like in PCR, can introduce errors
130
Ligases
Molecules that catalyze the joining of 3' hydroxy to the 5' phosphate of a nucleotide via AMP activation
131
Blunt end
When DNA is cleaved evenly without any sticky ends
132
Steps in a Li precipitation extraction of RNA
1)Li+ ions form a Li+-RNA complex which has a neutral charge letting it precipitate out of solution
2)RNA pellet is created after centrifugation while the DNA remains in the liquid and can be washed away
133
What does this depict?
Guanine
134
Product of the first round of PCR amplification
1)2 strands now becomes 4 strands
2) 2 template strands and 2 linear strands
NO PRODUCT
135
What are the four types of procedures used in reverse genetics what are their suitable hosts?
1)Knockout via plasmid insertion-plants
2)Knockout via HR-prokaryotes,yeast,mice and flies
3)CRISPR-Universal
4)Knckout via RNA interference-univerasl
136
Steps in DNA synthesis from mRNA using reverse Transcriptase
1)dT oliglio will anneal to mRNA poly A tail to generate a primer site
2)dNTPS are added via reverse transcriptase to form a complimentary DNA strand
3)Heat and RNAse activity digests the RNA template
4)DNA can be used as a template for PCR or a second strand can be synthesized to stabilize it.
137
Requirements for "good" quality DNA
1)Free from DNA altering contaminants
2)Appropriate length
3)Concentrated enough for down stream manipulation
138
Gain of function knockouts/knockdowns
1)Hypermorph-genetic mutation that leads to over production or excessive activity of a product via changes in promoters/regulatory genes
2)Neomorph- genetic mutation that gives rise to a new function such as expression of tissue types in new places
139
Template strand
Strand complimentary the coding strand that when read will produce the code of the coding strand. Read 3'-5'
140
Endonuclease
nuclease which cuts a strand of DNA internally at any position along the strand
141
Steps in DNA extraction using absorption chromatography
1)Crude lysate with low ph,high ionic strength and chaotrophic salt such as guanidinium
2)Centrifuge so nucleic acids bind to the silica membrane but excess material flows through
3)Wash away residual contaminates using a solution that favours DNA binding
4)Elute DNA with high PH and low ionic strength
142
Blunt end ligation
Ligation of strands of DNA which do not have overhangs meaning that there is no additional hydrogen bonds to stabilize the reaction
143
How to design a plasmid for RNAi
1)Design the sequence as promoter->Sense strand->spacer->antisense strand and have the entire sequence be read then folded
2)Design the sequence as FwdPromoter->insert->Reverse promoter and have the sequence be transcribed forward and reverse at the same time
144
What does this depict?
Adenine
145
Steps in ligation of 5' to 3'
1)Lysine in ligase forms a bond with ATP that causes ATP to release two phosphates and form the AMP-Ligase complex
2)AMP-ligase complex forms a bond with the 5' phosphate and activates it, the AMP binds to the 5' and the ligase is kicked off
3) 3'OH will attack activated 5' end and forms a bond kicking off the AMP
146
Commonly used exonucleases and their uses
Exonuclease 1-USed to remove single stranded DNA primers after PCR has synthesized double stranded cDNA
Ribonuclease- degrade RNA and removes RNA from DNA preps
DNAse1-Remove residual DNA for cDNA preps and used in DNA foot printing
147
Uptake of DNA by bacterial cell
Transformation
148
Forward primer
Reads the same as the 5'->3' target strand and will bind to the 3'->5' strand
149
Components of trizol
1. guanidiunium salt
2. phenol
3. buffer with low pH
BISC 357- Genetic Engineering
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