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What is the general protocol for purifying DNA?
- Disrupting the cell
- Centrifugate the lysate to separate the various cellular fractions
- add Proteinase K to degrade all of the proteins (including nucleases that can digest the DNA)
- Use various salts and organic solvents
What is the purpose of EDTA in DNA purification?
EDTA can be used to chelate metals and inhibit DNases
What is the purpose of adding Proteinase K in protein purification methods?
Proteinase K is able to degrade all of the proteins, including nucleases that can digest DNA
What are the various ways in which one can purify crudely obtained DNA?
- Salt-out the DNA with Potassium Acetate, centrifugate, and alcohol precipitation
- Place on a Cessium Chloride Gradient and spun in an ultracentrifugePurified with an Anion Exchange Column that binds to DNA's negatively charged backbone
Briefly describe Phenol-Chloroform Extraction
A method to purify DNA
Use of organic solvents such as Phenol, Chloroform, and Isoamyl Alcohol (to reduce foaming) in a 25:24:1 ratio
Phenol of the upper phase (aqueous) will contain DNA and RNA, while the Chloroform of the lower phase (organic) will contain proteins and aggregates
This method will produce hazardous waste, and residual OrganSolv ruin downstream techniques (such as PCR)
Briefly describe the following two methods: 1) Alkaline Lysis, and 2) Silica-Based Methods
Both are DNA purification methods
Alkaline Lysis: Uses SDS to break up phospholipid bilayer and NaOH to dissolve structural proteins
Silica-Based Methods: allows the selective absorption of DNA to beads in the presence of chaotropic salts (molecules able to disrupt H+ bonding, examples are Phenol, SDS, Urea, Ethanol)
Briefly describe the Miniprep
- Method of DNA purification use to specifically purify Plasmids (the method is also called Plasmid Minipreparation)
- Based upon the Alkaline Lysis technique, and also involves Silica-Based Columns
How is Plasmid DNA obtained from a Miniprep quantified?
Typically quantified by a spectrophotometer, plate reader, or Nanodrop at 260nm
What does an A260 of 1.0 = ?
A260 of 1.0 = 50 ug/ml of DNA
Why is RNA less stable than DNA?
RNA contains an additional 2' hydroxyl group that promotes degradation; due to this RNases (which are special endoribonucleases) do not require metals and are therefore not inhibited by metal chelating (removing) agents such as EDTA
How is a research able to prevent RNases from destroying RNA samples?
- Bake glassware at 280 °C overnight
- Treat water with diethyl pyrocarbonate (DEPC)Use molecular biology grade chemicals (B-ME)Wear gloves during all procedures
What properties does Guanidium isothiocyanate have, and what method(s) is it used in?
Guanidium isothiocyanate is able to denature proteins and inactivate intracellular RNases
Used in RNA purification
mRNA takes up less than ___% of RNA in the cell
mRNA takes up less than 5% of RNA in the cell
What is a useful property of mRNA?
mRNA contain polyA Tails which can be taken advantage of for purification columns that contain polyT tails which are able to hybridize (similar to how nickel binds to histidines on a protein affinity column)
What is the important distinctions that separate Agarose Gel Electrophoresis (AGE) from a protein electrophoresis (IE Acrylamide Gel)
AGE is used primarily for DNA purification as DNA is much heavier than proteins (Mw of a single base pair ~650kDa, while an amino acid is ~112kDa)
Acrylamide gels are only used for DNA sample less than ~200 base pairs
A very useful feature of DNA that aids in ease of running Agarose Gels is that DNA and RNA naturally have _______.
A very useful feature of DNA that aids in ease of running Agarose Gels is that DNA and RNA naturally have the same charge-to-mass ratios due to built in negative charges from their phosphate backbones
How does the conformation of DNA effect Agarose Gel results?
- Supercoiled DNA will usually run the quickest
- Linear DNA is typically slower than supercoiled, though that depends on the degree of supercoiling
- Open Circular DNA (aka "nicked DNA"), which are plasmids with only a single ssDNA cut, will typically run the slowest
How does the percentage of Agarose effect running the gels?
The higher the percentage of Agarose, the smaller the pores
1.2% to 1.5% agarose is used for DNA fragments less than 1000bp
0.7% to 1% agarose is used for larger DNA bands between 1000bp and 5000bp
If you wished to run a DNA sample greater than 5000bp's, would you be able to run it on a normal Agarose Gel?
No; You would be required to use Pulse Field Gel Electrophoresis which varies the current direction from multiple electrodes to increase sample separation
Can RNA be run on Agarose Gels?
Yes, but Formaldehyde must be added to keep the RNA from denaturing and allowing it to migrate properly
Briefly describe UV Shadowing
UV Shadowing is a DNA visualization technique performed after Agarose Gel Electrophoresis
Involves placing gel directly under UV, and the DNA (which absorbs at 260nm) will show as shadows in the gel
Requires large amounts of DNA!
What is the most common method of DNA visualization on an Agarose Gel?
Ethidium Bromide (EtBr) Staining is the most common method of DNA visualization following AGE as it is cheap and very sensitive
EtBr intercalates DNA, and is incorporated into agarose prior to polymerization or soaked after electrophoresis
Can achieve nanogram detection
Is a carcinogen
What are alternatives to using Ethidium Bromide?
Use of special dyes such as SYBR Green
or Methylene Blue
- Both bind to DNA and are safer than EtBr
- Not nearly as sensitive as EtBr, can only detect in the microgram range
Is Ethidium Bromide the most sensitive method for DNA visualization of Agarose Gels?
No, Autoradiography which uses radioisotopes such as 32P incorporated into the DNA (or other probes) and detect DNA via X-ray films
Time consuming, may use hazardous materials, requires special film
Why might one use Agarose Gels as a purification step as opposed to SDS-PAGE?
- DNA does not require boiling or denaturing
- Only requires very small amounts of sample (nanograms)
How is one able to extract DNA from Agarose Gels?
Gel Extraction using a razor blade on a UV transilluminator, DNA can then be purified using same silica column from Miniprep
Freeze Squeeze method where the band is frozen and squeezed in a parafilm to remove the agarose from the DNA sample
What is the general process/step-by-step of Recombinant DNA techniques
- Isolate the gene of interest and generate fragments (can be genomic DNA or cDNA)
- Insert the gene into a vector (such as a plasmid) to generate a composite molecule with both vector sequences and the gene of interest
- Introduce the composite molecule into living cells for propagation
- Selection process to obtain only the clones that contain the gene of interest
What is a Replicon?
The composite recombinant DNA molecule containing the gene of interest
What are Clones?
Identical copies of a single original cell
What are Restriction Enzymes?
Restriction enzymes are a special type of endoribonucleases that are DNA cutting enzymes responsible for bacteriophage restriction (Phage only able to replicate within most previous host)
What differentiates Type I, II, and III Restriction Enzymes?
Type I and III bind to DNA and digest it at positions where adenines are methylated
Type II bind to specific DNA sequences, and are thus much more useful
Where do Restriction Enzymes typically recognize and bind?
Generally recognize 4, 6, 8, or 12 nucleotides and are often dimers and therefore cut at palindromic DNA (such as 5'-GATC-3')
What is Palindromic DNA? Give an example
DNA that is the same sequence in both compliments
Which Restriction Enzyme is most used (based upon number of base pairs recognized)?
Typically the ones that recognize 6 base pairs are chosen as six nucleotides occur at random only every 4096 nucleotides, which is much less than the frequency of 4 base pairs
Why are Restriction Enzymes that recognize four base pairs typically not used?
A four-base sequence does not allow much specificity because statistically all four base sequences will occur every 256 base pairs if the DNA is 50% GC base pairs and 50% AT base pairs
How do cells prevent Restriction Enzymes from cleaving their own DNA?
Cells prevent their restriction endonucleases from cleaving their own DNA by methylating their DNA sequences
How are Blunt Cleavage Termini produced?
Occurs when digestion within the recognized DNA sequence by Restriction Enzymes occurs on both DNA strands in the center of the recognition site
How are Overhang Cleavages produced?
Occurs when Restriction Enzymes cleave at staggered positions on each DNA strand
What are the primary benefits of Blunt Ends vs Sticky Ends?
Blunt Ends: offer cloning flexibility
Sticky Ends: improve cloning efficiency and specificity
_______ are restriction enzymes from different species that digest at the exact same sequences in the exact same manner
Isoschizomers are restriction enzymes from different species that digest at the exact same sequences in the exact same manner
_______ are restriction enzymes from different species that digest at the exact same sequences but cut in different manners (often due to a difference in the overhangs)
Neoschizomers are restriction enzymes from different species that digest at the exact same sequences but cut in different manners (often due to a difference in the overhangs)
What is the purpose of Flanking Bases?
Most Restriction Enzymes need two or more flaking bases placed at the 5’ end of the restriction enzyme cut site (usually a GC) to bind to the DNA and digest is efficiently
How would the presence of BSA effect Restriction Enzymes?
BSA is able to stabilize restriction enzymes and improve their efficiency as well as precent the enzymes from sticking to the pipettes and microcentrifuge tubes
What is Star Activity, and how might it become increased?
Star Activity is the occurrence of Restriction Enzymes cutting at an incorrect sequence
Chances increase with prolonged reaction times, incorrect buffers used, Metal cofactors present, improper DNA/enzyme ratios, or presence of Volume Excluders (such as Glycerol)
What are the major benefits of DNA Polymerases?
- 5' → 3 downstream exonuclease activity for primer removal, digest DNA or RNA, or repair damaged DNA
- 3' → 5' proofreading exonuclease activity (repair misincorporated base pair)
What is a Klenow Fragment? When is it used?
- it is a DNA Polymerase I that has been cleaved by Protease Subtilisin
- Retained its 5’→3’ polymerase activity and 3’→5’ exonuclease activity, but does not retain the 5’→3’ exonuclease activity
Often used to fill in 5' sticky ends, copy ssDNA, or prepare probes
What are the characteristics of TAQ Polymerase and Pfu?
- TAQ Polymerase is able to withstand high tempuratures, is very efficient at copying DNA, but lacks 3' → 5' proofreading activity; able to add A overhangs
- Pfu has a very slow copy rate, but excellent proofreading capabilities; produces blunt ends
Sometimes the two are combined to maintain high rate of synthesis and a low mutation rate
What are the characteristics of T4 DNA Polymerase?
- Used to fill in 5' overhangs via standard DNA synthesis
- Chew back 3' overhangs via 3'→5' exonuclease activity
How does DNA Ligase function?
Ligates two pieces of DNA by using ATP to catalyze a covalent intermediate with DNA's phosphate backbone, which can then be joined to a nearby phosphate from another DNA strand
How would a research be able to ligate two pieces of DNA without DNA Ligase and skip use of restriction enzyme digestion?
Use TOPO TA Cloning
- A vector containing T overhangs attached to topoisomerase is mixed with an insert with A overhangs (such as a PCR product, TAQ can add Adenines)
- The two sequences anneal, topoisomerase seals phosphodiester backbone and disassociates