DNA Flashcards
What is the restriction modification system of EcoRI?
EcoRI methyltransferase methylates the second A of the GAATTC sequence; Eco RI restriction endonuclease activity cuts between the T and C of the PALINDROMIC sequence if A is not methylated; this results in staggered cuts which leave sticky ends
What are the restriction enzyme types?
Type 1, R+M; Type 2, R; Type 3, R
How does a type 1 RE function?
A specific sequence is recognized, and a random sequence is cut hundreds of bases away; this protein contains both a restriction component and marking/modification component, so it is difficult to isolate just the restriction activity
How does a Type 2 RE function?
A specific sequence is recongized (4-8 bp in length, palindromic) by a single protein WITHIN THAT SEQUENCE
How does a type 3 RE function?
A specific sequence is recognized, and a cut is made 25bp away; we can isolate the RE, but multi-subunit protein is problematic for purification
Remember Type II enzymes may recognized 4, 6, or 8bp enzymes; cut WITHIN the palindromic sequence–will create 5’ overhangs, 3’ overhangs, and blunt ends; they also require specific conditions such as pH, temperature, Mg2+ or NaCl
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What is star activity?
It is relaxed specifity resulting from improper conditions for the enzymes;
What are the generic ingredients for RE reactions?
10U or 1uL of enzyme, 1g DNA, 5 uL of 10X NEBuffer, BSA–add to a final concentration of 100ug/mL (1X?); top reaction up to 50uL; incubation time is usually 1 hour, but if enzyme added is really concentrated it can be much shorter (5 min maybe); temperature of the reaction is enzyme-dependent
How would one run a digest that uses two enzymes?
Either through a double digest, but only if the enzymes are compatible in their needs (especially in buffers)?, or run one reaction, stop and change buffer, then run the second reaction (or heat kill the first reaction by heating to 65C for 5 minutes, then changing the conditions to make it favourable to the other enzyme)
Why is DNA run on AGAROSE gel?
Because DNA segments are far larger than proteins, so they require larger holes for separate; small DNA segments (
Describe the general procedure of running an agarose gel
agarose is poured into tray and comb is added; gel is immersed in buffer (usually TAE, TBE, or SB); DNA+dye+glycerol is added into the wells; Current of 5-35mV/cm is applied to draw DNA (-ve) towards the anode (+); if you’re running on an SB gel you can run at 5X higher voltage (this will make it get hotter but that’s okay, the picture will still develop)
How do we visualize DNA on agarose gel?
Use EtBr+UV light–> orange bands; EtBr fits between DNA bands and glows more brightly there than when surrounded by water molecules;
Or SyBr+Blue light–>green bands
Remember:
Downside of EtBr: it’s a mutagen
• Since it fits between DNA, it can produce frame shift mutations in the user’s DNA because it stretches the gel and doesn’t let it line up properly
○ So people have moved to SyBr green, which works in the same way, except it doesn’t permeate through the cell membranes easily
○ EtBr is cheaper, so that’s why some people might not use SyBr green
§ Also if you already have a UV light transmitter, you may just want to stick with EtBr
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How do we solve the issue of resolving bands of DNA >20kbp long on agarose gel?
Above this length, DNA will not separate based on size due to reptation; so we need pulse field gel electrophoresis which will switch the direction of the run to counter reptation; therefore, fragments up to 1Mb can be resolved
PFGE can be used to create “fingerprints” for strains of bacteria–cut bacterial chromosome with rare enzyme and run on PFGE
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How are foodborne illness tracked?
PFGE
Miniprep generic procedure
culture is spun and pelleted to remove broth; cells are resuspended in Tris, EDTA, Glucose or Salt; Cells are lysed by NaOH, SDS, boiling, or lysozymes; solution is treated to ppt debris; (acidic potassium acetate needed if NaOH was used, not needed if sample was denatured by boiling); debris is spun out and plasmid remains in supernatant
What is in a PCR reaction mixture?
1X Taq buffer (10X Taq buffer, take 5uL and put in 50 uL reaction mixture); sterile deonized water; 0.2mM dNTP mix (take 5uL of 2mM dNTP mix and put in 50 uL reaction); 0.1-1 uM of Primer I and Primer II; 1.25U/50uL of DNA Taq polymerase; 25 mM MgCl2* VERY IMPORTANT FOR TAQ ACTIVITY; IF EDTA OR A CHELATING AGENT IS PRESENT IT WILL BIND UP MG SO TAQ CANNOT WORK*; Template DNA; 10pg-1ug
Per template, how many more dNTPs, primers, and enzymes do you need?
10 billion times more dNTPs; 10 million times more primers; 100 thousand more enzymes
Remember that the amount of initial primer put into PCR solution determines the maximum amplification possible
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Remember that the amount of enzymes becomes limiting after ~20 cycles
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What are useful alterations of Taq?
Make enzyme more processive–>copy longer regions or copy faster; hot-start taq–>make it only have activity at 70C, either by incorporating it into a ball of wax, binding it to antibody, or making it cold sensitive; also making it more tolerant of improper conditions (such as Mg2+)–>it will still amplify in sub-optimal conditions
Remember that annealing temperature is 5c below Tm
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What are the negative controls in a PCR reaction?
A mixture of everything, but with no template DNA
What is a gradient PCR machine useful for?
Finding the best annealing temperature
What are PCR applications?
Primers can be designed to give different sizes for function/nonfunctional genes; primers can be usd to amplify ONLY functional/nonfunctional genes; primers can be designed to give a product that is cut/not cut with a RE to reflect the presence of a disesed gene; PCR can be used to amplify a diseased region for sequencing
What is the PCR application Mutagenesis?
A primer with a single nucleotide mismatch, or small insertion or deletion, is amplified and cloned into a vector and studied
How does PCR sequencing work?
copies are amplified, but dNTP analogs lacking a 3’ OH are introduced into the mix, which will create products of different length (and coloured) and the sequence can be deduced
How does RT-PCR work?
It amplifies a ssDNA piece from RNA (reverse transcriptase converts RNA–>ssDNA)
What is qPCR and what is is used for?
Quantitative PCR and it is used to monitor the mplification of genes; it is compared to a known control. If more products are produced relative to the control, we know there is an over expression of that gene; if there is less, then their is an under expression (good for testing disease vs non-diseased states)
PCR is a DNA “photocopier”–>amplification through actual polymerization, and not cloning
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