PCR PRINCIPLES SOS! - PCR (Dec/16/2008 )
HI everyone!!! if any could help with this I will really apprecite it. I had some troubles amplifying buccal cell samples for PCR-RFLP, so I had to make some changes: I added to the reaction many extra things: Amonium sulfate, 2-mercaptoethanol, EDTA (Chelating agent), BSA (known helper) and MgCl2 (essential for DNApolymerase) instead of 10X buffer taq and it worked for 30% of all the samples, 100% for blood samples (even with 10X buffer taq). I have searched in many books, internet links, even a doctor but he couldnt tell me whats the function of all the PCR aditives, I know it works but I dont know why. I know Mg is an coenzyme and EDTA is a ion collector but the others?? I looked up the 10X buffer taq and it has Nonidet and KCl??
please help me out. Thanks!!!!!
please help me out. Thanks!!!!!
EDTA is deadly for PCR as it captures the needed Mg2+ as you wrote yourself. A little bit (e.g. in the TE buffer for template DNA) is okay, but larger amounts really stops the PCR reaction. Why don't you use the provided Taq buffer? Generally it is the best you can do, except in difficult template DNAs and special cases where some additives (to the provided buffer) can be helpful, except EDTA of course.
Which additives and their function, here a simple google search will do the job. Or a search here, many or all of the compounds are explained and discussed in old threads.
Thank you for answering!!! well its a little difficult to find specific function, I have all day long searching and there are some papers but I have to pay for them so it doesnt help at all even with abstracts. I have no possitive results with taq buffer for buccal cell extract PCR, and one doctor gave me the new buffer, he got it from glasgow university but he doesnt know the functions at all (a little embarrasing I guess). I know many are just for GC-rich sequences and primer specificity but there is not such scientific explanation, just everybody make a guess, maybe the explanation its too simple to put it into a investigation paper, thats why I enter this forum. But thank you anyway!
For crude samples, your best bet is to dilute the sample much more than you would guess. Try again with 100x and 1000x dilutions of your buccal samples in the normal buffer. Try adding 5% betaine if you think there is high GC present. You have too many variables, by far, to make progress in optimizing them. I would go in the other direction-- use a master mix containing your enzyme and dNTPs and buffer in one tube. When it's working and you have time to optimize on cost (e.g.) then go for the individual components.
Thanks!! well the first thing I did was a 1:100 dilution but it didnt work, thats why I worked with the DNA extracted direct from mouthswabs and at least 30% worked. The thing is that at the first time I worked with blood-DNA and it worked beautiful but when I changed to mouthswabs and saliva everything just got negative. My last resource was the use adjuvants but BSA didnt worked and in a desperate condition I tried the buffer 11X (with all the adjuvants) and it worked but I dont have more time to test each one of the adjuvants, so Ive heard that its the total combination that works but still dont know the effect of the adjuvants in PCR, thats all the problem. What is the role of the PCR adjuvants. Thanks!!!
BUFFER 11X
Tris Hcl pH8,8 45mM
Ammonium Sulfate 11mM
MgCl2 4,5mM
2-Mercaptoethanol 6.7mM
EDTA 4,4mM
BSA 113ng/ul
Some adjunts that do know.
BSA - a protein sacrifice that is adsorbed to plastic surface of the PCR tube. BSA thus increases the availability of the DNA polymerase to the PCR reaction, as less DNA polymerase is stuck on the PCR tube.
glycerol - disrupts water structure. Apparently it makes the water structure more cell like, as a cell cytosol isn't water but an protein fill solution.. which can have gel like behaviour. It basically makes the DNA polymerase "happier".
reducing agents - (DTT) prevents oxidative damage to the DNA polymerase. Increase half life. only important for long range PCR.. ie >15kb
betaine - disrupts water structure, allows DNA to melt at a lower temperature. Only use with GC rich template
Mg2+ - required for DNA polymerase activity. Polymerase specificity is also lowered with Mg2+ concentration. However high Mg2+ concentration >500uM inhibits the DNA polymerase Also require as dNTP chelates Mg2+. As a result if you increase the dNTP concentration, you have to also increase Mg2+ concentration
Salt concentration - high salt PCR buffer tends to select for shorter PCR product, as longer PCR products take longer to melt. Converse low salt PCR buffers are better when amplifying long PCR products
DMSO - also disrupts water structure. Melt secondary structure of DNA template. (ssDNA can anneal to itself) Make the polymerase job easier.
EDTA - Can help clean up the PCR signal by reducing background. Makes the signal sharper. It increase specificity by chelating contaminating divalent ions (eg Mn2+, Co2+). However too much and it kills the PCR reaction. Personally have never seen a reason to use this. Varying the Mg2+ concentration is usually good enough.
PCR reaction volume - at this time point most PCR machines can not change the temperature of the PCR reaction volume instantaneously. this results in a significant lag when the temperatures are cycles. As a result PCR reactions are most efficient and accurate when the volume is small. As odd as it is, smaller often better. You can often see things when the volume is smaller. thus it is better to do 5x 20ul than 1x100ul.
Thermal stable Uracil-DNA glycosylase - breaks down CTP that has been deaminated by temperature. Miss incorperation of UMP into DNA causes stalling of the DNA polymerase upon detection of this missincoperation. As no DNA repair machinary is available in the in vitro PCR, this stalling ultimately causes the polymerase to fall off the template cause formation of truncated PCR products.
BSA - a protein sacrifice that is adsorbed to plastic surface of the PCR tube. BSA thus increases the availability of the DNA polymerase to the PCR reaction, as less DNA polymerase is stuck on the PCR tube.
glycerol - disrupts water structure. Apparently it makes the water structure more cell like, as a cell cytosol isn't water but an protein fill solution.. which can have gel like behaviour. It basically makes the DNA polymerase "happier".
reducing agents - (DTT) prevents oxidative damage to the DNA polymerase. Increase half life. only important for long range PCR.. ie >15kb
betaine - disrupts water structure, allows DNA to melt at a lower temperature. Only use with GC rich template
Mg2+ - required for DNA polymerase activity. Polymerase specificity is also lowered with Mg2+ concentration. However high Mg2+ concentration >500uM inhibits the DNA polymerase Also require as dNTP chelates Mg2+. As a result if you increase the dNTP concentration, you have to also increase Mg2+ concentration
Salt concentration - high salt PCR buffer tends to select for shorter PCR product, as longer PCR products take longer to melt. Converse low salt PCR buffers are better when amplifying long PCR products
DMSO - also disrupts water structure. Melt secondary structure of DNA template. (ssDNA can anneal to itself) Make the polymerase job easier.
EDTA - Can help clean up the PCR signal by reducing background. Makes the signal sharper. It increase specificity by chelating contaminating divalent ions (eg Mn2+, Co2+). However too much and it kills the PCR reaction. Personally have never seen a reason to use this. Varying the Mg2+ concentration is usually good enough.
PCR reaction volume - at this time point most PCR machines can not change the temperature of the PCR reaction volume instantaneously. this results in a significant lag when the temperatures are cycles. As a result PCR reactions are most efficient and accurate when the volume is small. As odd as it is, smaller often better. You can often see things when the volume is smaller. thus it is better to do 5x 20ul than 1x100ul.
Thermal stable Uracil-DNA glycosylase - breaks down CTP that has been deaminated by temperature. Miss incorperation of UMP into DNA causes stalling of the DNA polymerase upon detection of this missincoperation. As no DNA repair machinary is available in the in vitro PCR, this stalling ultimately causes the polymerase to fall off the template cause formation of truncated PCR products.
Hey!! Thank you very much!! Its great!! Do you have any papers that can backup that??
somewhere. I'm sorry. I don't have them on hand.
And ooo... so many spelling mistakes. I must have been quite tired when I wrote this.
And ooo... so many spelling mistakes. I must have been quite tired when I wrote this.
Thats Ok, Thanks!!!!