Hey dearest course mates! Hope all of you are doing fine. It’s already been a month and I miss you guys and TP so much! ((:
Okay, besides those irrelevant things, I am attached to a research company together with Lyn, so our information might be similar.
My company is focusing on Pharmacogenetics. Now, you might be asking what is Pharmacogenetics? Is it the combination of Pharmacology and Molecular Biology/Genetics?
The answer is yes. Basically, pharmacogenetics is the study of genetic variations (polymorphisms) in individuals that bring about a different response to the same drug. Thus, for example, person A might take the drug and benefitted from it, whereas person B is administered with the same drug, but suffered from adverse drug reaction.
The 3 main areas that are extensively studied for their genetic variations involves gene that codes for 1) receptors, 2) transporters and 3) drug metabolising enzyme. As we have studied previously in Pharmacology, drugs need to bind to receptors, need to be transported to a specific target/ receptor and also need to be metabolized by certain enzymes. Thus, if it happens to be that the genetic variation in an individual is the one affecting the receptor, transporter or metabolism enzyme, then, the person would show a different pharmacodynamic and pharmacokinetic response.
Okay let’s recall! Pharmacodynamics is the study of what the drug does to the body while pharmacokinetics is the study of what the body does to the drug (absorption, distribution, metabolism and excretion). Thus, with that, the person would either have a lack of an efficacious response or more side effects.
Now, do you know why the same medicine might work very well for your friend (eg) and not for you, or vice versa? :)
To summarise what I am suppose to carry out for my research, my main objective is to find out the genetic polymorphism of a certain transporter gene, between 3 different ethnics group: Chinese, Malay and Indians and detect significant polymorphism of the drug that might affect its transportation factors, thus affecting the drug level in the body. The type of polymorphism that I am suppose to find out on, is SNPs, which is a single nucleotide polymorphism.
What is SNP? It is a single base (A, T, G and C) change that is found throughout the genome. For eg ATT GCC TCA to ATT GCC TAA. So, how does one nucleotide/base affect the gene? Because 3 nucleotides make up a certain codon that expresses for a certain protein, a single base change would change the codon and will result in the expression of a different protein. However, not all SNPs are significant, as it is common among individuals and does not affect the expression of proteins. Thus, for those SNPs that are significant, we can detect it by analysing the gene sequence carrying the SNPs. This will be explained in further details in later post.
As for now, I would describe the first step that is needed to be carried out, that is primer optimization using test DNA.
Primer optimization
Primer optimization is done to find the appropriate optimum annealing temperatures (temperature at which primer binds to DNA) that is needed later during the PCR (polymerase chain reaction) to make multiple copies of specific target sequence to be analysed.
As most primers’ annealing temperature is between 55-65oC and most appropriate number of cycles is 30, I can start testing the primers at that temperature and cycle number.
Methods
1. Prepare a master mix of 13 times the stated amount (need only 12 tubes but 13 times to overcome pipetting errors)
(total: 12 tubes)
Component(s): H20, 10x Buffer, MgCl2 (25mM), dNTPs (10mM), Forward Primer (10µM), Reverse Primer (10µM), Taq polymerase, Test DNA
Total Sample x 1 (µL): 6.9, 1, 0.6, 0.3, 0.2, 0.2, 0.3, 0.5 respectively. Total: 10µL
Sample x 13 (µL): 89.7, 13, 7.8, 3.9, 2.6, 2.6, 3.9, 6.5 respectively. Total: 130µL
[ Sorry I had to put it this way. Blogger doesnt allow to put table, right? I cant seem to copy and paste my table from word. :( ]
2. Prepare an ice-box.
3. Collect the different reagents and place them in the icebox. (Taq polymerase is to be collected last after adding all the other reagents as it will denature at room temperature or less than 20 degree Celsius if it is removed for too long)
4. Prepare a strip of 12 tubes and label them 1-12
5. Add in all the reagents into the master mix tube.
6. Place the remaining reagents back into the respective fridges.
7. Spin the master mix tube down for a few seconds to ensure all reagent is mixed
8. Pipette 10µL from the master mix into each of the 12 tubes.
9. Spin down the tubes again for a few seconds, to also remove all air bubbles.
10. Place the 12 tubes into the Thermogradient (thermocycler)
11. Place 4 empty tubes at each of the 4 corners to balance it.
12. Programme it at 55-65oC and carry out for 30 cycles.
Conditions of Thermogradient
Step 1: 95oC x 3min
Step 2: 94oC x 45s
Step 3: 55-65oC x 45s
Step 4: 72oC x 45oC
Step 5: Repeat steps 2-5 x 29 (total 30 cycles)
Step 6: 72oC x 7min
Step 7: 15oC x Infinity
-End-
This reaction takes about 1 and a half hours, so to not waste time, agarose gel is to be made (2nd step). I will explain about it in my next entry so my post wouldn’t be too lengthy ya, and before I bore you guys further. :)
So this is it, any queries, feel free to ask, I won’t bite. Hehe. Take good care of yourself and I’ll see you soon on the 25th!
Best regards,
Liyanah Zaffre
0607718D
TG02
Week 4 ( OMG 1 month already! ) :)
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12 comments:
July 18, 2008 at 8:17 PM
Hello Liyanah
Yoz, we miss u 2 ... we all r meeting nxt friday yar ... Btw i have some questions to ask you :)
1. Can you explain the specific types of receptor, transporter and drug metabolising enzyme and is mechanism of action in the application you are researching in?
2.When carrying out research on genetic polymorphism on the different races, what are the other factors you will have to keep constant to minimise varaitions in results? (e.g diet, lifestyle) ... plz explain..
3. What happens if there are more than a single nucleotide polymorphism? e.g. 2 nucleotide changes... will it affect experiment? will it make disease worse?
4. When using primer optimisation, when the temperature is rising to 55-65 degree Celsius, before reaching tje optimal temperature, will it already start to amplify? If yes will it lead to non specific products? What are ways to troubleshoot if non-specific products are produced?
5. What are the other factors to consider when doing primer optimization? (e.g salt conc, pH)
6. Why carry out 30 cycles of PCR? Why not 29 or 31?
7. What is the significance of using a thermogradient?
8. Are you going to run gel electrophoresis later on? If yes, why use agarose and not polyacramide? What is disadvantages and advantages of polyacramide and agarose?
Thanks for answering my queries ... Some questions may haf no answers u need not answer them :)
From: Ma Xianwei Benjamin
Class: tg01
0606181F
July 19, 2008 at 2:49 PM
Hi Liyanah
Juz one question.....why use 12 tubes?
Cya on coming fri =D
Xin Ni
TG02
July 20, 2008 at 5:51 PM
To Benjamin:
Hey Benjamin!
Woahhh, you and han yang uh, are VERY good at posing many tricky questions uh! Can set exam papers ready! Hehe.
Anyway, okay here are the answers to your doubts:
1. The project/research that I am working on is focused on only a transporter gene thus, the single nucleotide polymorphism of this gene is only affecting the transporting factors of the drug to its target, whereby in this case, it will not affect the receptor or metabolism enzyme functions.
What happens is that, as you know membrane transporters are responsible for the absorption of drugs in the GI tract, excretion into urine or bile, transport across blood brain barrier and into sites of action, such as cardiovascular tissue, tumor cells,and infectious microorg. Accordingly, polymorphism of transporters will have an influence on the bioavailability of drugs, thus affecting the person’s response. I have not gone into detail on the gene I am researching on, and thus, I cant tell you where the snps are and how it can affect the response, but in general, it affects the bioavailability.
As I am not dealing with a gene that codes for a receptor or a metabolism enzyme, i cannot apply it in my research, but if you want to know a few examples on the current research on those, you can ask me okay. ((:
2. Okay, firstly, the reason why 3 different ethnic groups are used in this study is that the people of the same race tend to have similar snps at certain part of gene that might affect their response to drugs. But, in this case, we usually conduct on a random basis as yes, lifestyles and diet could affect a person response to a drug, BUT that is not what we are looking for. We want to know the snps at the gene that affects the response, and this is genetically inherited. Of course, when conducting the experiment, everything else must be constant like the DNA concentration, Taq polymerase concentration and etc.
3. When there are 2 nucleotide changes, then it is no longer a SNP, as the name implies it is a SINGLE nucleotide change. However, it is still a polymorphism (there are many types of polymorphism actually but I am only concentrating on SNPs). And when that happens, it might or might not even affect the response of the person to the drug as some change might not be significant, some change might be significant. And that’s why we do population studies to see which polymorphisms are the novel ones.
4. 55-65 degree Celsius is like only a ‘guess’ of the range of temperature that most primers will anneal. So, we can try it out at 55-65 degree Celsius and see if we got bright and sharp bands (will be discussed further in future post). But there are some who doesn’t anneal at 55-65, as it might anneal at a higher or lower temperatures. This will then results to non specific binding or no bands at all. And if it happens that this happens, it will signal to us, to change the temperature either higher or lower till sharp bands are produced.
5. As primer optimisation is to find out the optimum annealing temperatures, the number of cycles needed for the primers to amplify, the salt concentration (MgCL2), and the pH. These are the factors we have to look at when conducting the experiment.
6. Oh, actually, what I have written down is just an example of the first try to amplify a primer. As we don’t know the optimum temp and the no. Of cycles the primer would amplify, we try it on the common optimum annealing temp and cycles eg 30 and see if we got bands. However, yes, some primers do amplify at 29 or even 31 cycles, but then again , depends on the bands produced to alter the conditions.
7. A Thermogradient is actually a type of thermocycler, but it has a few more advantages than just a normal thermocycler. Basically, what a normal thermocycler does is to perform the PCR reaction: denaturation, annealing, extension, amplification. We just need to key in the conditions we want to set for the primer and machine will do all the work. But the difference between a normal thermocycler and a Thermogradient is that, the Thermogradient can amplify the primers at a range of temp eg 55-65, but the normal thermocycler can only amplify at one temperature eg 58 degree Celsius.
8. Yes, after primer optimization, i need to run gel electrophoresis to visualize the bands. Why agarose is used and more about gel electrophoresis will be covered all on the next post, so stay tuned. Haha ((:
Okay, phew. It feels as though I just complete a term test paper on my sip. Hehe. Not that it is bad, its good actually, test my understanding on the whole thing. ((:
Liyanah Zaffre
0607718D
TG02
July 20, 2008 at 10:30 PM
To Xin Ni:
Hey Xin Ni!
When the temperature gradient is set up, it gets distributed across the 12 columns in the gradient cycler. (Gradient cycler has 12 x 8wells to put in the tubes btw) So we generally use the whole range, but sometimes later on, if necessary, we can use fewer tubes once exact temperature is known.
Liyanah Zaffre
0607718D
TG02
July 20, 2008 at 11:42 PM
To Liyanah
Haha, thanks for asnwering my questions. It has indeed enhanced my understanding on primer optimisation and thanks for putting so much efforts in answering the questions :)
From: Ben
July 21, 2008 at 8:48 AM
Woah, looks like you love your workplace... Lolz.
Anyway, I have a question or two for you:
1) Could you list some examples of what cause SNP?
2) And oh, I see you used a 10x buffer for the preparation of the master mix...
What is the purpose of the buffer and why not other concentration volume, other than 10x?
That's all to my questions, thanks for taking your time in looking through them.
And yes, hope to see you soon too =)
Many thanks
Quan Jun
TG02
Group 08
Posted: 21 July 2008
July 22, 2008 at 10:52 PM
July 22, 2008 at 10:54 PM
To QJ:
love my workplace? err, maybe, maybe not. haha. no la, its fine laa :)
1. SNPs are actually genetically inherited, so there are no causes to it actually. Just depending if you inherit either a heterozygous or homozygous dominant of the mutant allele.
2. The 10x buffer used is to maintain the optimum pH and salt concentration for the DNA and the taq polymerase.
actually the initial concentration volume of the buffer doesnt really matter, because depending on the company that you pruchase your buffer from it can be 10x or even 50x. We just have to ensure that the final buffer concentration when preparing the master mix is
1x.
Liyanah Zaffre
0607718D
TG02
July 23, 2008 at 12:39 AM
hey liyanah
when you say spin in steps 7 and 9, do you use a centrifuge machine, or just a vortex? and will there be any difference if i use either one?
thanks
rusydiana
July 23, 2008 at 1:24 AM
To Rusydiana:
hello! how come i never see you around although we like quite near. haha.
anw, umm, what i mean by spinning down is using either a mini centrifuge (if using the strip tubes)or the big centrifuge we used in our labs.
Yes, there is a difference in using either vortex or a centrifuge. We dont normally vortex as samples contain enzymes eg taq, thus enzymes are senstive and cannot be vortex.
Liyanah Zaffre
0607718D
TG02
July 23, 2008 at 8:50 PM
hey, you have a really interesting job. Anyway, i'm interested to know the results of your ethics test. Oh, and lets assume that 2 people of different races mate (chinese and malay), will the offspring's pharmacodynamic and pharmacokinetic response be more similar to the chinese or the malay? And will the pharmacodynamic and pharmacokinetic response be affected by gender?
-cornelyus
July 24, 2008 at 9:40 PM
To cornelyus:
eh hello!
umm, the pharmacodynamic and pharmacokinetic effects will only be affected/ different if person have a novel SNP that affect the response. This SNP is genetically inherited.
so back to your question, if the person is mixed, it actually depends on the gene she inherits, the allele which is more dominant will affect and show the response.
and no, it has not been reported that gender affects the genetically inherited SNPs.
Liyanah Zaffre
0607718D
TG02
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