Subject: PCR Techiques
Hi all! =) This is Lyn here. I will be the first in my group to share with you guys (and my group mates) about the things that i have done this first week of my attachment.
I'm attached to the research lab doing research relating to pharmacogenetics. My MP will be on that as well. Pharmacogenetics is basically the study of how the variability of the genetics of individuals affect the reponse to drug dosage. This is because, if an individual has a variant in his/ her gene, he/ she might need a lowerd drug dose as compared to an healthy individual with no variants. Thus, pharmacogenetics enables the prescription of the correct drug dose to the patient so as not to result in drug toxicity.
Bascially, what I do in the lab for this one week was preparing the PCR products using different primers, cast 1.5% and 2% agarose gel to carry out gel electrophoresis and the PCR which includes primer optimisation. Doing primer optimisation is actually to find out the optimum temperature whereby primers anneal to the DNA sample and result in bright sharp thick bands. I also get to do gel check for the PCR products that are prepared. It is similar to what we were shown during Mgen or Mbio practical sessions. However, in the lab, they use this machine known as the Gel-Doc to visualise the bands. In this machine, you place your completed agarose gel from electrophoresis process and turn on the UV so as to 'bring out' the bands. This machine is connected to a computer whereby there's a programme where you can print the video print of your agarose gel out. It comes out as a photo.
I also learnt about purification and sequencing. Purification is done actually to remove the excess primers and dNTPs that would interfere with the sequencing procedures. In purification, Exo-SAP is added to the PCR products. Exo = exonuclease 1: it cleaves and remove the excess primers. SAP = shrimp alkaline phosphatase: it cleaves and dephosphorylates the dNTPs so that they are unable to bind and carry out elongation.
Sequencing is done to determine the actual sequence of the DNA that is prodcued during the PCR that is complementary to the template DNA strand. This is done by adding the primers and the dye that contains dNTPs, ddNTPs and Taq polymerase. These ddNTPs are fluorescently labelled so that they can be detected and the sequence of the DNA strand complementary to the temlate DNA strand can be known. After the addition of the reagents needed, the samples are sent to the analyser, whereby the analyser will analyse the different ddNTPs that binds and also produce the peaks.
These are bascially what I have been doing for the first week of attachement.
Have fun at work for SIP. Take care everyone. =)
-Lyn-
posted by THE CODEC 5 @ 11:02 PM
17 Comments:
Hi there, hope you have enjoyed your first week of the attachment.
I'm actually quite interested in what you are doing, which is pharmacogenetics. Anyway, i have some questions to ask about.
You mention that you are researching on pharmacogenetics but i'm sure that is pharmacogenetics already established? As in is it proven that variants in genetic does make a different? And what do you all look out for in the gene? Is it the presence or absence of certain DNA sequence that is affecting the respond to drugs or something else?
Thanks!
Chew Yu Mei
Tg01
Hi!
Basically, studies on pharmacogenetics are already established. As in, there are researchers in the world doig studies on this. And from the reviews and articles I was given to read on, it shows that in actual fact, a variant in cetain important genes does have an impact on the absorption or metablism or excretion of a drug. Furthermore, presence of a variant for a certain gene can lead to toxicity or even death due to ADR.
In order to identify the variant, we do PCR, purification and sequencing. However, firstly we have to know the sqeunece of the healthy individual. From there, we are able to see the difference in the healthy and patient's DNA squenece. Further experiments will then be done to see which drug dosage is suitable for the patient. For this part, I'm not taught yet.
Yar. That's about it. Hope I answered your doubts and understand what i explained. =)
Lyn
TGO2
Hey..
whats the difference between the machine known as Gel-Doc with the machines that we use in school?
Andika Putra
TG01
hey babe. your first week sounds interesting.
anyway, could you please explain a bit more to me what you meant by "analyser will analyse the different ddNTPs that binds and also produce the peaks"?
thank you!
Nurathirah
Tg07
oops, i meant TG01!
why do they call SAP SAP? Is it because its really from shrimp?
--cornelyus--
Hi Lyn!
I would like to find out from u more about the primer optimization. How do you do it? Do you all use software? Thanks a lot =)
Jean Leong
TG02
To the bmt, the machine use we use to visualise the bands from the agarose gel in school is much simplier. As in, you just have to on it and place your ge on it and you can visualise the bands. As for the Gel-Doc that is used in the lab I'm in, it's more advance and with more functions. The Gel-Doc is basically connected to the computer so you visualise your bands in the computer itself. You can also adjust the light intensity and magnification of how the gel appears in the computer. The Gel-Doc also uses the UV to amplify the bands for us to visualise. The good thing about this machine is that since it's connected to a computer, you can get the image of the bands printed out. After which, you can analyse the bands.
Hope i answered your question.
Lyn TG02
To hellomedtech,sorry if i made it sound confusing to you.
What i meant was the analyser will detect the different ddNTPs that binds. As we have learnt, when a ddNTP happens to bind to the DNA strand, no other dNTPs or ddNTPs can bind. Thus, the process stops. As the ddNTPs are fluorescently labelled, they can be detected and the sequence of the DNA strand complementary to the temlate DNA strand can be known. The analyser will detect the different ddNTPs that binds and terminates the DNA strand. After the whole sequencing process is done, a file will be produced which contains different peaks of different colour. This tells you the DNA sequence of the DNA strand. We learnt this in Mbio last semester.
Lyn
TG02
To deincredibles,
primer optimisation is also known as the PCR. That is we place samples containing one primer- forward and reverse into the PCR machine to optimise it. What i meant by optimise is that you set a range of temperatures for different samples. This is done in a run and done in the Thermo-gradient. As the temperature for each sample will be different, their annealing temperature will be different as well. This enables us to find out the optimum annealing temperature for that particular primer.
Oh .. we do use softwares but I'm not so sure what they are called. We use software for genotyping. I haven't acutally done genotyping though.
Lyn
TG02
To thatperson,
SAP stands for shrimp alkaline phosphatase. SAP catalyzes the release of 5'- and 3'-phosphate groups from DNA, RNA and nucleotides. It also removes phosphate groups from proteins.
Regarding whether it's from shrimp, the source of SAP is Arctic shrimp Pandalus borealis.
Hope i answered your questions on whether SAP is from shrimp.
Lyn
TG02
To bmt, for more information and details on how to use the Gel Doc, you can refer to this website http://www.calpoly.edu/~bio/ubl/protocols_files/geldocdet.htm .
=)
Lyn
TG02
Hello Lyn,
I am very interested in knowing more about your project, as mine is somewhat similar to yours. I would like to enquire about some things:
1. What are the specific steps in making your primers ( with regards to the size of the primer you are making)
2. What is the principl of Gel-Dpc in aiding visualisation of the bands (e.g isit based on colour)
Thanks for answering my questions
Hi tg01 group 2.
Er .. May i know you are?? 'cause you didn't state your identity.
Well, actually my lab does not produce or 'manufacture' the primers. One of the staff will choose a particular primer needed from a programme. This programme enables you to key in the fragment you want to focus on or amplify and after which, it gives you a range of different primers for you to choose from. As for the manufacturing of the primers, I think the lab I am in send the sequence of the primer to an external lab where they manufacture it.
The Gel Doc is actually a machine whereby you place your agarsose gel that had undergone gel electrophoresis in. As this machine is linked to a computer and has a 'camera' in it. It enables you to adjust the light intensity, magnification and resolution of the gel and bands to aid in better visualisation of your bands. After which, there is a transilluminator in the machine where UV is being exposed onto the gel. After exposure of UV, the bands will be visible. You can do minor adjustment again and after that you can print out the photo that has your bands on the agarose gel.
For more details on the operation of the Gel Doc, you can refer to this website: http://www.calpoly.edu/~bio/ubl/protocols_files/geldocdet.htm
Thanks.
Lyn
Tg02
Hello Lyn,
I am very interested in knowing more about your project, as mine is somewhat similar to yours. I would like to enquire about some things:
1. What are the specific steps in making your primers ( with regards to the size of the primer you are making)
2. What is the principl of Gel-Dpc in aiding visualisation of the bands (e.g isit based on colour)
Thanks for answering my questions
yea, thanks for answering my quesion
By: Ma xianwei benjamin (forgot state my name)
class:tg01
Hi Lyn,
You've mentioned that an individual with a variant in his or her gene need lowered drug dose compared to healthy individual with no variants. Why is that so? What is the variant in gene?
Sharon
Tg01
Hi sip.
A variant in genes mean that you have somepart of your gene being different from any healthy individual. This place you at a risk of getting certain disease or cancer. Most of it is due to single nucleotide polymorphism (varitation whereby DNA differ by a single base or nucleotide).
It is usually more serious and palce you at a higher risk if the variation is in genes of drug-metabolising enzymes. This is because it will affect the metabolism of the adminstered drug. Since it will affect the metabolism of drugs, you have to decrease the drug dose. If not, it will lead to toxicity or serious death.
Hope i answered your question. =)
Lyn
TG02
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