Week 4 ( OMG 1 month already! ) :)

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 3 explanations...

hey everybody.. i suppose it would e easier if i answer everybody's queries in a post rather than in the comments page. anyways, here goes...

to Athirah:
contamination do happen and that's why good pipetting skills is very important. i always try not to let the tip of the pipette touch the wells as it will introduce other contaminants into the wells. however, according to a colleague of mine, the test is specific for mycoplasma pneumonia antibodies and other bacteria are not easily detectable.

to Xin Yi:
interdeterminate means you can't really tell if it is positive or negative. that's why it is indicated as a plus-minus sign. however we do not report as plus-minus. we try to compare the results of one well with the result of the well next or after it. using a ruler to measure the compactness is rather ridiculous actually. so what i was taught was, to observe the tightness of the ring. in other words, which ring appears darker or bolder. the lest tighter ring will be the positive end-point titre. i hope that answers your question S:.
presence of a least compact ring should still be reported and not ignored as it could indicate presence of weaker mycoplasma antibodies. it is simply to be on the safer side lah actually.

to Liyanah:
i dont really miss you but i know you're probably getting skinnier now cos you miss me too much. right? hah!
anyways, the samples are diluted to achieve a titre. a titre is like, series of dilution. we carry out a titre to find out how weak or how strong the antibodies are. if all the dilution is the same, or if everything is done as a neat (1:1), then it would defeat the purpose. we wouldnt be able to tell how "dangerous" the antibodies are. or how wide the disease has spread.
we don't use the awesome multi channel pipettes in serology but we do use them in hematology labs to prepare the reagents in the morning but i shall not go into that for now. haha. reason being, we have to pipette different amounts of reagents or samples each time and multi channel pipette usually gives the same amount of solution each time we pipette.

to Lee Jin:
sensitised particles basically means that the particles are pre-coated with antigens. we learnt this in hematology i think. usually, they come in lyophilised form in the test kits. lyophilised means powdered form. we learnt that in LMQA if i'm not mistaken. lyophilised substances are usually not ready-to-use. therefore, we have to prepare them ourselves by adding the required amount of diluent.

to Han Yang:
- regarding your first question, i'm not very sure why. it's just the way the lab works. we receive samples from hospitals, clinics, etc. and they usually collect samples in the morning, thus we receive them in the afternoon.
- some examples of automated tests are Candida virus tests, Epstein-Barr virus tests which tests for nasopharyngeal carcinoma, Varicella-Zoster tests which is the main cause of chicken pox, and many more. there're really A LOT. we usually carry out these tests using a machine called the EVOLIS and it works using the ELISA method.
- for the third question, i can't really answer that because it is what given in the test kit. probably, rabbit's serum has the same reaction as human serum towards the specific viral antibodies.
- a positive result will show agglutination THROUGHOUT. presence of a button or a ring shows that the sensitised particles are not able to agglutinate due to absence of the specific antibodies and therefore they SETTLE at the bottom of the well. when they settle at the bottom, they form either a compact button or a ring, depending on the presence of the antibodies or the strength of the antibodies if they are present.
- yes, you may assume that the most compact ring is negative as the wells before that shows a less compact ring. however, that doesnt mean the patient is free from infection. due to the less compact ring observed in the wells before, it does indicate presence of the specific antibodies.
- do you mean aseptic environment, for example, close to a bunsen burner or something? sadly, we don't practise that in our lab. but the pipette tips and microwells used are sterile before we use them. and of course, we put on gloves too to protect ourselves and prevent introduction of our bacteria into the wells.
- the reason why some tests remain manual is, there are probably no machines invented yet to carry out such tests. however, special tests such as this MPA tests are usually carried out on certain samples as requested by the hospitals/ clinics. they're usually less than 50 per day. human error is inevitable, thus, secondary checks are very important and are usually done by someone more experienced. we usually do 4-5 samples at one time and it usually take less than half an hour. during the incubation time, of course we carry out other tests on other samples as not to waste time.
- i'm not sure if there are any confirmatory tests. what i know and was taught, the MPA test is specific to test for presence of mycoplasma pneumoniae antibodies. after carrying out the test, we record the results and the results are sent out to respective hospitals/clinics.

wow, that was long.. i hope i've answered all 8 or probably more of your questions. heh..

to Quan Jun:
haha. you're weird. nobody would mistake 1 - 9 as 1 -6 ... cos then, it would only be 5 hours of work.
i think your question is kind of similar to someone's question. well, as i said, a button means that the particles settled at the bottom of the well and not agglutinate. my supervisor told me that we should look out for agglutination THROUGHOUT the well and not settling of the particles in a button or a ring. i know it's a bit confusing. i was confused the first time too but i was explained as such so yea.. if wou try to disrupt the button, the particles would dissolve and still NOT form any agglutination.

to Farhanah:
for serology lab, the screening test that we usually do is the Venereal Disease test, followed by the VD titre. i don't think we have any other screening tests. the confirmatory test would be the TPHA (treponema pallidum haemagglutination) test. other screening or confirmatory manual tests include ATG-AMC (anti-thyroglobulin and anti-microsomal antibodies) test, ASO (anti-streptolysin O) test, IM (infectious mononucleosis) test, WWF (Widal and Weil Felix) & Brucella test.

thank you all for the great response. i'm very sorry if my explanations arent that clear or it still leaves you clueless but i've really tried my best to explain to you guys as what was taught to me during my stay in the serology lab for the first 2 weeks. if you still wanna ask me anything, go ahead. keep asking. it's good.

see you all C: