SHINE like the MOON; And the Sun behind

Friday, April 18, 2008

I just realized during the short span of 3 days i missed out alot! well actually more than 3 days... Things like robin's bday, keith's bday and more...
well there is more to it than wad is seen on the surface
to keep it simple: life is not rosy as it seems for everyone.

Well just want wish robin and keith a happy belated bday! =D

so where did i spend my 3 days?

Went to biopolis again for another genetics workshop. It's abt viral detection, for this case influenza.

Now before the normal stuff lets have some technical talk. ^^
The influenza virus is in short, our flu virus. If you have noticed, nobody seems to ever gain immunity from it or has ever developed a reliable vaccine for it. Ever wonder why?

Viruses such as chickenpox, measles and hepatitis B are all basically made of a protein capsid(de outer covering) and rna inside which carries genetic information. Their RNA is a long long strand that is linear. When the virus infects a host cell, this rna will be the one that instructs de host cell to stop producing proteins for itself but rather, make de viral parts and construct more viruses.

But in flu virus, the rna is not linear and it is in the form of 8 segments. Each segment control a particular part of the virus to be produced, such as the matrix protein in the capsid. But because the rna is arranged in 8 segments, there will be more chances for mutation when replicating in the cell. A minute change in any one of the segments will cause a change in the property of the virus, for example, its matrix protein changes. Then a new subtype or an entirely new type of flu virus is produced. This is why flu virus mutates so fast and why we cannot find a reliable vaccine for it.

So lets examine the classification of the flu virus.
There are 3 kinds of flu
influenza A, influenza B, influenza C

influenza A is the most diverse in all 3 families. It mutates very quickly, have high virulence(contagious) thus it has many variations and it infects both animal and humans. Alot of flu epidemics are all caused by influenza A:
Spanish Flu
Hongkong Flu
Asian Flu
Avian Flu

Influenza B almost only infects humans and the only other animal it infects is the seal. It mutates 3 times slower than type A and thus is less genetically diverse.

Influenza C is most least understood and unlike A or B, it has 7 rna segments instead of the usual 8. It infects humans and pigs and can cause severe illness. but it is very uncommon to find influenza C.

How do we name the flu virus types?
we heard recently that the Avian flu was named H5N1. So how did they give it this boring name?

H is actually HA and is a glycoprotein on the surface of the capsid. It is called hemagglutinin. This helps the virus to get into the cell by binding onto the receptors of the cell. there are alot of types of HA glycoprotein, ranging from H1 to H16.

N is actually NA and is also a glycoprotein on the surface of the capsid. It is an enzyme call neuraminidase. It helps the virus to get out of its host cell after reproducing. There are 137 types and 9 subtypes of NA.

So if a flu virus has HA 5 and NA 1, then the virus will be called H5N1, which is your bird flu.

So now we know the structure of the flu virus, how do we see if a person has the flu?

1st we use a throat swab or phlegm sample and put it into a small eppendorf tube and let it incubate awhile. Then we add some enzymes into it to kill break down all the cells and virus in it into protein(from the cells and the virus coat), rna(from the viruses) and dna(from your cells). Then we add Dnase and proteases in it, filter, to get rid of the dna and the proteins and wad we left is the viral rna.

Then we do reverse transcription PCR(polymerase chain reaction) to amplify(make copies) the viral rna into large quantities of corresponding DNA for testing. Reverse transcription is the opposite of transcription. Transcription as we all know, is making RNA from DNA. Reverse Transcription is the making of DNA from RNA. Ingenious right? i know. =P

Why do we need DNA and not RNA in our testing? firstly, RNA is less chemically stable and will break down easily(as it is only one strand, the other side left open). Secondly, when we are to detect the virus later, we are going to use a detector called ethidium bromide that will only bind to DNA and not RNA. Thus, if RNA were to be used for testing, no results will be shown. Thirdly, to amplify the DNa we would use DNA polymerase, to amplify RNA we would use RNA polymerase. The difference is that RNA polymerase is less accurate in making copies then DNA polymerase, and will make alot of mistakes, causing inaccurate results.

so wad is PCR anw?
PCR is polymerase chain reaction. It uses DNA polymerase, which replicates DNA, to create many more strands of a particular segment of DNA you want to amplify. In the case of reverse transcription PCR, it is amplifying DNA from RNA.
There are a few components in PCR.
Buffer
Enzyme mix
DNA/RNA template
Primer mix
DNA nucleotides

The buffer is actually basically water with magnesium ions in it. DNA polymerase is an enzyme that has an active site that is activated by the presence of magnesium ions. The higher the concentration of Mg ions, the faster the reaction, but the less accurate the product DNAs would be.

Enzyme mix is your enzymes required in the reaction. In this reaction the enzymes are Reverse Transcriptase and DNA polymerase(TAQ polymerase to be specific). Why Reverse Transcriptase? To perform reverse transcription you have to have reverse transcriptase, and then from the DNA produced you amplify it with the TAQ pplymerase. An important thing to know is the optimum temperature of which the enzymes work. Reverse Transcriptase works best in around 50 degrees celsius. TAQ polymerase on the other hands, is taken from a hot springs bacteria called thermophillus aquaticus. Thus it would only start working in high temperatures such as 95 degrees celsius, in which the RNA polymerase would denature.

The Primer mix we can say is one of the most important things in the whole concoction. Earlier said, we were going to amplify a certain sequence, only around 195 nucleotides, and not the whole strand of Viral RNA. To make copies of this particular part of the nucleic acid, we would not use enzymes and cut it or wad, as it would be impractical since enzymes are specific in where they would cut and in every PCR we may want a different section amplified. This is where the primers come in. The primers are single stranded DNA that are around 3 to 5 nucleotides in length. It nucleotide sequence is corresponding to sequence at the sides of the section you want to amplify. for example the DNA is ATGCG........GTACG, where the .... part is where you want to amplify. The primer would be TACGC and CATGC, where TACGC is the start primer and CATGC is the end primer. Notice that the start primer corresponds to the start of the sequence and the end primer corresponds to the end of the sequence.(note: A binds to T and vice versa, and C binds to G and vice versa too) The start and end primer would then bind to the part of the DNA or RNA to be amplified and serves as markers for the polymerases to start and end copying.

The DNA/RNA template is the RNA or DNA we want to amplify. For our case, the viral RNA we obtained by the throat swab. The more template the faster the reaction would be at the beginning.

The DNA nucleotides, or DNTPs for short, are the basic building blocks for the DNA(the bases and the phosphate backbone). The polymerases need these to construct new DNA, linking and bonding the DNTPs together to form DNA as they progress in the reaction.

So we add optimal amounts of all these together, go through several cycles of heating and cooling which is regulated by a machine, and finally end up with the product, alot of the particular DNA we want!

There were 3 specific places of the RNA we wanted to amplify, one was the H5 gene, second was the matrix protein for flu A gene, and the last was the matrix protein for flu B gene.

So we take the DNA produced and conduct a test called gel electrophoresis. So there would be 4 results possible. first is positive for flu A H5 subtype, second is flu A, third is flu B and fourth is negative. This is because we test for flu matrix protein gene and the H5 gene. The H5 gene is only possessed by the flu A but not flu B, and not all flu A has the H5 protein gene(there may be H4, H1 etc...). So for flu B you would not see the flu A matrix gene nor the H5 gene but just the flu B matrix gene. And for negative, it would mean you have no flu, or have flu C, which is very rare.

Just like that we will determine whether we have the flu virus. Like all experiments, this one also has a chance of screwing up, especially the PCR part. So we cannot trust the results fully.

***End of Techno talk****

Whew i didn;t expect it to be so long! the technical part actually postponed this post for a day! I had to type it halfway and save it to drafts to continue the next day... hahas okok so now its the normal stuff le.

Well as seen from the technical stuff, the whole thing is a boring and arduous process. Well got someone say before, its not the show that we pay for, but the company. Yes, make alot of new frens and they were very nice ppl. Curiously, the last time i went, the frens made were from VJC and NUSHS, and now, they are from VS and NUSHS, we close indeed. The NUS ppl also know kylie, hahas =PP

Well my lab partner, shes from india! her name is Sneha =DD

The first half of bench one and two, lesser than 3 them

Saw this funny poster on the wall, appears to be koped from talking cock

The supervising scientist, we disturb him take his pic... hahas and he will tell us to sew lab coat buttons if we break the rules.

He do his worm experiment, no relation to us.

Liquid nitro!

Went out with the VS frens for lunch.

The lecturer. some guy from japan. He talk english very slow and jerky, but better than last time de peng jirong. no china accent. hahas. He very weird. He come to the lab to talk to us, then i letting my frens hear a song in my phone then he suddenly disturb and ask, "Can you live without phone?" then talk alot of crap shit. hahas then he ask my partner why she come to imcb and wad she want to be in the future. Then he ask wad why she take bio and why she like bio, walau eh so extra.

Saw something funny in the mrt.... wad is this man doing? secretly fcuking the guy beside ah? lol

Me and Jin Meng

Vera! =D

The gels left from the electrophoresis.

Our legacy we left at imcb.... the KCPSS, RGS, RI, MGS one all very nice hor! All have my participation one eh! KCP of course is i draw la, MGS also i help them draw, RI i give suggestions abt the color and RGS i help them ink and color! woohooo =PPP

The sereneness of the last day.... =(

Future Doctor eh.... =X

Hahas so after all that went back to school on friday afternoon to meet dex and gang. then went to football. After everybody else go home, dex and I went J8 to eat and drink. Then went home. Chem wokshop today was fun... blog abt it nextime... no time upload pics from phone =P Heheheheheheeeh

Everybody's been through alot, so stop being so selfish

stop looking down
brightness will break through, and SHINE @ 8:58 AM