the 7th week of SIP =)
Hi people! Hope you guys are doing fine for SIP. =)
I'm Lyn and yes, I am back once again after 7 weeks of attachment. I know almost everyone is saying this but still, time really flies.
In my very first post, I had already mentioned briefly on what I do in the lab that I am in. This time round, I shall share with you guys on blasting. As I am in a research lab, I won't be changing departments now and then. Thus, the things that I do are more or less the same.
Blasting
Ok. Blasting is a step that the lab I'm in does after the sequencing step. Sequencing is done by a DNA analyser. After the whole sequencing procedure is done, the trace files for a particular primer is retrived by injecting your thumbdrive into the desktop that is linked to the DNA analyser to save them. Usually, you have to will retrieve 2 trace files in total for a primer. This is because we do sequencing for both forward and reverse.
Trace files are the end result that you get after sequencing is done. By transferring the trace files and opening them in sequencing programmes, you can get the electropheragram of each DNA sequence. In the lab I am in, the sequencing programmes they use are Sequencing Analysis version 5.2 and SeqScape version 2.5 (Applied Biosystems, USA).
As these programmes enables one to view the electropheragram, we are able to bast. Blasting means detecting SNPs in the different DNA sequence. By doing blasting, we are able to find out any known or new SNPs in the gene. Blasting is actually quite an eye-straining step to do. However, there are no alternatives to it. One has to look through all the DNA sequences to detect the SNPs.
How one detect SNP(s) is bascically through scanning the whole DNA sequence of each different DNA. A SNP is shown on the electropheragram as two peaks at the same base nucleotide position. Usually, only one peak is seen.
Sometimes, blasting is made tougher when there are a lot of 'noises' in the DNA sequence. 'Noise' in this case is peaks formed by excess reagents that interfere with sequencing. Usually these 'noises' will affect the jugdement of whether the peak is a SNP. This is because, some of the 'noises' are of such high peaks that one may think or assume that they are SNPs. According to what I'm taught, one can confirm a peak is a SNP when the peak is almost the same height as the peak of the correct base, or at least 2/3 the height of the peak of the correct base.
By comparing with the reference sequence of the gene, one can detect and confirm the polymorphisms in a gene. Reference sequence of a cetain gene most of the time shows the correct DNA sequence that is found in the majority of the healthy population.
When a SNP is found, one will record down or comment on the position of the SNP and also the change in base mucleotides, in the obtained reference sequence of the gene. Reference sequences of different genes can be found at the UCSC genome browser webiste (http://genome.ucsc.edu/).
That's all. I shall share with you guys more on what I am doing in my lab the next time i post again. Hope what i explained was clear.
Take care everyone and have fun! =)
Lyn
0611027D
TG02
posted by THE CODEC 5 @ 10:51 PM
13 Comments:
Wow, it all sounds very deep. So there are some stuff that i don't get.
'According to what I'm taught, one can confirm a peak is a SNP when the peak is almost the same height as the peak of the correct base, or at least 2/3 the height of the peak of the correct base.'
What do you mean by the correct base?
So will the SNP peak exceed the height of the peak of the correct base?
Thank you =)!
-Li Ping-
TG o2
hi! =)
Erm .. For example, a sequence 'AATTGC'. There is a SNP found at the 3rd position. And take for example it the base changes to a G. So we will denote it as 'T>G' which means a change in base from a T to a G.
This is shown in electropheragram, where at the 3rd postition, there will be two peaks present. One of which is the one belonging to T while the other one belongs to G.
So, for example the peak colour for T is red and the one for G is blue, in the electropheragram at the 3rd position, there will be both red and blue peaks. However, the blue peak will be about the same height as the red peak or 2/3 the height of the red peak. It will not exceed the height of the red peak.
Yar. Hope i answered your question and the way i explain isn't confusing. =)
Lyn
Hello Lyn,
I have understand alot from reading your post. Hence i have only 2 questions that i would like to ask
1) What does the electrogram tells you (e.g. what is the x-axis and y-axis og the electrogram)
2) What are some of the reagents that can affect blasting?
Thanks!!
From: Benjamin Ma
Class: TG01
0606181F
Hi Lyn.
What is SNPs? And if SNPs in found, how it affects human?
Hardina
To hardina:
Oh .. SNP stands for single nucleotide polymorphism. They are genetic variations in the genes where there is a change in a single nucleotide or base in the DNA.
The presence of SNPs can affect the DNA sequence and depending on the location of the SNPs, they may or may not affect the function of cell, organ or the entire body.
Some SNPs may even increase the risk of one getting a particular disease (example: cancer) or affect certain drug response while others cause alteration of the function of the gene and affect the protein expression.
Certain SNPs that do not alter the gene function, acts as biological markers whereby they help to detect disease on the human genome map.
Hope my explaination helps you understand what is a SNP and what it does.
Lyn
your attachment sounds very complicated. haha
but anyways, i was just wondering, what happens once you detected a SNP? do you record it down? will further experiments will be done? how will it affect the person whose DNA you are using?
thx =)
Mayafirhana
TG02
To: Benjamin
On the electropheragram, the x-axis shows the peak intensity and the y-axis shows the nucleotide (A/T/G/C)and the nucleotide position.
Usually, blasting is affect by the presence of noise. Noise are the result of excess primers or DNA and also due to improper purification and precipitation. However, the major cause is contamination.
Hope I answered your questions.
Lyn
To: Maya
Oh .. Actually, when a SNP is detected, we will insert a comment on the reference sequence of the particular gene.
An example will be, -110G>A. This means at the base position 110 before the start codon, there is a change in nucleotide from a G to an A.
For introns and exons, the SNPs will be labelled differently. If it is found in the intron, it will be label as IVS7-4T>A, which means the SNP is located at 4 nucleotide before the 7th intron and there is a change from T to an A. If a SNP is found in the exon, it will be labelled as 1309G>A. This means that the SNP is found at the 1309th position from the start codon and there is a change of base from a G to an A.
After blasting, and all the SNPs are found, genotyping will be done. I will be covering on this in my subsequent post.
In actual fact, for blasting, we use the DNA of healthy individuals. That means individuals who are free from cancer. Nothing will happen to them actually. So don't worry. =)We just use the DNA to detect SNPs. That's all.
Hope my explanation makes you understand better.
Lyn
helo there, nice post, quite easy to understand except that i didnt noe wads SNPs at first until Hardina asked =)
i would like to ask a qn,
u said that blasting is an 'eye-straining' step, how do u view it? through a microscope or computer screen?
thanks.
raihana~
To: raihana
Hi! =) Yar. It's an eye-straining process 'cause you have to stare at the computer screen and scroll down to detect any SNPs if possible.
Lyn
Hi Lyn,
If an SNP is detected in the introns itself, is it significant for the purpose of MP? Because intron can be spliced away or go through alternative splicing?
Also, if the SNP does not affect the final amino acid encoded, it is also not significant rite?
thanks =)
Jean Leong
TG02
To: Jean
Hi. Erm .. There are actually two types of SNPs. One of them is the synonymous SNP and the other is the non-synonymous SNP. For synonymous SNP, although there is a change in nucleotide, the protein they encode will be the same. For example, for a codon TGT which codes for cysteine, if there is a SNP that changes the T at the 3rd position to a C, the codon will now become TGC. However, TGC codes for cysteine as well. Thus there's no change in amino acid encoded despite the presence of a SNP.
On the other hand, for a non-synonymous SNP, a change in nucletide in a codon will result in a change in amino acid encoded. For example, TGC which codes for cysteine. When there is a SNP present at the C position and the C changes to G, the codon will now become TGG which codes for tryptophan; another amino acid.
Since both of them codes for the same amino acid, it is usually not significant. However, sometimes the tRNA concentration will be different. For example, the tRNA concentration of TGT is higher than that of TGC. This will affect the translation rate. But such cases is quite rare.
Yar. SNPs can be found not only at the exonic region but also at the intronic region. In the project that im doing, im actually focusing on the exonic regions. However, the intronic regions are important as well. If a SNP is found in the intron and near the splicing site, it will change the splicing variant. Also, if the SNP in the intron and it is in linkage to another functional SNP, this is significant.
Hope I answered your doubts. =)
Lyn
heys Lyn,
Yep, thanks for clearing my doubts =)
Jean Leong
TG02
Post a Comment
Subscribe to Post Comments [Atom]
<< Home