jamescorden, on Mar 3 2010, 01:46 PM, said:
Sorry but that's confused me again.
How do you extract mRNA from rodents and human samples directly without extracting DNA? We extract DNA first, then RNA, dont we? I've just realised I'm really poor at Genetics and Molecular Biology
This is the whole thing:
People are dying of fever(haemorrhagic) because of an outbreak of possibly a virus. A guy gets samples from rodents and patient specimens and he performs RT-PCR on them(HOW? how to extract RNA?)
, with positive controls (dont get this bit!)
consisting of genomes from viruses known to cause this fever. Nucleotide sequencing reaction (which is this? how we do it?)
were then performed on small fragments of these RT-PCR products. Now, there is an accident and origin of sample was destroyed.
I now need to determine identities of viral genome amplicons. I have been provided a diagram of sequencing gel, labelled A-G.
This is what I did:
From the diagram of sequencing gel, I found my sequence and did a BLAST Search on NCBI Website and got a Virus with 79.8 Score.
how do i know this virus is reponsible for the outbreak? what do i look for?
COULD ANYONE HELP ME PLEASE? I REALLY WANT TO LEARN THIS AND UNDERSTAND. MY FRIENDS ARE JUST WORRIED ABOUT THE ACTUAL MARKS, BUT I REALLY WANT TO UNDERSTAND THIS BIT AS ITS CRUCIAL AND MUST BE SIMPLE TO UNDERSTAND. THANKS A LOT FOR ALL THE PEOPLE WHO REPLIED ABOVE.
First, by RT-PCR, do you mean real-time PCR or reverse-transcriptase PCR?
I will assume reverse transcriptase for this response (I assumed real time above).
You can purify RNA by a number of methods, and it's not dependent on DNA purification. Positive controls are known samples used to compare the products of the unknown samples. The sequencing reaction, which I'm not as familiar with, is likely done to verify sequence and match to the database to differentiate between species that have similar but not exact sequences.
The original sample may not be necessary if the cDNA product was archived. That can be used as PCR template if a second reaction is needed, which can then be used as template for a sequencing reaction.
When you do your blast search, what are the % identity and similarity? If it's a 100% (or close to it) match, there's a good chance that is your virus.
Anyway, that's a pretty general answer. Without more specific questions, I can't give any more specific answers. I don't think the details of the method of PCR are as important as figuring out the sequence and blasting it for the identity. You could use regular PCR, but might suffer some loss in sensitivity. That is, you may not get a product to sequence without amplifying the signal by first using reverse-transcriptase.