Quick Question !

Genetics as it applies to evolution, molecular biology, and medical aspects.

Moderators: honeev, Leonid, amiradm, BioTeam

Post Reply
RachBio
Garter
Garter
Posts: 9
Joined: Tue Dec 30, 2008 4:48 pm

Quick Question !

Post by RachBio » Tue Dec 30, 2008 4:53 pm

Hi everyone!

I hope this is in the right section - i will copy it into the Genetics section too incase i am wrong!

I have been asked to research some things on the yeast S.Cerevisiae.

I need to find out the techniques that can be used to identify Novel Genes involved in Filament formation in S.Cerevisiae....

So far, the 2 that i think i have found are RT-PCR and Northern Blotting.

Are there any others that can be used?

Also what is the best method? i have found information that supports both of these methods, but a) i dont know if these are the only two techniques, and b) how do i choose which is correct for what i want?

Thank you in advance for any help anyone can give me, itis greatly appreciated!

User avatar
MrMistery
Inland Taipan
Inland Taipan
Posts: 6832
Joined: Thu Mar 03, 2005 10:18 pm
Location: Romania(small and unimportant country)
Contact:

Post by MrMistery » Tue Dec 30, 2008 10:12 pm

pretty much nobody does northern blotting anymore. RT-PCR can do the same Northern blotting can, only with more genes. And there are also microarrays, that do the same thing with many many more genes.
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter

RachBio
Garter
Garter
Posts: 9
Joined: Tue Dec 30, 2008 4:48 pm

Re: Quick Question !

Post by RachBio » Tue Dec 30, 2008 10:56 pm

Girlfriend said something silly here.

RachBio
Garter
Garter
Posts: 9
Joined: Tue Dec 30, 2008 4:48 pm

Post by RachBio » Wed Dec 31, 2008 1:13 am

Hello everyone, sorry about my girlfriend Rachel, i've been having some issues with a Genetic assignment and i think she just wanted to help me by asking here. She doesn't actually know anything about Genetics, so the problem i'm having trouble with wasn't phrased very well here, bless her :/

I've never thought of using a forum to help with Genetic issues.. but i'll stick around and help others out if i can afterwards :D

Anyway, my issue is this:

I'm asked what method i would use to discover a new gene in S.Cerevisiae that's involved in filiment formation.
Looking through my notes, my lectures seem to want us to say Northen Blotting and RT-PCR, and for checking for regulatory genes, Differential RNA Display or Microarrays.

Problem is, i've never done any practical work with these techniques, and my notes on all four techniques are really vague :/

I mean, i can understand how you can identify a specific gene or RFLP using hybridization, or RT-PCR with specific probes - but how do you find a brand new gene involved with filiment formation that has no homologue?

I think it's something to do with RNA that's made in yeast which isn't making filiments, and comparing it to RNA that's made in yeast which is undergoing filiment formation.

But without having something to hybridize, how can i compair the two yeast-forms RNA to find the genes being expressed in the filiment form?

Thanks a lot guys, really appreciate it! :]

Jonesy
Garter
Garter
Posts: 3
Joined: Wed Dec 31, 2008 1:29 am

Post by Jonesy » Wed Dec 31, 2008 1:34 am

I made a new account.
Looking around, this website looks awesome! I just hope people don't only come here when they want answers. I'll certainly hang about to help students doing GCSE's or A Levels if i can.
Once again, thank you MrMistery for your help. If you hadn't replied i wouldn't be here :D

User avatar
GreenDog
Coral
Coral
Posts: 126
Joined: Sun Mar 27, 2005 8:37 am

Post by GreenDog » Wed Dec 31, 2008 8:45 am

I think you got it right.
Compare RNA from wild type yeast with yeast with defects in filament formation.
You can do a "subtraction library"; extract RNA from both populations, create cDNA, and RT the driver, add adaptors marked with different colors to the two kinds of cDNA. Hybridize, remove hybridized DNA and you'll get the differentiallly expressing genes.
"When In Danger Or In Doubt Run In Circles Scream And Shout"
Lawrence J. Peter

Jonesy
Garter
Garter
Posts: 3
Joined: Wed Dec 31, 2008 1:29 am

Post by Jonesy » Wed Dec 31, 2008 2:53 pm

Thanks for your help GreenDog, but i think i have more questions now than before :P

Did you mean defects to filiment formation? Because that would make sense to me. If it can't get enough nitrogen, it defects to filiment formation.
If you mean, find a yeast cell which has defects in it's filiment formation, that's something else i guess.

I've never heard of a subtraction library before - which is great as i now have a new search term to do more research on :) Thank you!

Extract RNA from both regular and filiment-forming yeast.

I understand that - one will have mRNA used to make filiment-making-protiens, other won't.

Create cDNA.

I understand that. We're wanting to find the gene that made this mRNA. Gotcha.

RT the driver.

I don't really know what a driver is. I'm guessing it's what my lectures call primers. Short fragments of DNA/RNA which starts enzymes like RTase or Polymerase off.
Even then, i don't really understand why you would Reverse Transcribe it. I must be confuzed somewhere.

Add adaptors marked with different colors to the two kinds of cDNA.

Never heard of an adaptor before. I think it may have been used to talk about converting blunt-end restriction sites into stick-end'ed restriction sites.. but that has nothing to do with the next part, hybridization.

Hybridize, remove hybridized DNA and you'll get the differentiallly expressing genes.

Well, i can understand if you take your cDNA from both samples, you would want to compair them, and if you want to see them your going to have to hybridize something florecent/radiolabeled to it.

But what i expected was to have two runs of something simlar to gel electrophoresis, with bands in different places.
Then all the genes involved in filiment formation are simply the bands in the filiment-formation run, and not in the regular yeast run.

But how do i hybridize markers to these bands, when i don't even know what's there!? >_<

Do i just add some small marker, which marks *everything* - or would that make one blurry line, and not bands (to inspecific).

Ahh, why am i being asked questions on things i've never been taught/seen before. :'(

Jonesy
Garter
Garter
Posts: 3
Joined: Wed Dec 31, 2008 1:29 am

Re: Quick Question !

Post by Jonesy » Wed Dec 31, 2008 2:58 pm

Oooh, i think i get what you mean now!

Instead of compairing the cDNa by eye, like you would with gel electrophoresis, you simply hybridize the cDNA from the regular yeast with the cDNA from the filiment forming yeast (which has been blotted and fixed) together (use the reverse strand of the cDNA from the regular yeast, and add markers).

Anything that has hybridized will mean it's present in both regular and filiment forming, and thus irrelevent.
Anything that's left is only found in filiment forming.

Label all remaning cDNA, and there are your cDNA bands which contain novel genes.

Some how go from cDNA to genomic DNA (that's probibly in my notes somewhere) and bobs your uncle, you have your genes involved in filimentation.


Right? :/ Or have i just invented my own technique? :P

Post Reply

Who is online

Users browsing this forum: No registered users and 1 guest