unknown seq out of heterogeneous sample - obligate plant parasite (May/06/2008 )

Dear all,

I am working with a novel, obligate plant-parasite belonging to a not well known group of organisms. I have no chance of getting it in pure culture, cannot isolate single cells without contamination of plant, fungi and bacteria.

I want to get some sequences for phylogenetic studies, but all I have until now is a small DNA fragment (1kb) which is in a highly variable region of the rDNA. I have designed specific primers to get into a less variable region and they work well, but only within the known seq. I tried six different "universal" primers in the region of interest, but none of it works with my primers. Tm are similar (+/- 2°C) and the rest of the PCR-conditions should be optimal enough to give a product. Previously I tried to clone and seq products obtained with two different "universal" primer pairs but all I got were seq of contamination.

So I am making crazy the whole university at the moment which "alternative" methods there are to find out the seq of my region of interest.
I was told that a MDA would solve my problem...but I think this would enrich the contaminating DNA too
I was told to by a genomic walking kit, but these are very very expensive keeping my budget in mind
I was told to use degenerate universal primers, but I dont know which positions I should degenerate (and I think I cannot order a NNNNNN-primer)
I was told to use inverse PCR, which is at least inexpensive, but I was told that it will be very difficult and I would need lots of luck to get at least a part of my seq of interest


please share your expertise with me and tell me which method would you suggest???? Or are there other methods which I could try?

g

You may find some of the techniques outlined here useful:

Garcia-Chapa M, Batlle A, Rekab D, Rosquete MR, Firrao G.
PCR-mediated whole genome amplification of phytoplasmas.
J Microbiol Methods. 2004 Feb;56(2):231-42.
PMID: 14744452

Huang CL, Ho KC.
Isolation and characterization of the ATP-binding cassette (ABC) transporter system genes from loofah witches' broom phytoplasma.
DNA Seq. 2007 Oct;18(5):347-56.
PMID: 17654010

Cimerman A, Arnaud G, Foissac X.
Stolbur phytoplasma genome survey achieved using a suppression subtractive hybridization approach with high specificity.
Appl Environ Microbiol. 2006 May;72(5):3274-83.
PMID: 16672467

Oshima K, Kakizawa S, Nishigawa H, Jung HY, Wei W, Suzuki S, Arashida R, Nakata D, Miyata S, Ugaki M, Namba S.
Reductive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma.
Nat Genet. 2004 Jan;36(1):27-9. Epub 2003 Dec 7.
PMID: 14661021

Tell us more about the organism/host. Inverse PCR is not difficult, but you are likely to run into regions which are highly conserved rDNA regions, which can't then be expanded due to similarity to other ribosomal regions. You best bet is probably subtractive hybridization followed by random cloning. You could also try to pull out DNA with the probe you have by binding it to a bead (biotinylated primers, e.g.) and cloning the fished out fragments. You'd need to shear your DNA first to 4-10Kb fragments. End repair and cloning would finish it up.

You could try some gyrB universal primers. I believe there is also a set of rpoB universal primers in widespread use.

Knight TF Jr.
Reclassification of Mesoplasma pleciae as Acholeplasma pleciae comb. nov. on the basis of 16S rRNA and gyrB gene sequence data.
Int J Syst Evol Microbiol. 2004 Nov;54(Pt 6):1951-2.
PMID: 15545416

Volokhov DV, Neverov AA, George J, Kong H, Liu SX, Anderson C, Davidson MK, Chizhikov V.
Genetic analysis of housekeeping genes of members of the genus Acholeplasma: phylogeny and complementary molecular markers to the 16S rRNA gene.
Mol Phylogenet Evol. 2007 Aug;44(2):699-710. Epub 2006 Dec 19.
PMID: 17267242

'Rolling Circle Amplification' (RCA) is one method for 'hopeless' cases with small amounts of DNA in large amounts of foreign DNA and small parts of known DNA sequence.
Perhaps it works in your case.

V. V. Demidov: Rolling-circle amplification in DNA diagnostics: the power of simplicity. Expert Rev. Mol. Diagn. 2(6), (2002)

phage434 and hobglobin thanks a lot! I will check the literature...

I am a bit lost, as I am working in a Taxonomy-lab where the most sophisticated molecular method applied is a cloning kit
so subtractive hybridization, biotinylated primers and even inverse PCR sound very very dangerous to me

but I wont hesitate to ask more questions (but will have a look at the literature first)

g