There are many observations of ITS polymophisms and many speculations about lateral gene transfer between related species of bacteria

Gürtler V. The role of recombination and mutation in 16S-23S rDNA spacer

rearrangements. Gene. 1999 Sep 30;238(1):241-52. PubMed PMID: 10571000.

Milyutina IA, Bobrova VK, Matveeva EV, Schaad NW, Troitsky AV. Intragenomic

heterogeneity of the 16S rRNA-23S rRNA internal transcribed spacer among

Pseudomonas syringae and Pseudomonas fluorescens strains. FEMS Microbiol Lett.

2004 Oct 1;239(1):17-23

It can be related to better adaptation of the bacteria to different econiches or to evolution speed of the genome (some differences in ITS can be explained by transposable elements)

Hi Mio

I agree with Alex and you can go through some recent papers in different systems. There are several mechanisms that are responsible for such variations in the clones. There are two ways ; 1) clone the sequences directly from the genomic DNA or use a library of the organism...2) do sequencing of multiple clones....the variations which you see at same sites in many clones might be exisitng in the ITS variants in the genome and some can be generated during PCR perse .

bye

Alex，Ajay

Thank you for your comments. Shay if I clone sequences directly from genomic DNA , as you recommended, how do I target on ITS region? I am confused.

Miao, you can PCR the ITS and clone then for sequencing. The number of clones depends on number of ribosomal loci in your bacteria. Usually it is from 2 (including most of proteobacteria) to 9. Sometimes they are different in size, but if not, you should sequence more clones than expected number of loci. Simple calculation show that for 2 loci you can sequence >=5 clones, for 9 loci you need to sequence 30 clones per strain to get each locus with 95% probability. Of cause, some loci can have poor PCR-ability comparing to others.

Hi Miao

1) If genome of your organism of interest is sequenced it is easy. You can select the restriction enzymes flanking the ITS region...Use the genomic DNA...digest withe two restriction sites...resolve them on gel and isoate the frgaments in the expected regions and clone in a vector digected with compatible enzymes

2) If some body (or an Institute) has a genomic library of the organism..use it to screen clones containing ITS by hybridization...and sequence the clones of interest

3) The above methods will be difficult...and as Alex mentioned above the PCR based amplification, followed by cloning and sequencing of multiple clones will be simle and quick..

but you'll have to be carefull to identify in the end the true representative sequences and not paralogs or result of horizontal gene transfers..

so based on the available resources you plan the experiments....if executed carefully both the approaches will be useful

bye

Ajay Saini