L-H PCR Urgent!

[Alkendi]

Dear All:

I really need your help with this, please reply as soon as you read the massage !!!!!!!!!!!!!!!

I read about the L-H PCR (Length Heterogenecity PCR) which is an amplification of 16S rDNA gene and separates it based on the length of the gene. The question is how the 16S rDNA gene is having different length and how this works with the primers. I amplified 16S rDNA gene using 27f and 1492R primers and when I ran them on a gel they all lined up in one raw having the same length. I am totally confused!!!!

 

please help me out with this if you know any thing about it.

[Bluenoise]

According to this abstract

 

"In length heterogeneity PCR (LH-PCR) a fluorescently labeled primer is used to determine the relative amounts of amplified sequences originating from different microorganisms. Labeled fragments are separated by gel electrophoresis and detected by laser-induced fluorescence with an automated gene sequencer. We used LH-PCR to evaluate the composition of the soil microbial community. Four soils, which differed in terms of soil type and/or crop management practice, were studied. Previous data for microbial biomass, nitrogen and carbon contents, and nitrogen mineralization rates suggested that the microbial characteristics of these soils were different. One site received two different treatments: no-till and conventional till perennial ryegrass. The other sites were no-till continuous grass plots at separate locations with different soil types. Community composition was characterized by assessing the natural length heterogeneity in eubacterial sequences amplified from the 5' domain of the 16S rRNA gene and by determining fatty acid methyl ester (FAME) profiles. We found that LH-PCR results were reproducible. Both methods distinguished the three sites. The most abundant bacterial community members, based on cloned LH-PCR products, were members of the beta subclass of the class Proteobacteria, the Cytophaga-Flexibacter-Bacteriodes group, and the high-G+C-content gram-positive bacterial group. Strong correlations were found between LH-PCR results and FAME results. We found that the LH-PCR method is an efficient, reliable, and highly reproducible method that should be a useful tool in future assessments of microbial community composition."

 

Ritchie NJ, Schutter ME, Dick RP, Myrold DD, Use of length heterogeneity PCR and fatty acid methyl ester profiles to characterize microbial communities in soil. Appl Environ Microbiol. 2000 Apr;66(4):1668-75.

 

 

The lengths varry between organism. So I see a few possibilites.

 

1. You're using the same organism for all amplifications (ie one organism is over represented in your sample)

2. The organism are very closely related

3. The conditions of your pcr aren't right and your amplifying something else instead.

 

Why don't you use the primers and compare it to sequence data (if available) from the organism your checking to determine the expected sizes.

 

If you tell me the size of the band your getting I maybe able to help...

[Alkendi]

Thanks for your reply . The mistake I did is I thought that this gene has the same length among all bacteria!!!! if this was true then LH PCR will not be applicable.

The gel I used to separate the 16S rDNA gene (from different isolates) was based on the molecular weight so I got all the segments on the same line and when i sent the product for sequencing they returned the same segment length (~1500bp in length). If this gene is naturally different in length between bacteria then it should yield different positions on the gel (I used agarose gel). I remebered something, is the length of this gene differs among all bacteria or only eubcteria?

 

Thanks

your response is highly appreciated

[Bluenoise]

I remebered something, is the length of this gene differs among all bacteria or only eubcteria?

 

Well I have no idea. You'll probably have to do an extensive literature search to find that one out.

[Albert-LV]

ooookay here, I know it was urgent, but I figured out I'd post an answer because this page shows up in google when looking for LH-PCR...

 

you made several mistake :

first you amplified the full length gene, you'd better use the variable V3 region (Ghiglione, 2005) :

 

The primers used were w49 (5’-ACG GTC CAG ACT CCT ACG GG-3’;

Delbès et al. 1998) and w34 (5’-TTA CCG CGG CTG

CTG GCA C-3’; Lee et al. 1996), which amplify the

variable V3 region of the 16S rDNA (Escherichia coli

positions 329–533; Brosius et al. 1981).

 

then, from what you say, it seems you ran your PCR fragments on an agarose gel (or did u use a PAGE ?), you should run the fragments on a capillary systems, it's more accurate (no, it's required).