Help me do my job

[GenesForLife]

if they are talking about other algal contaminants, the idea is PCR + species specific probe hybridization.

I'ma learn what that means now!

well, 16S RNA can be used to identify a species, and you can have fluorescent probes et cetera for that, there is a technique called Rt-PCR which amplifies DNA prepared from the RNA of the cells from the culture. You can then carry out electrophoresis and carry out Southern Blotting, with the probe included, just to see if there are 16S rRNA sequences from other species showing up too.

1) Identify species marker for the non-contaminant organism.
2) Use Rt-PCR to amplify DNA from culture.
3) Separate by Agarose Gel Electrophoresis.
4) See if all the bands hybridize with the fluorescent probe or if just a few will.
5) If some bands go unhybridized they indicate contaminants.



I am going to check later to see if I can design primers that amplify a wide range of 16S rRNA primers from algae, btw, PCR + probe hybridization is a highly senstive assay and can detect contaminants from small samples of rRNA/DNA.

Please note, for evaluating rRNA samples you need to use Rt PCR (where Reverse transcriptase converts 16S rRNA to DNA, which then is amplified by PCR)

How quantitative is this? If it's +/- 10% or better, it's probably adequate for what these fellas want. Considered similar ideas but didn't know whether we could obtain quantitative results.

Ignoring specifics, and given novice technicians, what's a reasonable expectation for sample turnaround time? Will we be able to discriminate between contributions from contaminant bacteria and unwanted algae without too much further effort?

3 hours or so for excellent results, there of couse is another method called qRT-PCR which also quantifies contaminants and thus eliminates electrophoresis in cases, but this will require operations to be run with different primers for each contaminant, from what I know, and this could complicate things a lot. You may want to talk to qRT-PCR manufacturers to get some advice on the technique, since I haven't used it I cannot offer any advice.

Quantification could be done after electrophoresis, the gel can be melted, the DNA can be reisolated and used to calculate the amount of original contaminant DNA that was amplified since we know how many doublings have taken place to arrive at the amount recovered after electrophoresis. This is done by means of a simple assay using a spectrophotometer or colorimeter.

The glitch here is to identify what number of 16S rRNA copies have been produced by amplification based on the DNA concentration, if that challenge can be worked out then I see no reason why this cannot make a very good solution for your needs.

To distinguish between bacteria and algal contaminants too, the PCR protocol will require some changes too, I will get back to you on this once I have it sorted in my head.

[GenesForLife]

These references may help at the moment.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2607152/

PCR amplification of pure bacterial DNA is vital to the study of bacterial interactions with corals. Commonly used Bacteria-specific primers 8F and 27F paired with the universal primer 1492R amplify both eukaryotic and prokaryotic rRNA genes. An alternative primer set, 63F/1542R, is suggested to resolve this problem.

So it looks like there are already two listed primers that can amplify DNA of both prokaryotes and eukaryotes in general, so the problem of amplifying DNA is solved, the problem of quantifying DNA remains.

When is the deadline coming up, SS?

[ScholasticSpastic]

When is the deadline coming up, SS?

Ah, an easy question. There is no deadline that I'm aware of. I was asked for ideas, crapped in my pants (out of respect for my actual experience), then offered what I could come up with. None of what I came up with is particularly painless, so I suspect they'll be open to further suggestions and receptive to any new ideas I might bring them. The algae are growing right now, so I guess that's a pressing concern for the engineers doing the project. However, if they suffer difficulties due to their failure to take into account that algae will tend to colonize any open system containing light and nutrients I'm not actually going down with them.

Feel free to PM me your actual name if you want attribution for the species specific probe hybridization idea. As in: This is the guy who actually had the idea for this application in the case of this system. Assuming it's an option we can use to do what I want to do. Which is questionable, considering that the sad sacks have still to adequately frame their question.

[GenesForLife]

Right , here is the thing worked out for the moment, this will allow the estimation of contaminant algal DNA and bacterial DNA while also allowing you to estimate quantity of the DNA in your culture.

mass of unamplified 16S rRNA DNA = mass at end of amplification/number of cycles.

number of copies of 16S rRNA gene = mass of unamplified 16S rRNA gene/ mol.wt of 16S rRNA gene

the molecular weight can be calculated for a sequence or a sequence length thus.
http://www.changbioscience.com/genetics/mw.html

The workflow is...

1) Isolate DNA from culture
2) Amplify using PCR
3) Use specific probes for the algae of your species (one fluorescent colour) , other algae in general (other fluorescent colour) , and other bacteria in general (another colour) to make sure you isolate the right DNA for quantification, which can be done by the calculations above.

If you have everything cropping up in the same molecular weight zone you may have a problem with resolution, in which case you would want to carry out 2d electrophoresis to improve resolution, which is a pain in the rear. This is the first solution to what I think WILL be a problem with this approach.

OR

Amplify DNA in general, carry out affinity chromatography, as I've described in the diagram below, and use the equations at the top or the ideas for result interpretation I've put in the diagram, this one is a far more user friendly method, and requires less technician skill.




If you are in the mood for something really fancy, you could contact QIAGEN or someone for advice about direct quantitative PCR using a multi-probe system (IIRC they have such a thing) , all you need to do is extract DNA, measure it first, then add the probes and the primers, and go off for a coffee as the quantities are analysed real time and posted on a computer graph.

This one is very likely to be the least labour intensive and the most precise. This is called Multiplex PCR and you may want to write to them about it.

I have another variant enabling the use of Multiplex PCR to just quantitatively estimate contaminant DNA...

1) Isolate DNA ( measure total mass)
2) Pass through affinity chromatography column that retains DNA from the species being cultured.
3) Residual DNA will have to be contaminant DNA, this will be quantified by multiplex PCR.

Disadvantage - contamination values are absolute and not relative to standards, the establishment of standards will take time.

[GenesForLife]

That much I can come up with off the top of my head. I've done affinity chromatography in the past, and it isn't difficult to do, the procurement of the column is something they or you will have to look at when dealing with this proposal.

[GenesForLife]

Sorry, you should want to try contacting Agilent and not QIAGEN <facepalm>

[GenesForLife]

Of course there are other possible methods, look at this , for instance...

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC368316/

We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with “universal” primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.

I think this approach could be applied to bacterial and algal contaminants, too, but this isn't quantitative.

[GenesForLife]

http://en.wikipedia.org/wiki/Real-time_ ... n_reaction

In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (Q-PCR/qPCR/qrt-PCR) or kinetic polymerase chain reaction (KPCR), is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of one or more specific sequences in a DNA sample.

The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in real time, a new approach compared to standard PCR, where the product of the reaction is detected at its end. Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target.

Now this , if you could get a system that can use several probes, will mean you can have all the probes in, the results from the probe that binds to algal species in general will include the DNA from the species being cultured. This has to be subtracted with the results for the algal species that is being cultured to calculate contaminant algal DNA, the bacterial thing is a simple, direct count. This is rather high investment (low compared to the MALDI setup ) but very efficient, precise, and convenient, and it is even possible to have automatic quantification.