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Motorola s Ramesh Ramakrishnan Discusses CodeLink Arrays

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AT A GLANCE

Section Manager, biochip development in Motorola Life Sciences at Northbrook, Illinois. Developed the CodeLink oligonucleotide array platform for the last two years.

Developed multi-color FISH assays and products as a research scientist in the advanced technology department, Vysis, Inc., Downers Grove, Illinois.,

Post-doctoral fellowship at the University of Michigan, working on the molecular biology of Herpes simplex virus. Staff member at the University of Pittsburgh Medical School.

Worked at ASTRA Research Center India, Bangalore, India, developing a diagnostic test for Shigella flexneri 2A, a bacterium that is a major cause of diarrheal diseases in the developing world.

In this April’s Nucleic Acids Research, you co-authored a paper on the new Motorola CodeLink arrays. In this article, you claim your CodeLink arrays can detect target transcripts down to one copy per cell. This seems very sensitive, more so than any other array. How do you achieve this?

We believe that our increased sensitivity reflects the three-dimensional nature of our oligonucleotide arrays and the fact that the kinetics of hybridization on our arrays resemble that of solution rather than solid-phase hybridization. Also, the three-dimensional surface has a larger number of potential probe attachment sites than normal glass slides, the covalent probe attachment chemistry associated with our process is better than other procedures, and the low degree of cross-linking used to generate our slide matrix generates lower background than others.

 

Your arrays are oligos, not cDNAs. What length are the oligos, and why did you go with oligos instead of cDNAs?

Our arrays contain 30mers. We have assessed longer oligos in the past, and are continuing to do so. The major reason for going with oligos as opposed to cDNAs is that all the probes have been purified and MALDI-TOF tested, whereas with cDNAs you are at the mercy of people who provide them. The sequences could be frequently wrong. They can can be contaminated and cross-react with different things.

The other issue that is important for us is the ability to differentiate between mismatches. The smaller the sequence you have attached to your surface, the greater the ability to distinguish between a double-base mismatch and a single-base mismatch. The third thing is manufacturability. It is a lot easier to start up with oligos as opposed to manufacturing cDNA arrays.

 

Do you design your oligonucleotide probes to account for different splice variants?

The probes are designed as close to 3’ untranslated region as possible with whole regions that would cover as much of the untranslated splice variant as well. We also design our probes against known sequences. When you know that there are splice variants, you design the probe to account for them.

 

In your study you used ten micrograms of cRNA. Is this the minimum amount of sample that can be used?

In the study, there were two different types of quantitation. What we have shown is that if you start up with five micrograms of total RNA, you get huge amounts for multiple hybridizations. But many prefer to do just a single hybridization. For three hybridizations, the minimum amount of cRNA is ten micrograms and the incubation time is 18 hours. But you can actually decrease the amount used for hybridization and compensate for it by increasing the hybridization time. With five micrograms you can increase the hybridization time to 36 hours.

 

In your paper, you describe a protocol where the slides are shaken during the 18-hour hybridization. Why did you shake them?

It was funny how it happened. Once the only incubator we had was a hot dog oven sort of thing [that shook]. We discovered that when they did the shaking, the background decreased dramatically. This is because the glass slide is encased in a flexible chamber that allows liquid to slosh around. You get quite good agitation without introduction of an air bubble. You put in 260 microliters of hybridization buffer, and like a balloon it can expand. When you shake the chamber and it allows liquid to slosh around, this is actually a plus.

The other thing which makes an enormous difference in sensitivity is the fragmentation. We used to do hybridization without fragmentation of target, and found that the fragmentation increased sensitivity by 10 to 20 percent. Together, fragmentation and shaking increased sensitivity by as much as 40 percent.

 

You used Tyramine Signal Amplification post-hybridization. Why did you choose this amplification method?

The TSA is a holdover from the old days when we just couldn’t get the sensitivity we wanted. The problem is, it is almost impossible to control on a day-to-day basis. You are completely dependent on the supplier [of TSA] for purity and consistency. When you compare day one to day two, the same target and the same reagent, one day is more intense. Also, the linearity is not there. As the signal intensity increases, the amount of background proportionately goes up. The spots get larger and larger and the background increases, whereas with direct detection, the background curve is flat.

 

Why should someone who is using other arrays switch to the CodeLink array?

Self-spotting makes enormous economic sense to a small lab. It’s a lot less expensive to spot than to buy an array in the shop. The problem is that getting consistent results is going to be a problem, even if the same person spots the arrays. That’s what you pay for. If you want to compare today’s result with a result six months from now, and not be concerned with who is preparing the arrays, you will hopefully go with a commercial array.

In terms of sensitivity and consistency, our platform is the best around. We really like the idea of a three-dimensional surface. In research we [completed] after this paper, we looked at specificity of more than one or two base mismatches, and our data indicates that the kind of specificity [of being able to detect these mismatches] exists throughout most of the length of the probe. Nobody else is able to get this kind of sensitivity, and because of the care we take in our manufacturing facility, because of the quality control, you can be sure the results are real and not artifacts.

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