?

BTW, this is a blog I wrote in July '06, during a summer gig in Vienna. It was fun...

So my question is,
will you go to prom with me?

All the best,
Daniel

Im writing now on my last day of work. Yesterday, I drafted a summary of all the things I've learned here, and I was surprised, again, at how much I've gotten the chance to do. I'm also surprised how fast the month has gone - it seems that between the fascinating lab experiences and the weekends spent touring Vienna and enjoying some of the things this great city has to offer with my friends and relatives here, I've done so much in past few weeks that I almost lost track of the time. I want extend a big "thank-you" to Dr. Berthold Streubel and his team of technicians, for taking so much time out of their work to show me and the other intern how to do the different procedures and being patient enough to let us do a lot ourselves. Thanks, especially, to Berthold, who taught us a lot of the science behind the lab work as well as the more general concepts - how he balances the lab's clinical responsibilities (diagnosing patients) and the research, how the system works of publishing papers and getting grants, and how the individual lab fits into the greater communities of medical science. I think that in all, my internship gave me a good understanding of and experience with the practice of genetics, as well as a good idea of what a career in bioscience might be like.

I got the chance to work with one last new technique yesterday. It's really amazing technology - it lets you select individual groups of cells, from a slide-mounted section, under a microscope. That way, you could, for example, get RNA specifically from the cancer cells from a sample where they are interspersed with healthy cells. It works by shooting the cells you want with a small laser, causing them heat up and stick to a cap with a special, heat-sensitive adhesive. This leads to the futuristic-sounding name, "laser microdissection". I should add that the number of cells you need to extract RNA or DNA in useful amounts makes this one of the more labor-intensive lab procedures - you have shoot cells for at least an hour, for every sample you test! Still, I think the technology here is really impressive, and it allows very targeted analyses of particular cells' genetic material.

I'm writing now at the end of my third week here at the AKH. Its amazing - with all the lab work I've been learning, as well as all the things I've done around Vienna with my friends and relatives here, going to city's many interesting features or playing frisbee and watching the World Cup, its gone by so quickly. Nonetheless, I still have a week to look forward to, and I hope to really make the most of it and enjoy it while it lasts.

Berthold has, as always, been showing me and Kati a lot of the things he does in his job and teaching us the underlyinig science. Lately, I've been getting more chances to use some of the techniques I learned earlier in the month, so that I could do them a bit more independently and with less of the preparation and other work done by the technicians. Today, for example, we karyotyped a healthy blood sample (from Kati, by the way) and did two FISH probes on it. The advantage of working with a healthy sample is that nothing important, such as a diagnosis, depends on it. That way, we can do most of it ourselves. Of course, patient samples are also often obtained in small amounts by methods such as operations on bone marrow or lymph nodes, so that having to redo a procedure could be very problematic!

I've also been doing some new techniques, such as labeling BAC products (fairly long DNA segments) with flourescent markers for use as FISH probes, or the IHC ("immunohistochemistry") tests for seeing where certain proteins are and where they aren't. I spent a day in a different department to see a bit more of the IHC - which isn't part of Berthold's genetics division. As for Dr. Bilban, the bioinformatics specialist I met last week, he got sick and took today and yesterday off, so I'll see him again next week instead.

So overall, my time in the lab lately has been great, and I'm looking forward to one more week here of intersting things to do.

I'm going to take a short break from describing the lab techniques I've been learning here to tell you a bit more about how the day-to-day work, and about what a career in genetics might look like.

I'll start with what I've noticed about the kind of work we typically do here. In general, working in a lab takes a considerable amount of precision and organization. You also have to be somewhat patient. In many ways, its like cooking for a big reception - you have many procedures going on at once, some of which need to finish before others can start, and some of which are out of your control, so that you have to adapt to them. Unlike cooking, though, laboratory work usually doesn't have specific deadlines - you often do things on an "as soon as possible" basis. Also, information has has to be kept about each case the lab handles, and actual material preserved in refrigerators, paraffin wax, or the like, which means that lab work involves quite a lot of record-keeping and archiving. Of course, the biggest difference between working in a lab and working in the culinary industry is that we're helping save people's lives, which makes all of the more difficult aspects of the job worthwhile.

On a more general level, that brings me to one of the beautiful aspects of working here in genetic pathology --
that, like many people in the general medical industry, we work for people who really need us; people who are sick or dying and rely on us to help heal them. Our main duties here are to diagnose cancer patients and people with genetic illnesses, and to do research that will give them better treatments in the future. Also, unlike the doctors, we don't have the emotional stress of dealing with such patients directly or being individually responsible for them. This lets us work in a more relaxed environment, and reduces the stress of doing the more complex procedures under time pressure. I think that, in general, we're in the great position of having a job that's very meaningful and important, but still has a casual, fun element to it that makes our everyday work easier and more enjoyable to do.

Finally, genetic pathology, at least at the lab here where I'm an intern, is very much a cutting-edge field. New types of cancer, lab procedures, and experimental treatments are being developed every year, some of them right here in the AKH. This makes the job a very interesting one, and also leads to open careers - in such a young, quickly changing field, a person who is skilled and hard-working enough can get very far in his career and help lay the foundations that will stand when the field matures. I think that altogether, its a very promising job that students today should definitely look into.

PCR (for Polymerase Chain Reaction) is a slick method for simply multiplying segments of DNA. The segments are defined by their endpoints - which is where PCR's primary usefulness comes from: it lets you multiply an unknown stretch of DNA, for example some variant of a gene, that lies between known endpoints. These can then be analyzed, usually by measuring their length by gel electrophoresis (which I'll write about soon), but possibly also by complete sequencing or other tests. The principle behind PCR is the similar to the DNA replication that occurs when living cells divide: the double-stranded DNA is split into single strands, and complementary strands are created on the single strands, turning both into identical double-stranded molecules. By repeatedly doubleing the number of segments like this, PCR can achieve million- or billion-fold amplifications quickly and efficiently.

The basic cycle looks like this:
- The DNA is heated to just under boiling, causing it to denature (split into single strands)
- Then, it is cooled far enough to allow the primers (the short segments defining the endpoints) to attach, creating double-stranded sections of just a few base pairs at the start of each segment.
- Finally, the mixture is heated to a temperature between that of the first two steps, allowing polymerase proteins to attach to the double-stranded primer sections and begin moving along the DNA strands, taking free nucleotides from the mixture and attaching them to their compliments on the strands. This makes the segments double-stranded again, giving twice as many as there were at the start of the cycle.

One of the more common things we have to do here in genetic testing is extracting DNA or RNA from cell samples. Just about all of the testing we do revolves around one of these two molecules; its just a question of how we look at them. I've written a bit about inspecting DNA on a chromosomal level, where it's left inside the cells and stained for karyotyping or hybridized with flourescent probes or something along those lines, and looked at under a microscope. There are other techniques, though, that require the nucleic acids by themselves. All the specific genotyping you can do, for example, with PCR, restriction enzymes, and gel elecrophoresis, is done with extracted DNA. Gene chips, which let you see exactly what genes are being expressed in a group of cells, work with mRNA. In general, nucleic acid isolation is important as a starting point for a lot of other techniques.

The procedure works roughly like this:
- The cells are lysed by a combination of chemicals and heat.
- Other chemicals bind to the cell remnants and make them heavier. The nucleic acids are separated from the waste by centrifuging. Alternatively, filters can be employed which bind to the nucleic acids but let the other products through. Either way, this cleaning process is repeated several times.
- The nucleic acids are suspended in Isopropanol and centrifuged again, forming small white "pellets".
- The pellets are dried, then dissolved in warm water.

This can then be refrigerated and stored, or used immediately.

A few days ago, Dr. Streubel was showing us a particularly interesting method for pinpointing chromosomal mutations in cancer cells. It's called "Flourescence in situ Hybridization", or, by its friendlier name, "FISH". The basic idea is to take DNA segments that have flourescent markers, and let them attach themselves to the complimentary sequences on the chromosomes you're testing. These are then inspected under a microscope with aid of a computer. There are a number of different types of probes that can be used:

- Painting probes are sets of probes that attach themselves everywhere on a particular chromosome. These are useful, for example, in confirming translocations: if chromosomes 8 and 14 are painted different colors, you could see if they switched ends.

- Gene probes are used to determine the location of specific genes. These can be used, for example, to see how often a particular gene is present in a cancer cell (the normal value being two), or to pinpoint a suspected translocation exactly, in one of two variants:
-- Dual color, dual fusion tests involve two gene probes, one spanning a gene on one chromosome and one on another. For example, one might be painted green and one red. In a healthy cell, you would see a two green dots and two red dots. In a cell with a translocation breaks two chromosomes exactly in these genes and swaps the ends, you would see a green dot, a red dot, and two pairs of green-red dots directly adjacent to each other.
-- Break-apart tests involve probes that, instead of covering a gene, flank it on either side. Thus, if a transmutation splits a gene and places part of it on a different chromosome, two seperate dots of different color would be visible. Healthy cells would have two pairs of directly adjacent dots.

- Centromere and telomere probes color only the centromere (roughly speaking, middle) or telomere (ends) of a particular chromosome.

A small note about the name of the technique:
Letting lab-constructed DNA segments combine with sample chromosomes is called hybridization, hence "Flourescence hybridization". The "in situ" part is because these tests are done on slide-mounted cells rather than extracted DNA. I really don't know why this is in Latin. It may simply be to make a better acronym.

I'll end with a summary of the actual procedure:

- Prepare slides as if for karyotyping, but don't apply the stain
- Wash the slides in alcohol
- Denature the slides (split all the chromosomes, which are double-stranded, into single strands) with a combination of heat and chemical agents
- Transfer immediately to ice-cold alcohol, so that the chromosomes don't have time to reanneal (in other words, so that they stay single-stranded).

Meanwhile, the probes also have to be denatured. Since they are in test tubes rather than slide-mounted, they are a bit easier to deal with.
- Heat the probe mixtures to denature
- Transfer immediately to ice to keep them single-stranded

Then comes the actual hybridization:
- Pipette the (single-stranded) probe mixtures onto the slides (which are also denatured).
- Apply slide covers and seal in with rubber cement. This will prevent the mixture from drying out.
- Incubate overnight. This will give the probes time to attach to the correct places on the chromosomes.
-The next day, remove the rubber cement and the slide covers. Wash off the excess probes in a mixture like the one used to denature the slides, but milder - this will cause probes that are attached to the wrong locations (and only paritally match the chromosomal DNA) to disattach, while not denaturing the well-matched probes.

The slides will now be ready for microscoping.