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An Introduction to Genetic Engineering : Desmond S. T. Nicholl

03/18/07

  06:00:28 am, by Nimble   , 2012 words  
Categories: Reviews, Books, Science

An Introduction to Genetic Engineering : Desmond S. T. Nicholl

Link: http://www.amazon.ca/gp/product/0521004713?ie=UTF8&tag=thecerealkill-20&linkCode=as2&camp=15121&creative=330641&creativeASIN=0521004713

This was a pretty interesting book. Note, though, that it is in no way an introduction to biology at all. As a matter of fact, if you don't have at least the highest level of high school biology or equivalent hobbyist reading under your belt, you might as well skip to the short discussion chapter right near the end of the book :)

The most surprising feature of the book is that it's a surprisingly practical book. If you wanted to clone a fragment of DNA, or sequence it, or determine which bacteria took up the segment of DNA that you wanted them to, there's enough practical information in here to do it. However, lest you think this be some cookbook of doom for armchair geneticists, it's pretty unglamorous hard work to do such things, including very strict matters of cleanliness (if you're trying to amplify DNA, for example, you can imagine how anything containing DNA getting in contact with your work could prove to be a problem), hours of chilling, heating up, growing, mixing just the right dilutions of enzymes to chop things up into the right pieces, running gels, working with radioactive phosphorous (just for marking things! this isn't even about creating radioactive creatures!)...

...I'm not saying that it's not nifty, just that I'm glad I'm not doing it.

The book starts off introducing a few concepts and tools, and even here, often interjects practical advice, for example, when you would choose agarose gel or polyacrylamide gel (the latter is better for sequencing small pieces).

There are some differences between bacteria (prokaryotes) and nearly everything else (eukaryotes, which includes everything from yeast to people) that make a big difference in genetic engineering. One thing that makes eukaryotes a little complicated is that when the DNA is copied into RNA, there are actually pieces that get snipped out, called introns. This shortened RNA is termed mRNA, or "messenger RNA", and to make pieces of DNA that you can put into bacteria, which do not have introns or an intron-removing facility, you need to make DNA out of that shorter mRNA instead.

They talk about methods for handling and extracting DNA and RNA, involving salt washes and ethanol (yes, you have to get RNA right stinking drunk to get it to precipitate). The amounts of DNA and RNA are often incredibly tiny, so there are ways of labeling it with fluorescent dyes like ethidium bromide or radioactive isotopes like H(3) (tritium - hydrogen with two extra neutrons) or radioactive phosphates. These can be incorporated in any number of ways - while making a copy of a strand, or by nicking it and using DNA polymerase I (apparently, this will start replacement and repair of one of the strands at the nicked spot)

The "tools of the trade" chapter talks about things such as restriction enzymes in great detail. Restriction enzymes basically chop the DNA at certain sequences of DNA letters, and it can chop it either straight across, or leave a small "tail" on each side. These are extremely important tools in the trade, since it not only lets you chop things up for DNA sequencing, but lets you splice sequences into bacteria or other cells by putting the complementary parts of the "tails" onto those sequences. This lets you grow the bacteria so that you have more of the DNA and/or protein you're looking for, whether for research, or for commercial interests like producing insulin.

Polymerases help build or copy DNA, and ligases help join things together.

Bacteria and apparently some yeasts have a plasmid - a circular piece of DNA that's separate from their main DNA. Bacteria sometimes trade or copy pieces of these, though not always, and they are usually more changeable pieces that do things like confer antibiotic resistance. They are also a great spot to stick a piece of DNA that you are trying to clone.

Note that when they refer to cloning in genetic engineering, by and large they are just talking about DNA. Cloning entire organisms (like Dolly the sheep) is a highly uncommon undertaking.

One way of getting DNA into a bacterium, and indeed into many organisms, is to co-opt viruses to do so. Virus DNA/RNA often consists of DNA for making the virus parts and for forcing the cells that they infect to make them, in addition to other nasty payloads from time to time. If the gene you are trying to clone is small enough, you can snip out part of the virus and put in a packaged version of your gene instead, then let the virus loose to infect the bacteria.

Note that this technique is also a possible delivery mechanism for gene therapy cures. Cystic fibrosis is a problem with ion transport, and can make for extremely thick, sticky mucous, making it hard to breath. Adenoviruses often infect humans, causing mild respiratory illnesses. That makes them possible vectors for inhaled gene therapy, though this has been tricky to accomplish. The University of Iowa has a Center for Gene Therapy, as do many other places.

There are cosmids and phagemids and all sorts of things. There are even "artificial chromosomes" that can be put into yeast and bacteria. On top of this, there are brute force methods, like injecting the material directly, or coating tiny pellets with the genetic material in question and then shooting, shotgun style at a petri dish, with special apparatus.

Some of the products you can use that are commercially available are provided. A lot of things that researchers used to have to do entirely themselves are being bundled up and mass-produced. This is the same kind of stepping-up of technology that helped computers finally get off the ground, because it lets you get to trying to answer the questions you want to ask faster, or even just making it possible.

Cloning strategies are covered in detail, as is one of the biggest advances in genetic engineering, the polymerase chain reaction, or PCR for short, which won Kary Mullis the 1993 Nobel Prize (oddly enough, for chemistry). It lets you multiply your DNA in a solution, by heating to denature the DNA (heat it so that it unravels), then adding polymerase to copy, over and over, without using a living organism.

One of the big improvements in the heat/cool/add polymerase cycle that was discovered was a polymerase coming from the bacterium Thermus aquaticus. This bacterium can survive high temperatures. It turns out that its polymerase can, too. So now, you can heat to denature the DNA, cool it down, but you don't need to add any fresh polymerase, because it survived the heating. This lets the reaction be done in a machine in a more unsupervised manner, which can only be a bad thing for folks who really, really like babysitting machines for hours on end.

Now the organisms you're using in genetic engineering don't always end up carrying the gene you want. There are techniques to help you select which ones. One simple, common technique is to use antibiotic resistance genes (not for genes resistant to more recent antibiotics!), put them in with the sequence you want, and then kill off the non-carriers with antibiotics.

One really neat technique uses X-gal, which is a great hairy transvestite.

No it isn't. It's a molecule which can be chopped up by the same enzyme that chops things into simple sugars. When X-gal gets chopped up by this same enzyme, it reacts to form a blue stain. You can use these blue stains to determine which of the bacteria are producing the enzyme, which you either made accompany the gene you're inserting (in which case you want the blue-stained colonies), or disrupted with your DNA-chopping action (in which case you don't).

There are other techniques such as attaching radioactive labels onto antibodies, etc.

Once you've got your gene cloned, you will want to analyze them. Sometimes you may need to further tell whether you've got the right clone, since some techniques (you can't use X-gal for everything) can only distinguish both kinds, but not tell you which is which. Techniques such as HART (hybrid arrest translation) and HRT (hybrid release translation) can tell you which is which.

Sometimes, you want to cut down the amount of gene you're looking at in the first place. Maybe your 19 Kb (Kb = kilobase = 1000 letters) gene is actually too big, so you can chop things into fragments with the aforementioned restriction enzymes, and see whether there are actually any sites within the gene that match a sequence to chop up. If there aren't, you might be able to use that information to do an extra chop. Really, the smaller the amount of DNA you have to sequence, the much, much easier.

This check is often done in a blotting apparatus. Only the original technique, the Southern blot, was actually named after anyone; in this case, Ed Southern. In typical scientist oddness, there is now also a Northern blot and a Western blot.

The latter part of the book gets a little more out of the laboratory into the larger projects and implications. Sequencing projects of various organisms are described, in addition to the more famous Human Genome Project.

The discussion about how much DNA is "junk DNA" is treated here. There are many, many repeats in the human, and indeed in most non-bacterial, genomes. The moderately repetitive sequences cause problems when trying to sequence, since you can often only work on short stretches and are trying to get computers to paste them together into larger sequences. The percentage that actually codes for proteins is estimated at 3%. This is not to say that it is all "junk" per se - some of it could be structural, or protective (I've read myself that big stretches of C-G electrically attract damage away from other parts), but it's not necessarily sensical in and of itself.

They go over examples, some contentious, some not, of applications of genetic engineering, such as finding better ways to get insulin. Some vaccines are derived by genetic engineering techniques - heptatitis B vaccine comes from getting plain old yeast to surface the same proteins found on the coat of hepatitis B itself.

On to discussions of genetic diseases in humans, disease pedigrees and how some particular diseases have been traced to problems in particular genes. The Online Mendelian Inheritance in Man web site lets you search for such things, even with keywords like "alzheimer", but unless you're a practising genetics professional, you probably won't enjoy the output all that much.

The medical future of gene therapy is considered, by doing things like removing cells, treating them externally and putting them back in, or internally via viruses (efficient but make sure there are no viable real viruses in there) or things like lipoplexes (less effective than viruses, but also less apt to trigger an immune response).

The public's response to genetic engineering varies, but there seems to be a definite pattern to what the public finds disagreeable. If it's done in the name of medicine, there seems to be little contention, since the end is seen to definitely justify the means. If it's done in the name of food, then it's much more contentious, witness the GMO furors and the complaints of "frankenfoods", though secondary products are little problem: for example, using genetic engineering to produce things for cheesemaking like rennet does not seem to make the cheese itself be considered a GMO product. Also, genetic engineering on animals does not, perhaps surprisingly, seem to raise the same furor as genetic engineering on plants. Perhaps it's because plants are so slutty (they are!).

...

It's a neat book, if you've got some biology background to you (I do, though as a hobby), albeit not a casual read. I hope they keep updating it - I'll probably want to buy a revised copy in 5-10 years. So much will have changed. I learned a lot about the practicalities, and how much more patience and care is required for doing genetic engineering in the lab than I'd ever be able to offer :)

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