Genetic Engineering
David A. Jackson, Robert H. Symons, and Paul Berg, Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of Escherichia Coli, 1972
To most people, genetic engineering is practically magic. The wizard, possessed of arcane knowledge, toils in a laboratory and emerges with an organism altered by methods entirely incomprehensible to the ordinary person. The reality is much more mundane. The tools of genetic engineers are clumsy, time consuming, and mostly borrowed from nature rather than designed from scratch.
The basic tools of genetic engineering detect, isolate, and measure DNA. Centrifuges and filters can separate DNA from other substances and sort DNA fragments by size. Radioactive label molecules allow scientists to track particular samples of DNA through a series of steps. Electron microscopes can even create images of individual DNA molecules, showing the rough shapes (linear, coiled, circular, branched, etc.). All of these are observational tools: they don't alter DNA, just allow researchers to figure out what goes on in their test tubes.
The tools that allow researchers to manipulate DNA are almost all enzymes taken from bacteria and other living organisms, extracted and refined using basic tools similar to those used to study DNA. Each enzyme does one particular thing and no other: restriction enzymes cut DNA strands wherever a particular target sequence occurs; λ exonuclease snips base pairs from the 5' ends of DNA strands; terminal transferase adds new base pairs to the 3' ends of DNA strands; DNA Polymerase I replaces missing complementary DNA; DNA ligase repairs a specific type of break in DNA strands; exonuclease III converts one type of break into another. And so on, and so forth. The researchers know what these enzymes do, but for the most part don't know how they do their job. (Figuring that out requires a long study of the structure of the protein, as with Haemoglobin.)
By carefully putting samples of a particular enzyme into a test tube of DNA, at the temperature and pH it operates best at, allowing time for it to work, then separating out the DNA again, you can make one small change to a sample of DNA molecules. Well, to most of them, anyway. Every step in the process is statistical; there is always some small percentage of DNA that remains unaffected, or is changed in the wrong way. With additional filtering and centrifuging, these defective molecules can sometimes be removed before you go on to the next step, but this is not always possible.
Paul Berg and company figured out how to use newly discovered enzymes to perform what may well have been the first act of genetic engineering. It had recently been discovered that viruses insert their DNA into host cells, where it then merges with the cell's DNA. Berg worked out a general method for inserting arbitrary DNA into a virus, which could then insert it into a living cell, there to be expressed and passed on to the cell's descendants.
For his initial experiments, Berg chose to use Simian Virus 40, which infects monkey cells with few side effects. SV40 only has a short loop of DNA, short enough for Berg to find a restriction enzyme (restriction endonuclease RI) that breaks it in exactly one location (because RI's target base pair sequence, GAATTC, occurs only once in SV40). He put some broken SV40 DNA into a test tube with terminal transferase and a supply of adenine (the A base pair), which added a string of As to both ends of the DNA. Then he prepared a sample of the DNA he wanted to insert into SV40, using terminal transferase to add thymine (the T base pair, complementary to A) to both ends of it. When mixed, the adenine ends of the SV40 stuck to the thymine ends of the arbitrary DNA, and the result was a test tube full of SV40 with a bit of extra DNA spliced in.
I've skipped several steps in this (a bunch of repair enzymes are needed to finish the bonding, for example), but that's the basic idea: isolate the bit of DNA you want using restriction enzymes, build "sticky ends" onto it, build corresponding sticky ends on the target DNA, and mix the two samples together. The whole process requires the application of about five or six different enzymes, not including the work to isolate the bits of DNA you want. It's important to note that genetic engineering does not (yet) involve designing new genes. Heck, we don't even know what most "known" genes do. For now, the best we can do is move a gene whose function is known (otherwise what's the point?) from one place to another. Even then, if it's put in the wrong place it won't do anything except possibly kill your experimental organism. Genetic engineering isn't magic.
Berg and many of his colleagues quickly realized the potential dangers of genetic engineering (Berg already had the tools to insert a toxin-producing gene into E. Coli, potentially producing a new type of food-poisoning bacteria), and worked to establish guidelines for experimenters in the field. The first commercial application of genetic engineering was the production of human insulin using bacteria, starting in 1982.
To most people, genetic engineering is practically magic. The wizard, possessed of arcane knowledge, toils in a laboratory and emerges with an organism altered by methods entirely incomprehensible to the ordinary person. The reality is much more mundane. The tools of genetic engineers are clumsy, time consuming, and mostly borrowed from nature rather than designed from scratch.
The basic tools of genetic engineering detect, isolate, and measure DNA. Centrifuges and filters can separate DNA from other substances and sort DNA fragments by size. Radioactive label molecules allow scientists to track particular samples of DNA through a series of steps. Electron microscopes can even create images of individual DNA molecules, showing the rough shapes (linear, coiled, circular, branched, etc.). All of these are observational tools: they don't alter DNA, just allow researchers to figure out what goes on in their test tubes.
The tools that allow researchers to manipulate DNA are almost all enzymes taken from bacteria and other living organisms, extracted and refined using basic tools similar to those used to study DNA. Each enzyme does one particular thing and no other: restriction enzymes cut DNA strands wherever a particular target sequence occurs; λ exonuclease snips base pairs from the 5' ends of DNA strands; terminal transferase adds new base pairs to the 3' ends of DNA strands; DNA Polymerase I replaces missing complementary DNA; DNA ligase repairs a specific type of break in DNA strands; exonuclease III converts one type of break into another. And so on, and so forth. The researchers know what these enzymes do, but for the most part don't know how they do their job. (Figuring that out requires a long study of the structure of the protein, as with Haemoglobin.)
By carefully putting samples of a particular enzyme into a test tube of DNA, at the temperature and pH it operates best at, allowing time for it to work, then separating out the DNA again, you can make one small change to a sample of DNA molecules. Well, to most of them, anyway. Every step in the process is statistical; there is always some small percentage of DNA that remains unaffected, or is changed in the wrong way. With additional filtering and centrifuging, these defective molecules can sometimes be removed before you go on to the next step, but this is not always possible.
Paul Berg and company figured out how to use newly discovered enzymes to perform what may well have been the first act of genetic engineering. It had recently been discovered that viruses insert their DNA into host cells, where it then merges with the cell's DNA. Berg worked out a general method for inserting arbitrary DNA into a virus, which could then insert it into a living cell, there to be expressed and passed on to the cell's descendants.
For his initial experiments, Berg chose to use Simian Virus 40, which infects monkey cells with few side effects. SV40 only has a short loop of DNA, short enough for Berg to find a restriction enzyme (restriction endonuclease RI) that breaks it in exactly one location (because RI's target base pair sequence, GAATTC, occurs only once in SV40). He put some broken SV40 DNA into a test tube with terminal transferase and a supply of adenine (the A base pair), which added a string of As to both ends of the DNA. Then he prepared a sample of the DNA he wanted to insert into SV40, using terminal transferase to add thymine (the T base pair, complementary to A) to both ends of it. When mixed, the adenine ends of the SV40 stuck to the thymine ends of the arbitrary DNA, and the result was a test tube full of SV40 with a bit of extra DNA spliced in.
I've skipped several steps in this (a bunch of repair enzymes are needed to finish the bonding, for example), but that's the basic idea: isolate the bit of DNA you want using restriction enzymes, build "sticky ends" onto it, build corresponding sticky ends on the target DNA, and mix the two samples together. The whole process requires the application of about five or six different enzymes, not including the work to isolate the bits of DNA you want. It's important to note that genetic engineering does not (yet) involve designing new genes. Heck, we don't even know what most "known" genes do. For now, the best we can do is move a gene whose function is known (otherwise what's the point?) from one place to another. Even then, if it's put in the wrong place it won't do anything except possibly kill your experimental organism. Genetic engineering isn't magic.
Berg and many of his colleagues quickly realized the potential dangers of genetic engineering (Berg already had the tools to insert a toxin-producing gene into E. Coli, potentially producing a new type of food-poisoning bacteria), and worked to establish guidelines for experimenters in the field. The first commercial application of genetic engineering was the production of human insulin using bacteria, starting in 1982.
posted by JeremyHussell @ 11:10 a.m.
0 comments
0 Comments:
Post a Comment
<< Home