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Recombinant DNA technology

Recombinant DNA technology - C H A P T E R 6...

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C H A P T E R 6 RECOMBINANT-DNA METHODOLOGY Restriction Analysis Gels and Electrophoresis Blotting Restriction Fragment-Length Polymorphism Cloning Sequencing Mutagenesis Polymerase Chain Reaction Much of what we know about the regulation of information flow (gene expression) has been made possible by the ability to manipulate the struc- tures of DNA, RNA, and proteins and see how this affects their function. The ability to manipulate DNA (recombinant-DNA methods) has gener- ated a new language filled with strange-sounding acronyms that are easy to understand if you know what they mean but impossible to understand if you don’t. Understand? RESTRICTION ANALYSIS Restriction enzymes are sequence-specific endonucleases that cut double-stranded DNA at specific sites. 61
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Most useful restriction enzymes cut DNA at specific recognition sites, usually four to six nucleotides in length. There can be multiple restriction sites for a single endonuclease within a given piece of DNA, there can be only one (a unique restriction site), or there can be none. It all depends on the sequence of the specific piece of DNA in question. Cutting with restriction endonucleases is very useful for moving spe- cific pieces of DNA around from place to place. It’s also a useful way to name pieces of DNA. For example, a piece of DNA that is cut from a bigger piece of DNA is often named by size and given a surname that corresponds to the two restriction enzymes that did the cutting—the 0.3-kb EcoRI-BamHI fragment. Restriction enzymes themselves are named for the bacterial strains from which they were initially isolated. A restriction map shows the location of restriction sites in a given DNA sequence. When digested with two (or more) restriction enzymes at the same time, most large pieces of DNA give a specific pattern of different-sized DNA fragments depending on the distance separating the different cleav- age sites. These different fragments can then be separated by size on an agarose gel. By working backward (biochemists are good at this) from the sizes of the different DNA fragments, it is possible to construct a map that locates the different restriction sites along a given piece of DNA. For example, if we cut the 3.6-kb piece of DNA in Fig. 6-1 with SmaI, we would see two bands on the agarose gel—1.9 and 1.7 kb. This would tell us that the SmaI site is very near the middle of the fragment. We could start constructing our map by putting the 1.7-kb fragment on the left side or the right side—it doesn’t matter, and we can’t know which is right (or left). In Fig. 6-1, the DNA is arbitrarily put down with the smaller frag- ment on the right. If we cut with BamHI, we get fragments that are 0.9 and 2.7 kb. Again we wouldn’t know whether to put the BamHI site on the right or left of the map, but here it does matter because we already have the SmaI site on the map. The way to decide where to put the BamHI site is to cut with both BamHI and SmaI. Let’s say that you get fragments of 0.9, 1.0, and 1.7 kb. Notice that the 1.7-kb fragment is the
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Recombinant DNA technology - C H A P T E R 6...

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