barcode scanning in asp.net Figure 13.25 Restriction map from electrophoresis of a restriction endonuclease in Software

Generator QR Code ISO/IEC18004 in Software Figure 13.25 Restriction map from electrophoresis of a restriction endonuclease

Figure 13.25 Restriction map from electrophoresis of a restriction endonuclease
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digest. (a) Original piece of DNA, showing restriction sites marked by A. (b) Agarose gels showing bands of total and partial restriction digests. Asterisks mark radioactive bands produced by end-labeled segments. At the left is the scale of molecular weight markers in base pairs (e.g., 800 bp, 700 bp). The total digest produces fragments that are 400, 200, 100, and 50 bp and the 200 and 100 bp fragments are end labeled. The partial digest yields six additional bands.
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Tamarin: Principles of Genetics, Seventh Edition
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III. Molecular Genetics
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13. Genomics, Biotechnology, and Recombinant DNA
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Restriction Mapping
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Constructing a Restriction Map
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How do we construct a restriction map Figure 13.25 shows a hypothetical piece of DNA cut by restriction enzyme A. Below this map is a diagram of the electrophoresed digest on agarose gels, which are usually used because their porosity allows DNA fragments of relatively large size to move. The restriction enzyme makes three cuts in the DNA, generating four fragments that are 200, 50, 400, and 100 base-pairs long. The banding pattern on the gel at the left in gure 13.25b is the result of the electrophoresing of that digest. (Note that smaller segments move faster than larger segments.) The sizes of the segments are determined by comparison with standards of known size (not shown, although the scale is indicated on the left). The gel does not reveal the order of these segments on the chromosome. Several methods can be used to determine the exact order of the restriction segments on the original piece of DNA. Before restriction enzyme digestion, the 5 ends of the DNA can be labeled radioactively with 32P using the enzyme polynucleotide kinase. Since the enzyme is acting on double-stranded DNA, both ends will be labeled. Upon electrophoresis after digestion of the DNA in gure 13.25, the 200-base-pair and 100-base-pair (bp) bands will be labeled radioactively, indicating that these segments are the termini of that piece of DNA. However, we still don t know the order of the middle pieces. The order of the other segments can be determined by slowing down the digestion process to produce a partial digest. If the reaction is cooled or allowed to proceed for only a short time, not all restriction sites will be cut. Some pieces of DNA will not be cut at all, some will be cut once, some twice, and some cut at all three restriction sites. The result of electrophoresis of this partial digest is seen at the right in gure 13.25b. From this gel, we can reconstruct the segment order. This gel contains the four original segments plus six new segments, each containing at least one uncut restriction site. From the total digest gel, we know that the 200 and 100 bp segments are on the outside because they were labeled radioactively. This means the 50 and 400 bp segments are on the inside. In the partial digest, we nd a 250 bp segment but not a 150 bp segment, which tells us that the 50 bp segment lies just inside and next to the 200 bp terminus ( g. 13.26b). There is a 500 bp segment but not a 600 bp segment, which tells us that the 400 bp segment lies adjacent to the 100 bp terminus ( g. 13.26c). An unlabeled 450 bp segment con rms that the 400 and 50 bp segments are adjacent and internal in the DNA. We thus unequivocally reconstruct the original DNA (compare g. 13.26e with g. 13.25a), creating a map of sites of restriction enzyme recognition regions separated by known lengths of DNA.
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In practice, restriction mapping is usually done with several different restriction enzymes. Figure 13.27 is a map of the DNA of gure 13.25, with the recognition sites of a second endonuclease, B, included. Using the same methodology just outlined, we can show that the order of the B segments is 350, 250, and 150 base pairs arising from two cuts by endonuclease B. What we do not know is how to overlay the two maps. Do the B segments run left to right or right to left with respect to the A segments ( g. 13.27a and b) We can determine the unequivocal order by digesting a sample of the original DNA with both enzymes simultaneously, thus producing a double digest. The two orders shown in gure 13.27a and b are used to make different predictions about the double digest. From the rst order (a), we predict a 200 bp end segment, radioactively labeled. From the second order (b), we predict that the labeled 200 bp segment will be cut back to 150 base pairs: there should not be a labeled 200 bp segment. The double digest shows a labeled 200 bp segment, indicating order (a). All other aspects of order (a) are consistent with the double digest. Restriction mapping thus provides us with a physical map of a piece of DNA, showing restriction endonuclease sites separated by known lengths of DNA.This technique
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