Lecture Notes 4.B

B1c algorithm 1 step 1 generate multiple

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Unformatted text preview: nce 2 ⇥ 30 + (n 3) ⇥ 20 = 140. ¶10. Ligation is used to remove the nicks in the backbone. B.1.c Algorithm ¶1. Step 1: Generate multiple representations of all possible paths through the graph. ¶2. This is done by combining the oligos for the edges with the oligos for the complements of the vertices in a single ligation reaction. ¶3. Step 2: Amplify the concentration of paths beginning with vin and ending with vout . ¶4. This is done by PCR using vin and vout as primers. Remember that denaturation separates the sense and antisense strands. 234 CHAPTER IV. MOLECULAR COMPUTATION PCR moves in the 30 direction from vin on the sense strand, and in the 30 direction from vout on the antisense strand. ¶5. We now have paths of all sorts from vin to vout . ¶6. Step 3: Only paths with the correct length are retained. For n = 7 this is 140bp. ¶7. This is accomplished by gel electrophoresis. The band corresponding to 140bp is determined by comparison with a marker lane. The DNA is extracted from this band and ampliﬁed by PCR. ¶8. Gel electrophoresis and PCR are repeated to get a su cient quantity. ¶9. We now have paths from vin to vout , but they might not be Hamiltonian. ¶10. Step 4 (a nity puriﬁcation): Select for paths that contain all the vertices (and are thus necessarily Hamiltonian). This is done by selecting all those that contain v1 , and of those, all that contain v2 , and so forth. ¶11. To do select for vi , ﬁrst heat the solution to separate the strands. Then add the vi bound to a magnetic bead. Rehybridize, and use a magnet to extract all the paths containing vi . Repeat for each vertex. ¶12. Step 5: If there any paths left, they are Hamiltonian. There...
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This document was uploaded on 03/14/2014 for the course COSC 494/594 at University of Tennessee.

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