Almost any method used to screen a library will identify several clones some of

Almost any method used to screen a library will

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Almost any method used to screen a library will identify several clones, some of which will be false positives that do not contain the gene of interest; several screening methods may be needed to determine which clones actually contain the gene. 18.15 Genomic and cDNA libraries may be screened with a probe to find the gene of interest. Gly Val Arg Met Asp Trp Asn Tyr Glu Pro Leu Thr Ser Trp Glu Met Asn Gln Trp Phe Val Arg Ala (2 2 2 2 =16 possible sequences) (2 2 2 = 8 possible sequences) Known part of amino acid sequence Possible codons AGA AGG CGA CGC CGG CGU C C C A UUA UUG CUA CUC CUG CUU AGC AGU UCA UCC UCG UCU AGA AGG CGA CGC CGG CGU ACA ACC ACG ACU GUA GUC GUG GUU GCA GCC GCG GCU CCA CCC CCG CCU GAC GAU AAC AAU UAC UAU GAA GAG GAA GAG AAC AAU CAA CAG UUC UUU UGG AUG UGG UGG AUG GUA GUC GUG GUU GGA AUGGA U UGGAA U UA U GA G A C A UGGGA G AUGAA U CA G UGG GGC GGG GGU This sequence would make a better probe because there is less degeneracy than in the sequence at left. 18.16 A synthetic probe can be designed on the basis of the genetic code and the known amino acid sequence of the protein encoded by the gene of interest. Because of ambiguity in the code, the same protein can be encoded by several different DNA sequences, and probes consisting of all the possible DNA sequences must be synthesized. To minimize the number of sequences that must be synthesized, a region of the gene with minimal degeneracy is picked. Concepts A DNA library can be screened for a specific gene by using complementary probes that hybridize to the gene. Alternatively, the library can be cloned into an expression vector, and the gene can be located by examining the clones for the protein product of the gene.
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526 Chapter 18 Chromosome walking For many genes with important functions, no associated protein product is yet known. The biochemical bases of many human genetic diseases, for example, are still unknown. How could these genes be iso- lated? One approach is to fi rst determine the general loca- tion of the gene on the chromosome by using recombination frequencies derived from crosses or pedigrees (see p. 000 in Chapter 7). After the gene has been placed on a chromo- some map, neighboring genes that have already been cloned can be identi fi ed. With the use of a technique called chro- mosome walking ( F IGURE 18.17) , it is possible to move from these neighboring genes to the new gene of interest. The basis of chromosome walking is the fact that a genomic library consists of a set of overlapping DNA frag- ments (see Figure 18.13). We start with a cloned gene or DNA sequence that is close to the new gene of interest so that the walk will be as short as possible. One end of the clone of a neighboring gene (clone A in Figure 18.17) is used to make a complementary probe. This probe is used to screen the genomic library to fi nd a second clone (clone B) that overlaps with the fi rst and extends in the direction of the gene of interest. This second clone is isolated and puri- fi ed and a probe is prepared from its end. The second probe
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