Almost any method used to screen a library will identifyseveral clones, some of which will be false positives that do notcontain the gene of interest; several screening methods may beneeded to determine which clones actually contain the gene.18.15Genomic and cDNA libraries may be screenedwith a probe to find the gene of interest.◗GlyValArgMetAspTrpAsnTyrGluProLeuThrSerTrpGluMetAsnGlnTrpPheValArgAla(2222 =16 possible sequences)(222 = 8 possible sequences)Known part ofamino acid sequencePossiblecodonsAGAAGGCGACGCCGGCGUCCCAUUAUUGCUACUCCUGCUUAGCAGUUCAUCCUCGUCUAGAAGGCGACGCCGGCGUACAACCACGACUGUAGUCGUGGUUGCAGCCGCGGCUCCACCCCCGCCUGACGAUAACAAUUACUAUGAAGAGGAAGAGAACAAUCAACAGUUCUUUUGGAUGUGGUGGAUGGUAGUCGUGGUUGGAAUGGAUUGGAAUUAUGAGACAUGGGAGAUGAAUCAGUGGGGCGGGGGUThis sequence would makea better probe becausethere is less degeneracythan in the sequence at left.18.16A synthetic probe can be designed on the basis of thegenetic code and the known amino acid sequence of the proteinencoded by the gene of interest.Because of ambiguity in the code, thesame protein can be encoded by several different DNA sequences, andprobes consisting of all the possible DNA sequences must be synthesized.To minimize the number of sequences that must be synthesized, aregion of the gene with minimal degeneracy is picked.◗ConceptsA DNA library can be screened for a specific geneby using complementary probes that hybridize tothe gene. Alternatively, the library can be clonedinto an expression vector, and the gene can belocated by examining the clones for the proteinproduct of the gene.
526Chapter 18Chromosome walkingFor many genes with importantfunctions, no associated protein product is yet known. Thebiochemical bases of many human genetic diseases, forexample, are still unknown. How could these genes be iso-lated? One approach is to first determine the general loca-tion of the gene on the chromosome by using recombinationfrequencies derived from crosses or pedigrees (see p. 000 inChapter 7). After the gene has been placed on a chromo-some map, neighboring genes that have already been clonedcan be identified. With the use of a technique called chro-mosome walking(FIGURE18.17), it is possible to movefrom these neighboring genes to the new gene of interest.The basis of chromosome walking is the fact that agenomic library consists of a set ofoverlappingDNA frag-ments (see Figure 18.13). We start with a cloned gene orDNA sequence that is close to the new gene of interest sothat the “walk”will be as short as possible. One end of theclone of a neighboring gene (clone A in Figure 18.17) isused to make a complementary probe. This probe is used toscreen the genomic library to find a second clone (clone B)that overlaps with the first and extends in the direction ofthe gene of interest. This second clone is isolated and puri-fied and a probe is prepared from its end. The second probe