Qsm

UGGGAAAUGAAUCACUGG -

This sequence would make a better probe because there is less degeneracy than in the sequence at left.

(2X2X2 = 8 possible sequences)

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 isolated? One approach is to first determine the general location 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 chromosome map, neighboring genes that have already been cloned can be identified. With the use of a technique called chromosome walking (IFigure 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 fragments (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 find a second clone (clone B) that overlaps with the first and extends in the direction of the gene of interest. This second clone is isolated and purified and a probe is prepared from its end. The second probe is used to screen the library for a third clone (clone C) that overlaps with the second. In this way, one can walk systematically toward the gene of interest, one clone at a time. A number of important human genes and genes of other organisms have been found in this way.

Clone A

A probe complementary to the end of clone A is used to find overlapping clone B.

Clone B

| A probe complementary to the end of clone B is used to find overlapping clone C.

Clone C

^ A probe complementary to the end of clone C is used to find overlapping clone D containing gene of interest.

Clone D

Previously-cloned gene

Direction of walk

•Gene of interest

Conclusion: By making probes complementary to areas of overlap between cloned fragments in a genomic library, we can connect a gene of interest to a previously mapped, linked gene..

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