Almost any method used to screen a library will

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.

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