John M. S. Bartlett.pdf - Bio-Nica.info

Cloning Gene Family Members 493
Fig. 2. Gel electrophoresis analysis of PCR-amplified DNA. DNA isolated from a human
kidney cDNA library in bacteriophage λgt10 was amplified with degenerate primers up-1 (lanes
1, 5, and 6), up-2 (lanes 2, 7, and 8), down-1 (lanes 3, 5, and 7), and down-2 (lanes 4, 6, and 8).
Reactions containing 5 × 10 6 pfu of heat-denatured phage DNA, 100 pmol of degenerate primers,
and 1.5 mM MgCl 2 in a 100-µL volume were subject to 40 cycles of PCR amplification under
the following parameters: 94°C for 60 s, 48°C for 90 s, and 72°C for 60 s. After chloroform
extraction and ethanol precipitation, the DNA was resuspended in 20 µL of water, and 5 µL was
electrophoresed into a 4% NuSieve agarose gel in 1× TAE. The gel was stained with ethidium
bromide and photographed. The relative mobility of HaeIII digested φX174 DNA markers is
shown on the right. The bracket shows the size range of known members of this gene family
from the primers used.
for new homologs. Because the degenerate oligonucleotide primers are designed from
the sequence of the known gene family members, these primers will likely be biased
for those homologs. Aquaporin-1 is abundant in the capillaries around the salivary
glands and throughout the body, but absent in the salivary gland (16). To identify a
salivary homology of the Aquaporin gene family, we used a rat salivary gland cDNA
library that also contained Aquaporin-1 cDNAs, presumably from the surrounding
capillaries. We first amplified the cDNA library with an external set of degenerate
primers, digested the PCR-amplified DNAs with the restriction enzyme PstI (which
cuts between the NPA motifs of rat AQP1), and reamplified with an internal pair of
primers. We again digested with PstI to digest the rat AQP1 DNAs, then cloned and
sequenced the DNA fragments between 350 and 450 bp (9). This strategy would not
work if the resulting cDNA (AQP5) also contained a PstI site. By trying different
restriction enzymes that cut DNA infrequently (6–8 bp-recognition sites), a number
of new homologs will preferentially be identified. Alternatively, after cloning the
DNA products into bacterial expression vectors, bacterial colony lift hybridization
can be used to identify colonies containing inserts for known gene family members
(3,12,13).