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

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amplification approach with two degenerate primers to preferentially amplify desired
sequences.
2. It is possible to perform a PCR on first-strand cDNAs made from a small amount of
RNA and, in theory, from a single cell. Several single-stranded “minilibraries” can be
rapidly prepared and analyzed by PCR from a number of tissues at different stages of
development or cell cultures under different hormonal conditions. Therefore, PCR cloning
can potentially provide information about the timing of expression of an extremely rare
gene family member, or messenger RNA splicing variants, that may not be present in
a recombinant library.
3. Finally, the time and expense required to clone a gene should be considered. Relative
to conventional cloning methods, PCR cloning can be more rapid, less expensive, and
in some cases, the only feasible cloning strategy. It takes at least 4 d to screen 300,000
plaques from a λgt10 library. With PCR, an entire library containing 10 8 independent
recombinants (~5.4 ng DNA) can be screened in one reaction. Again, to ensure authenticity
of your PCR clones, you should either use the initial PCR clone to isolate a cDNA clone
from a library or sequence at least two clones from independent PCRs.
1.3. Degenerate Oligonucleotide Theory and Codon Usage
Because the genetic code is degenerate, primers targeted to particular amino acid
sequences must also be degenerate to encode the possible permutations in that sequence.
Thus, a primer to a six- amino acid sequence that has 64 possible permutations can
potentially recognize 64 different nucleotide sequences, one of which is to the target
gene. If two such primers are used in a PCR, then there are 64 × 64 or 4096 possible
permutations. The target DNA will be recognized by a small fraction (1/64) of both
primers, and the amplification product from that gene will increase exponentially.
However, some of the other 4095 possible permutations may recognize other gene
products. This disadvantage can be ameliorated by performing nested amplifications
and by using “guessmer” primers. A guessmer primer is made by considering the
preferential codon usage exhibited by many species and tissues (see Subheading 3.1.).
For instance, the four codons for alanine begin with GC. In the third position of
this codon, G is rarely used in humans (~10.3% of the time) or rats (~8.0%), but
often used in Escherichia coli (~35%) (5). This characteristic of codon usage may be
advantageously used when designing degenerate oligonucleotide primers.
1.4. Strategy for Cloning Aquaporin Gene Family Members
In a related methods chapter (3), I described the cloning by degenerate primer PCR
of Aquaporin-1 (formerly CHIP28) from a human fetal liver λgt11 cDNA library
starting with the first 35 amino acids from the N-terminus of the purified protein. A
full-length cDNA was subsequently isolated from an adult human bone marrow cDNA
library (4) and following expression in Xenopus shown to encode a water selective
channel (6). We now know that the Aquaporin family of molecular water channels
includes genes expressed in diverse species, including bacteria, yeast, plants, insects,
amphibians, and mammals (1,2,7). We have used degenerate oligonucleotide primers
designed to highly conserved amino acids between the different members of the
Aquaporin family to clone novel Aquaporin gene family cDNAs from rat brain (AQP4)
and salivary gland (AQP5) libraries (8,9). In Subheading 3., I will describe the creation
of a new set of degenerate primers that we have used to clone, by degenerate primer
PCR, Aquaporin homologs from a number of different tissues and species. Subheading 3.