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

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Fig. 1. Design of degenerate primers to amplify Aquaporin gene family members. Top, the
amino acid sequences of 10 MIP family proteins, including the S. cerevisiae FPS1 (23), E.
coli GlpF (24), α- and γ-tonoplast intrinsic proteins (TIP) of Arabidopsis thaliana (25), the
vasopressin-responsive water channel of rat renal collecting tubules (AQP2) (26), the major
intrinsic protein (MIP) of bovine lens fiber membranes (27), human Aquaporin-1 (4), turgor
responsive gene (TUR) 7a from Pisum stivum (28), the Drosophila neurogenic big brain protein
(29), and the Rhyzodium root Nodulin-26 peribacteroid membrane protein (30) were aligned
by the PILEUP program of progressive alignments (31) using a gap weight of 3.0 and a gap
length of 0.1 running on a VAX computer system. The two most highly conserved regions are
shown, separated by the number of intervening amino acids. The most highly conserved amino
acids are enclosed. Middle, below the aligned sequences, the consensus amino acid sequences
are shown. Bottom, from part of the consensus amino acid sequences, the degenerate nucleotide
sequences were determined (using the degenerate nucleotide alphabet from Table 1) followed
by the sequences for the degenerate oligonucleotide primers.
family, but they are also highly related to each other, with the conserved motif being
(T/S)GxxxNPAxx(F/L)G, that has been speculated to have resulted from an ancient
internal duplication in a primordial bacterial organism, because this repeat has persisted
in Aquaporin homologs from bacteria through plants and mammals (1,6,14). These two
regions are functionally related, both contributing to the formation of the water pore in
Aquaporin-1 (15).
2. The next step is to determine the number of permutations in the nucleotide sequence. There
are 192 permutations ([2 × 4] × 3 × 4 × 2) in the sequence 5′-YTN-ATH-GGN-GAR-3′,
which encodes the hypothetical amino acid sequence Leu-Ile-Gly-Glu. We can reduce the
degeneracy by making educated guesses in the nucleotide sequence, that is, by making a
guessmer. The 3′-end of a primer should contain all possible permutations in the amino
acid sequence because Taq DNA polymerase will not extend a prime with a mismatch at the
extending (3′) end. If the above primer was to a human gene, a potential guessmer would
be 5′-CTB-ATY-GGN-GAR-3′, which only contains 64 permutations. This guessmer is
proposed by taking into account the preferential codon usage for leucine and isoleucine
in humans (5).
3. The degeneracy of a primer can be reduced further by incorporating inosine residues in the
place of N. The advantages of using inosine-containing primers is that they have a reduced
number of permutations and that the inosine reportedly base pairs equally well with all