Three sets of nucleotide sequences were obtained via the cloning and sequencing of PCR products amplified using degenerate primers
for RT of Ty1-copia (10 sequences, clones Aesp1 to Aesp10), Ty3-gypsy (13 sequences, clones Aesp11 to Aesp23), and LINE (11 sequences, clones Aesp24 to Aesp34) retrotransposons (Supplementary Table S1).
(2000), where four degenerate primers
were used for genogroup I detection.
practice, to synthesize degenerate primer
pairs with each of the two
The original sequences were adapted to consistently amplify most of the Flavivirus members resulting in degenerate primers
PFlav-fAAR (TACAACATGATGGGAAAGAGAGAGAARAA from 9040 to 9068 of AF196835) and PFlavrKR (GTGTCCCAKCCRGCTGTGTCATC from positions 9305 to 9283 of AF196835).
The cloning strategy for isolating PAL promoter gene fragment from orchid (Dendrobium crumenatum) relied on the use of degenerate primer
corresponding to the conserved region of PAL protein.
An internal piece of CA sequence was obtained from 5-day-old aposymbiotic larvae, using the degenerate primers
dCA-F and dCA-R (Figs.
The wheat Cre3 (Lagudah et al., 1997) and maize Rp1-D (Collins et al., 1999) sequences were aligned and degenerate primer
sets were designed for conserved motifs in the NBS regions of these genes (Fig.
3' RACE reactions employing a degenerate primer
targeted to the conserved FMPERW motif of mitochondrial P450s produced two partial cDNA sequences that were designated DU 3 and DU 13, from the fruit fly species Drosophila arizonae.
Acquiring MSI1 gene's core fragment: According to the homology comparison results among other species' MSI1 gene, we designed 3 degenerate primers
using the primer 5.0 software.
DOP-PCR amplification was done in eppendorf tube with 20 Lofchromosome solution 5 L of 10x buffer (Takara Shuzou Siga Japan) 5 L of 25 mM MgCl2 (Takara Shuzou Siga Japan) 4 L of 10 mM dNTPs 4 L of 10 pM degenerate primer
[5'-CCGACTCGAGNNNNNNATGTGG-3'(6- MW)] (Telenius et al.
lutea was used as a template in standard PCR reactions using b-tubulin degenerate primer
pairs (5' 3') forward: GCTTCMRWGTCACCCACTCT and reverse: CTTGGGGTCGAACATCTGCTG.
The species-specific markers require only conventional PCR (with degenerate primer
sequences) which is readily available, rapid and inexpensive for molecular identification of species in question.
The degenerate primer
set we used here was originally developed by Ito et al.
Evaluation of a degenerate primer
for the PCR detection of human caliciviruses.
have been underlined and made bold.