for amplification of PAL promoter gene fragments
Sequences for 500 subcloned cDNAs from a single degenerate 3'RACE reaction were analyzed and provided insight into the utility of a degenerate primer
in terms of the maximum number of unique sequences that can be amplified simultaneously.
RT-PCR that used degenerate primers
against HPeVs, enteroviruses, and rhinoviruses identified 29 HPeVs, 72 enteroviruses, and 26 rhinoviruses, respectively.
Using the highly degenerate primers
P1 and P2, a cDNA fragment of 1,323 bp (accession number: EF636888) was obtained.
Those for cob cloning are "universal" degenerate primers
, and those for quantification are specific primers for N.
The consensus primers GP5/6 (and the extended version GP5+/6+) (19) and the degenerate primers
MY09/11 (and the modified version PGMY09/11) (20) are the most widely used primers.
A guide to the design and use of mismatched and degenerate primers
PCR using degenerate primers
reported to amplify a conserved region of the coat protein of potyviruses amplified a fragment of 33 bp.
We designed degenerate primers
for nested PCR screening on the basis of a multiple sequence alignment of the NS1 gene from human BuV, WUHARV parvovirus and MpBuV as follows: BuV-F1 (position 190-215 in MpBuV genome, 5'-TCAAWRTMACCTGGAAAGACrACAGA-3') and BuV-R1 (position 1503 1534, 5'-TCATTGGTTGTCATKAYWACTGGAGTTGGTTC-3') for the first PCR round, and BuV-F2 (position 980-1006, containing an equimolar mixture of 5'-AGAAAAATGGATGCTCCAAGATCCAGA-3' and 5'-AGAAAAATGGATGCTTGGTGAWCCWGA-3') and BuV-R2 (position 1444 1465, 5'-ATTGCTTGGCCACTCATGATKG-3') for the second PCR round.
targeting conserved gene regions were determined by alignment of published GluCl sequences from related species.
PCR fragments generated using the degenerate primers
were cloned into pCR[R]4-TOPO vector, transformed into E.
Consensus, highly degenerate primers
have been developed that can amplify a short fragment of the adenoviral DNA-polymerase gene.