MGG – see below for citation)
LINE Element Degenerate Primers
Pat Heslop-Harrison These work well in Hordeum, Allium, Oryza, Secale, Nicotiana and Antirrhinum. 5' RVNRANTTYCGNCCNATHTC 3' (named BEL1) encoding [E/D/K/N/S][E/D/N] FRPIS 3' TCYGTCCCCCTRGGRRACAG 5' (BEL2) encoding RQGDPLS (very similar to Andy's downstream primer) Bel1/Bel2 PCR products should be approximately 410bp.
Modified with changed specificity at the 3' end by Sybille Kubis (Kubis SE, Castilho AMMF, Vershinin AV, Heslop-Harrison JS. 2003. Retroelements, transposons and methylation status in the genome of oil palm (E laeis guineensis ) and the relationship to somaclonal variation. Plant Molecular Biology 52 : 69-79), work well in oil palm and Brassica (Alix K, Heslop-Harrison JS. 2004 . The diversity of retroelements in diploid and allotetraploid Brassica species. Plant Molecular Biology 54 : 895-909) and mosses, ferns and liverwords (Elsebeth Kolmos)
BEL1MF: 5'-RVNRANTTYCGNCCNATHAG-3' and
BEL2MR: 5'-GACARRGGRTCCCCCTGNCK-3'.
Andy Flavell These work well in Vicia . Upstream Primer: 5' CCNGGNCCNGAYGGNWT encoding PGPDG[IMF] Downstream Primer: 5' SWNARNGGRTCNCCYTG encoding QGDPLSP or: 5' SWNARNGGRCANCCYTG encoding QGCPLSP These primers together amplify a band of approximately 650bp.
Hill P, Burford D, Martin DMA and Flavell AJ Retrotransposon populations of Vicia species with varying genome size Molecular Genetics and Genomics Published online: 13 May 2005 DOI: 10.1007/s00438-005-1141-x Have published more primers based on Wright et al (1996): Reverse transcriptase (rt) gene fragments were amplified from LINE retrotransposons in Vicia DNAs by degenerate PCR using primers DVO144 (5-GGGATCCNGGNCCNGAYGGNWT-3) and 10712 (5-SWNARNGGRTCNCCYTG-3), which were derived from those described by Wright et al. (Wright DA, Ke N, Smalle J, Hauge BM, Goodman HM, Voytas DF (1996) Multiple non-LTR retrotransposons in the genome of Arabidopsis thaliana. Genetics 142:569–578): probably for protein amino acids DPGPDG and QGDPLSP
PCR Programmes
Typical Mix for PCR amplification: We use 50 ul for both running on a gel and cloning, 15 ul for analysis of amplification alone. We use a Biometra T-gradient PCR machine.
