According to this method, genomic DNA was digested with two frequently cutting restriction enzymes, which do not cut within telomeric repeats, resolved by pulsed field gel electrophoresis (PFGE), transferred onto a nylon membrane and hybridized with a telomeric probe.
Briefly genomic DNA was extracted from MEFs, digested with the frequently cutting restriction enzymes Hinf1 and Rsa1.
Although this study has relatively few sequence loci available for comparison, our findings suggest that a denser RAD marker scan, using a more frequently cutting restriction enzyme would interrogate more genome sequence and interrogate more sequence for comparative analyses.
We rather expect that this ITS is interspersed by nontelomeric sequences containing sites for frequently cutting restriction enzymes and appear in the gel as fragments below 4 kb like the ITS of birds [ 68].
Based on our positive experience with PstI-based genomic representations [ 25] we initially evaluated several combinations of PstI with different frequently cutting restriction enzymes (RE) as a complexity reduction approach for sorghum.
Re-mapping the reads that are deemed non-mapped in the initial mapping is necessary to avoid discarding a significant number of informative reads for an assay such as TM3C that uses a frequently cutting restriction enzyme (or enzymes) for digestion.
It is unclear if the BJ1 genome ever forms a circular molecule but if it does then cos sites are unlikely to be involved as digests with three infrequent cutting restriction enzymes (HindIII, EcoRV and EcoRI) followed by melting at 80°C failed to show any change in the number of bands compared to un-melted digests (data not shown).
The nucleotide sequences of each plasmid were analyzed using VectorNTI version 10.3 (Invitrogen, Carlsbad, CA) and a rare cutting restriction enzyme that cleaves the plasmid once was selected.
The PCR-amplified products were digested by the 4-bp cutting restriction enzyme TaqI, and the digests were separated by a 13% non-denaturing polyacrylamide gel.
The genome sequence of M. suis was validated by comparison of virtual fingerprint patterns with that of fragments derived from pulsed-field gel electrophoresis (PFGE) of rare cutting restriction enzymes as previously reported [76].
The cDNA was then digested by a 4-base cutting restriction enzyme NlaIII to generate the 3' cDNAs at about 140 bps [18] that fit with the 454 sequencing range.
