analysis was performed to establish the methylation status of the promoter region, to detect FMs that failed to amplify on PCR analysis and to differentiate females who were homozygous for a normal-sized FMR-1 allele from female PM/FM heterozygotes.
FMR1 Southern blot analysis is used both to characterize samples with numbers of CGG repeats too large to amplify by the PCR and to determine the methylation status of the gene (11).
For Southern blot analysis, 7-10 [micro]g of isolated DNA was digested with EcoRI and NruI and separated on an 8-g/L agarose gel containing Tris-acetate-EDTA buffer (40 mmol/L Tris-acetate and 1 mmol/L EDTA).
To determine the location of each 2b locus, PCR products generated from exons 1, 7, and 9 of 2b9 were used to probe the BAC clone Southern blots.
This was established by the failure of intron 1 and exon 9 primers to amplify from any of the BAC clones and by the lack of hybridization to the Southern blots using a 2fc2 exon 9 probe.
The expected sizes of the amplicons were ascertained by electrophoresis in agarose gels, and the identity of each PCR product was further verified by Southern blot
The most common method of FXS molecular diagnosis is Southern blot analysis supplemented by direct PCR amplification of the FMR1 CGG repeat (6).
Although Southern blot analysis detects large expansion mutations reliably, small premutation alleles cannot be distinguished from NL alleles in the high end of the range.
3) mtDNA overreplication Liver Patient 11 1133 (25) 225 (11) Patient 12 2059 (88) 409 (31) 9 controls 504 (99) (a) Southern blots were hybridized with mitochondrial (ND1) and nuclear (18S rRNA) probes, and the radioactive signals were quantified as described in the legend for Fig.
Our present results show that CVs of 20% for q-PCR and 37% for Southern blot could not be avoided in age-matched controls, regardless of the tissue studied.
The antibiotic resistance gene organization was also assessed by Southern blots
of genomic DNA cut by either XbaI, XhoI, HindIII or EcoRI by using as a probe the XbaI insert from recombinant plasmid pSTF3, comprising nearly the entire DT104 antibiotic resistance gene cluster, as described (13,14).
of HindIII-digested genomic DNA, using a ctxA probe of the 1996 O139 strain (AM258), showed three bands, and the Southern blots
of the PstI- and BglII-digested genomic DNA, also using the ctxA probe, showed only two bands.