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).
DNA Isolation and Southern Blot
In addition, the exon 1 and 6 products hybridized only to clone RP22-78A19 on the Southern blots
and not to RP23-430G 14.
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
Therefore, the DNA we used in the Southern blots
and PCR experiments was molluscan, not algal.
The Southern blots
thus imply heteromorphism for one or a few large tandem arrays of Lh1, which was confirmed by in situ hybridization.
The fluorescent ms-PCR assay described here requires 5- to 6-fold less patient DNA than Southern blot
analysis (1 [micro]g vs 5-6 [micro]g), with a turnaround time from genomic DNA to diagnostic test results of <2 working days compared with a 1-2 week turnaround time for Southern blots
In addition, PCR analysis is not appropriate for detection of stable integration of genes, especially in Agrobacterium-mediated transformation, and invariably provides less information than restriction analyses and Southern blots
. Southern detection from small amounts of tissue is desirable during the transformation process, for example, in confirmation of transgenic status of calli and shoots.
Individual Southern blots
with DNA cut by HindIII were probed with the nptII (Fig.
This was true whether the Southern blots
were analyzed by scanning densitometry of autoradiographs or by a PhosphorImager (patient 4).
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).