Application Of Ttge Analysis To The Detection Of Novel Cftr Mutations

Cystic fibrosis (CF) is one of the most common and life-shortening autosomal recessive disorders, with an incidence of 1 in every 2000-3000 Caucasians. It occurs less frequently among African-Americans (1 in 15,300)[17] and Hispanics. The disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.[18] Although it appears that the majority of CF mutant alleles carry the AF508 mutation, more than 900 CFTR mutations have been identified (Cystic Fi-brosis Genetic Analysis Consortium, 2003, http://www. genet.sickkids.on.ca). The overall mutation spectrum and the common mutations found in specific geographic or ethnic background varied significantly among different populations. Current molecular diagnoses are designed to detect 80-97% of CF chromosomes in Caucasians and Ashkenazi Jews. However, by using the pan-ethnic mutation panel, the detection rate for Hispanic CF patients is only about 58%.[19] To improve the mutation detection rate and to provide a more accurate risk assessment for

Hispanic CF patients, TTGE was developed for comprehensive mutational analysis.

Primers were designed for all 27 exons, including at least 20 bp into the flanking intron regions.[20] The sizes of the PCR products range from 260 bp for exon 23 to 862 bp for exon 13. The PCR and TTGE conditions have been described above and also in a previous publication.[20] Figure 2 shows the detection of homozygous and/or heterozygous mutations and/or polymorphisms in CFTR gene. Panel A is a heterozygous G386A (G85E) mutation in exon 3 showing four bands. The two upper bands are heteroduplexes, and the lower two bands are homodu-plexes, with the mutant homoduplex shift-up. Panel B is a heterozygous 875+40A>G polymorphism in intron 6A, showing similar four bands with the mutant homoduplex shift-down. Panel C is a heterozygous 124-146 del23 mutation showing two bands with the homoduplex deletion mutant shift-down. The mismatched heteroduplex bands are not formed, probably as a result of the large deletion. Panel D is a homozygous C2800T (Q890X) nonsense mutation in exon 5, showing single-band shift-up. Panel E shows a homozygous (lane 2) and a heterozygous (lane 3) 1285insTA mutation. The two homoduplex and the two heteroduplex bands are clearly separated.

These results indicate that TTGE banding patterns of homozygous and/or heterozygous mutations can be easily recognized. Using TTGE analysis, we have identified at least 22 novel mutations and 2 novel polymor-

Fig. 2 Detection of mutations in CFTR gene by TTGE analysis. Lanes 1 are wild-type DNA and lanes 2 and 3 are mutant DNAs. A) Heterozygous G386A (G85E) mutation in exon 3.

B) Heterozygous 875+40A>G polymorphism in intron 6a.

C) heterozygous 124-146del23 mutation in exon 1. D) homozygous mutation C2800T (Q890X) in exon 15. E) Homozygous (lane 2) and heterozygous (lane 3) 1285insTA in exon 8.

Fig. 2 Detection of mutations in CFTR gene by TTGE analysis. Lanes 1 are wild-type DNA and lanes 2 and 3 are mutant DNAs. A) Heterozygous G386A (G85E) mutation in exon 3.

B) Heterozygous 875+40A>G polymorphism in intron 6a.

C) heterozygous 124-146del23 mutation in exon 1. D) homozygous mutation C2800T (Q890X) in exon 15. E) Homozygous (lane 2) and heterozygous (lane 3) 1285insTA in exon 8.

phisms.[20,21] Sequencing of the 350 TTGE negative DNA fragments detected eight additional distinct mutations. These mutations are either located in the GC-rich region or close to the end of the DNA fragment, or nucleotide substitutions without the change in the number of hydrogen bondings. However, as soon as the TTGE condition is optimized for the melting domain in which the mutation located, the mutations were detected.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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