A haplotype framework for cystic fibrosis mutations in Iran.

This is the first comprehensive profile of cystic fibrosis transmembrane conductance regulator (CFTR) mutations and their corresponding haplotypes in the Iranian population. All of the 27 CFTR exons of 60 unrelated Iranian CF patients were sequenced to identify disease-causing mutations. Eleven core haplotypes of CFTR were identified by genotyping six high-frequency simple nucleotide polymorphisms. The carrier frequency of 2.5 in 100 (1 in 40) was estimated from the frequency of heterozygous patients and suggests that contrary to popular belief, cystic fibrosis may be a common, under-diagnosed disease in Iran. A heterogeneous mutation spectrum was observed at the CFTR locus in 60 cystic fibrosis (CF) patients from Iran. Twenty putative disease-causing mutations were identified on 64 (53%) of the 120 chromosomes. The five most common Iranian mutations together represented 37% of the expected mutated alleles. The most frequent mutation, DeltaF508 (p.F508del), represented only 16% of the expected mutated alleles. The next most frequent mutations were c.1677del2 (p.515fs) at 7.5%, c.4041C>G (p.N1303K) at 5.6%, c.2183AA>G (p.684fs) at 5%, and c.3661A>T (p.K1177X) at 2.5%. Three of the five most frequent Iranian mutations are not included in a commonly used panel of CF mutations, underscoring the importance of identifying geographic-specific mutations in this population.

Large-scale pyrosequencing of synthetic DNA: a comparison with results from Sanger dideoxy sequencing.

Pyrosequencing is a relatively recent method for sequencing short stretches of DNA. Because both Pyrosequencing and Sanger dideoxy sequencing were recently used to characterize and validate DNA molecular barcodes in a large yeast gene-deletion project, a meta-analysis of those data allow an excellent and timely opportunity for evaluating Pyrosequencing against the current gold standard, Sanger dideoxy sequencing. Starting with yeast genomic DNA, parallel PCR amplification methods were used to prepared 4747 short barcode-containing constructs from 6000 Saccharomyces cerevisiae gene-deletion strains. Pyrosequencing was optimized for average read lengths of 25-30 bases, which included in each case a 20-mer barcode sequence. Results were compared with sequence data obtained by the standard Sanger dideoxy chain termination method. In most cases, sequences obtained by Pyrosequencing and Sanger dideoxy sequencing were of comparable accuracy, and the overall rate of failure was similar. The DNA in the barcodes is derived from synthetic oligonucleotide sequences that were inserted into yeast-deletion-strain genomic DNA by homologous recombination and represents the most significant amount of DNA from a synthetic source that has been sequenced to date. Although more automation and quality control measures are needed, Pyrosequencing was shown to be a fast and convenient method for determining short stretches of DNA sequence.

Sequence-specific recognition of the major groove of RNA by deoxystreptamine.

Aminoglycoside antibiotics specifically interact with a variety of RNA sequences, and in particular with the decoding region of 16S ribosomal RNA in the aminoacyl tRNA acceptor site (A-site). Ring II of aminoglycosides (2-deoxystreptamine) is the most conserved element among aminoglycoside antibiotics that bind to the A-site. NMR structures of aminoglycoside-A-site RNA complexes suggested that the 2-deoxystreptamine core of aminoglycosides specifically recognizes (5')G-U(3') and potentially (5')G-G(3') or (5')U-G(3') steps in the major groove of RNA. Here, we show that isolated deoxystreptamine specifically interacts with G-U steps within the major groove of the A-site RNA. The bulge residue of A-site RNA is required to open the major groove for accommodation of deoxystreptamine. The chemical groups of deoxystreptamine presented to the RNA by the framework of the 6-carbon ring modulate RNA recognition.

Characterization of synthetic DNA bar codes in Saccharomyces cerevisiae gene-deletion strains.

Incorporation of strain-specific synthetic DNA tags into yeast Saccharomyces cerevisiae gene-deletion strains has enabled identification of gene functions by massively parallel growth rate analysis. However, it is important to confirm the sequences of these tags, because mutations introduced during construction could lead to significant errors in hybridization performance. To validate this experimental system, we sequenced 11,812 synthetic 20-mer molecular bar codes and adjacent sequences (>1.8 megabases synthetic DNA) by pyrosequencing and Sanger methods. At least 31% of the genome-integrated 20-mer tags contain differences from those originally synthesized. However, these mutations result in anomalous hybridization in only a small subset of strains, and the sequence information enables redesign of hybridization probes for arrays. The robust performance of the yeast gene-deletion dual oligonucleotide bar-code design in array hybridization validates the use of molecular bar codes in living cells for tracking their growth phenotype.