Automated DNA sequencing methods involving polymerase chain reaction

Polymerase chain reaction (PCR) as a method for preparing DNA templates has been used for several DNA sequencing applications. An in situ procedure for directly sequencing PCR products by the dideoxy-termination method has been developed by using fluorophore-labeled sequencing primers. Completed sequence reactions were combined and loaded into a single electrophoretic lane of a fluorescence-based DNA sequence analyzer. DNA targets devoid of a universal primer sequence could be sequenced with labeled universal primers by incorporating a universal primer sequence into the PCR product. With this method, the sequence of a 351-bp region in the bacteriophage lambda genome was fully analyzed in a single lane with automatic base identification accuracy of greater than 99%. An unknown sequence, 1.7 kb long, also was sequenced by this procedure, in combination with a "PCR gene walking" strategy. Comparison of the 1110 bases in overlapping sequence data from both strands yielded only two single-base ambiguities. Automated DNA sequence analysis of the highly polymorphic HLA-DQA-1 (alpha) region in the human genome can be performed with this simple methodology. Use of this PCR-sequencing method to analyze DNA extracted from a one-month-old blood sample from an individual who is heterozygous at this locus allowed unambiguous assignment of genotype.     

Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA.

The Lesch-Nyhan (LN) syndrome is a severe X chromosome-linked disease that results from a deficiency of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT). The mutations leading to the disease are heterogeneous and frequently arise as de novo events. We have identified nucleotide alterations in 15 independently arising HPRT-deficiency cases by direct DNA sequencing of in vitro amplified HPRT cDNA. We also demonstrate that the direct DNA sequence analysis can be automated, further simplifying the detection of new mutations at this locus. The mutations include DNA base substitutions, small DNA deletions, a single DNA base insertion, and errors in RNA splicing. The application of these procedures allows DNA diagnosis and carrier identification by the direct detection of the mutant alleles within individual families affected by LN.     

Synthesis, structure, and activity of diclofenac analogues as transthyretin amyloid fibril formation inhibitors.

Twelve analogues of diclofenac (1), a nonsteroidal antiinflammatory drug and known inhibitor of transthyretin (TTR) amyloid formation, were prepared and evaluated as TTR amyloid formation inhibitors. High activity was exhibited by five of the compounds. Structure-activity relationships reveal that a carboxylic acid is required for activity, but changes in its position as well as the positions of other substituents are tolerated. High-resolution X-ray crystal structures of four of the active compounds bound to TTR were obtained. These demonstrate the significant flexibility with which TTR can accommodate ligands within its two binding sites.     

Iodoamino acid content and distribution in normal and abnormal human thyroids.

Iodoamino acid content and distribution were measured in adenomas, nodular goiters, thyroiditis and carcinomas and compared to thyroids from patients who died suddenly and normal thyroid tissues originally adjacent to diseased tissue. Tissues were hydrolyzed with Pronase, derivatized and analyzed by gas chromatography as had previously been reported for rat thyroids. Considerable overlap in values was found among adenomas, nodular goiters and papillary and follicular carcinomas as compared to values in normal thyroid tissue, but low values were also found in several diseased tissues. MIT/DIT and T3/T4 ratios were essentially constant in most tissues, even when T4 values were low. MIT/DIT ratios were, however, high and iodothyronines undetectable in all Hurthle cell neoplasms, clearly differentiating them from other thyroid conditions.