Termination of DNA synthesis by novel 3'-modified-deoxyribonucleoside 5'-triphosphates.

Eight 3'-modified-dNTPs were synthesized and tested in two different DNA template assays for incorporation activity. From this enzymatic screen, two 3'-O-methyl-dNTPs were shown to terminate DNA syntheses mediated by a number of polymerases and may be used as alternative terminators in Sanger sequencing. 3'-O-(2-Nitrobenzyl)-dATP is a UV sensitive nucleotide and was shown to be incorporated by several thermostable DNA polymerases. Base specific termination and efficient photolytic removal of the 3'-protecting group was demonstrated. Following deprotection, DNA synthesis was reinitiated by the incorporation of natural nucleotides into DNA. The identification of this labile terminator and the demonstration of a one cycle stop-start DNA synthesis are initial steps in the development of a novel sequencing strategy.

A simple protocol for the automation of DNA cycle sequencing reactions and polymerase chain reactions.

Automated DNA sequencing methods using robotic workstations have been previously reported, however it is often an arduous task to import these technologies into a laboratory. We describe protocols making use of a Beckman Biomek 1000 robotic workstation to prepare polymerase chain reactions (PCRs) and "cycle sequencing" reactions to be performed in a Perkin Elmer Cetus System 9600 thermocycler. The combination of these two instruments allows a high throughput of PCR and DNA sequencing reactions. The programs described are freely available via anonymous file transfer protocol (FTP).

DNA typing and genetic mapping with trimeric and tetrameric tandem repeats.

Tandemly reiterated sequences represent a rich source of highly polymorphic markers for genetic linkage, mapping, and personal identification. Human trimeric and tetrameric short tandem repeats (STRs) were studied for informativeness, frequency, distribution, and suitability for DNA typing and genetic mapping. The STRs were highly polymorphic and inherited stably. A STR-based multiplex PCR for personal identification is described. It features fluorescent detection of amplified products on sequencing gels, specific allele identification, simultaneous detection of independent loci, and internal size standards. Variation in allele frequencies were explored for four U.S. populations. The three STR loci (chromosomes 4, 11, and X) used in the fluorescent multiplex PCR have a combined average individualization potential of 1/500 individuals. STR loci appear common, being found every 300-500 kb on the X chromosome. The combined frequency of polymorphic trimeric and tetrameric STRs could be as high as 1 locus/20 kb. The markers should be useful for genetic mapping, as they are sequence based, and can be multiplexed with the PCR. A method enabling rapid localization of STRs and determination of their flanking DNA sequences was developed, thus simplifying the identification of polymorphic STR loci. The ease by which STRs may be identified, as well as their genetic and physical mapping utility, give them the properties of useful sequence tagged sites (STSs) for the human genome initiative.

Multiplex DNA deletion detection and exon sequencing of the hypoxanthine phosphoribosyltransferase gene in Lesch-Nyhan families.

The Lesch-Nyhan (LN) syndrome is a genetically lethal human neurological disease that results from mutations that inactivate the hypoxanthine phosphoribosyltransferase (HPRT) gene. The elucidation of the complete DNA sequence of the human HPRT gene locus has enabled the construction of multiple oligonucleotide primer sets for the simultaneous in vitro amplification of all nine HPRT exons. The multiplex polymerase chain reaction provides a facile assay for the detection of HPRT exon deletions and the reaction products can be analyzed by direct automated fluorescent DNA sequencing to identify subtle alterations in the gene. Alterations have been identified in the HPRT genes from 15 independent LN cases, and 10 LN family studies were performed. The sequencing method uses solid supports and is sufficiently simple and sensitive to be a favored approach for LN diagnosis. LN heterozygotes can be diagnosed without reference to the affected male. In addition, these procedures will be useful for somatic mutagenesis studies.

Automated DNA sequencing of the human HPRT locus.

The complete sequence of 57 kb of the human HPRT locus has been determined using automated fluorescent DNA sequencing. The strategy employed increasingly directed sequencing methods: A randomly generated M13 library was sequenced to generate contiguous overlapping sets of sequences (contigs). M13 clones at the ends of these contigs were further sequenced using M13 (universal and reverse) and custom oligonucleotide primers to order the contigs and to complete the sequencing project. The human HPRT sequence includes 1676 bp 5' and 15,238 bp 3' to exons 1 and 9, respectively. The sequence contains 49 representatives of the Alu repeat, along with several other types of repetitive sequences. The Alu sequences exhibit a biased orientation, with those sequences in the first half of the locus oriented in the minus direction relative to transcription of the gene (3'----5' = 77%, P less than 0.005) and those sequences in the latter half of the locus oriented randomly (5'----3' = 67%, P less than 0.5). The development and performance of the sequencing strategy and the features of the human HPRT gene are presented.