As part of our efforts to construct a high-resolution physical map of human chromosome 4, we developed a systematic approach for efficiently generating large numbers of chromosome-specific sequence-tagged sites (STSs). In this paper, we describe how rate-limiting steps in our STS development were identified and overcome, and detail our current development strategy. We present information for 822 new human chromosome 4-specific STSs, including PCR amplification conditions and subchromosomal localization data, obtained by analysis of the STS with somatic cell hybrids containing different portions of human chromosome 4. Although most STSs presented here were developed from anonymous clones whose sequences were determined in this laboratory, several STSs were developed for genes and other DNA sequences that were previously mapped to chromosome 4. Our data indicate that the availability of DNA sequence for an STS locus, in addition to the sequences of the two PCR oligonucleotides, significantly increases the transfer of that STS by allowing investigators to select new oligonucleotides best suited to the standard conditions used in their laboratories.

Genomic Denaturing Gradient Gel Electrophoresis (gDGGE) provides an alternative to the standard method of restriction fragment length polymorphism (RFLP) analysis for identifying polymorphic sequence variation in genomic DNA. For gDGGE, genomic DNA is cleaved by restriction enzymes, separated in a polyacrylamide gel containing a gradient of DNA denaturants, and then transferred by electroblotting to nylon membranes. Unlike other applications of DGGE, gDGGE is not limited by the size of the probe and does not require probe sequence information. gDGGE can be used in conjunction with any unique DNA probe. Here we use gDGGE with probes from the proximal region of the long arm of human chromosome 21 to identify polymorphic DNA sequence variation in this segment of the chromosome. Our screening panel consisted of DNA from nine individuals, which was cleaved with five restriction enzymes and submitted to electrophoresis in two denaturing gradient conditions. We detected at least one potential polymorphism for nine of eleven probes that were tested. Two polymorphisms, one at D21S4 and one at D21S90, were characterized in detail. Our study demonstrates that gDGGE is a fast and efficient method for identifying polymorphisms that are useful for genetic linkage analysis.

DNA sequencing is presently performed almost exclusively by primed DNA synthesis in the presence of the 2' ,3'-dideoxy chain terminators (1), with best results being obtained with singlestranded templates and using small plasmid clones. For largescale sequencing projects, direct sequencing of cosmids is a more desirable strategy, since the sequence of a cosmid (approx. 40 kb of insert DNA) could be determined from a single DNA preparation without the need for mapping or subcloning of smaller fragments. Targeted priming of the sequencing reactions along the 40 kb DNA would further reduce sequencing redundancy and thus minimize the amount of work required to determine the entire sequence of a cosmid. However, there are still technical difficulties associated with direct sequencing of cosmids, and most new sequence information presently generated comes from experiments on smaller DNA clones. Our initial efforts to sequence larger (20-40 kb) DNA, using sequence-specific primers and the Applied Biosystems DyeDeoxy Terminator cycle sequencing strategy gave satisfactory results with a phage clone XHAFP33 (2), but sequencing data obtained from cosmids were inferior to those obtained from X DNA. This led us to consider possible improvements and we decided to investigate whether certain parameters in the cycle sequencing reactions, such as temperature and topology of template DNA, might affect the quality of sequencing results.Our cosmid clones came from a library containing genomic DNA from human chromosome 4 cloned in the vector sCos-1 (3). The library was constructed at the Los Alamos National Laboratory (Dr Larry Deaven, personal communication). A unique BamHI site in sCos-l is the cloning site, which is flanked by T3-and T7-promoter sequences, and insert DNA can be excised by using NotI or EcoRI restriction endonucleases. The sequence surrounding the BamHI cloning site in sCos-1, including the position of our new primers, is shown in Figure 1. Individual clones from the cosmid library were grown overnight in Terrific Broth (4), and cosmid DNA was purified from the culture using Qiagen Tip columns according to the manufacturer's protocol. The isolated cosmid DNA is a mixture of two circular forms, the supercoiled and the relaxed nicked-circle.

BackgroundTo identify variants likely responsible for Mendelian disorders among the three major ethnic groups in the Bronx that might be useful to include in genetic screening panels or whole exome sequencing filters and to estimate their likely prevalence in these populations.MethodsVariants from a high‐density oligonucleotide screen of 192 members from each of the three ethnic‐national populations (African Americans, Puerto Ricans, and Dominicans) were evaluated for overlap with next generation sequencing data. Variants were curated manually for clinical validity and utility using the American College of Medical Genetics (ACMG) scoring system. Additional variants were identified through literature review.ResultsA panel of 75 variants displaying autosomal dominant, autosomal recessive, autosomal recessive/digenic recessive, X‐linked recessive, and X‐linked dominant inheritance patterns representing 39 Mendelian disorders were identified among these populations.ConclusionScreening for a broader range of disorders could offer the benefits of early or presymptomatic diagnosis and reproductive choice.