Progress, and Applications
of the Human Genome Project
Sponsored by the U.S. Department of Energy Human Genome Program
Human Genome News Archive Edition
Human Genome News, April-June 1996; 7(6)
Santa Fe '96
Several speakers reported on the rapidly expanding work to develop new technologies for detecting specific DNA sequences. These technologies will be useful in identifying disease mutations and genetic typing of genes as well as for resequencing specific genomic regions. Many investigators are currently working on scaleup for diagnostic uses.
Charles Cantor [Boston University (BU)] described hybridization-based methods for isolating and profiling triplet-repeat DNA sequences and for general mutation detection. In one approach developed by Cassandra Smith (BU), target DNA is hybridized to an array of simple repeating sequences immobilized on magnetic microbeads and analyzed for mismatches (bulges) in the target or array. Detection of repeat length is by electrophoresis on an automated fluorescent gel reader. BU scientists are also developing chip-based methods with a detection system based on mass spectrometry, which may prove to be a practical method for comparing short sequences.
Deborah Nickerson (University of Washington, Seattle) discussed a method for scanning STSs from the physical map to develop polymorphic markers based on single-base changes. These markers will be useful for the high-throughput genotyping of human populations needed for studying complex traits, including diseases. An estimated 5 million single-base changes (1 in 600 bp) in the human genome lead to population diversity.
Nickerson's group screened 154 STSs from the Whitehead-MIT physical map and found that 1 in 4 was polymorphic (single-base variations), with variation frequency (heterozygosity) greater than 30%. To detect sequence variations in STSs, they used Polyphred software, which detects heterozygous positions in automated sequence traces and interfaces with Phil Green's programs Phred, Phrap, and Consed (email@example.com). Nickerson estimates that minimal scanning of long (>250 bp) STSs would yield a new genetic marker every 2 Mb and produce more than 1500 useful diallelic markers.
Commenting on the 107 bp of sequence entered into the public sequence databases for the relatively tiny 10-kb HIV genome, George Church (Harvard Medical School) pointed out that sequencing will not end once the first genome is finished. His laboratory is developing scaleable, sensitive, cost-efficient technologies for diagnostic resequencing of genomes, as well as for new sequencing efforts. Using available components, Church's group developed a system that eliminates probing and separates detection from the electrophoresis step. In collaborative efforts, the researchers have developed a rapid method to detect mass tags (using 400 different electrophores) on primers and clones or genomic target DNA. This method enables targeting of 100 spots per second.
Genetic Bar Codes
Mary Ann Brow (Third Wave Technologies) described a new thermostable, structure-specific enzyme that can detect mutations in clinically significant genes, including ß-globin, p53, and the Mycobacterium tuberculosis. The enzyme Cleavase recognizes and cuts secondary structures formed in the single strands of a DNA fragment following denaturation. Presence of a mutation is indicated by a change in fragment pattern near the mutation region. The Cleavase reaction (cleavase fragment length polymorphisms, called CFLPs) can thus detect and localize mutations. Like bar codes, cleavage patterns are reproducible and can be archived electronically.
Michael Pirrung (Duke University) discussed the preparation of high-density arrays of short DNA sequences (oligonucleotides) for arrayed primer extension (APEX) in mutation detection and sequencing. Pirrung's group has developed a superior new photoremovable group for light-directed DNA array synthesis. APEX uses analyte DNA as a template and synthesized DNA in an array as a primer. These arrays can be used effectively for comparison sequencing with APEX and for analysis of gene expression with mRNA templates and reverse transcriptase. Substitution, deletion, and insertion mutations have been detected.
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