Biomedical subjects

R J Lipshutz

Publications and source records attributed to R J Lipshutz.

5 recordsLinked to original sources

PubMed1996

Mapping genomic library clones using oligonucleotide arrays.

We have developed a high-density DNA probe array and accompanying biochemical and informatic methods to order clones from genomic libraries. This approach involves a series of enzymatic steps for capturing a set of short dispersed sequence markers scattered throughout a high-molecular-weight DNA. By this process, all the ambiguous sequences lying adjacent to a given Type IIS restriction site are ligated between two DNA adapters. These markers, once amplified and labeled by PCR, can be hybridized and detected on a high-density oligonucleotide array bearing probes complementary to all possible markers. The array is synthesized using light-directed combinatorial chemistry. For each clone in a genomic library, a characteristic set of sequence markers can be determined. On the basis of the similarity between the marker sets for each pair of clones, their relative overlap can be measured. The library can be sequentially ordered into a contig map using this overlap information. This new methodology does not require gel-based methods or prior sequence information and involves manipulations that should allow for easy adaptation to automated processing and data collection.

Base Sequence↗

PubMed1995

Using oligonucleotide probe arrays to access genetic diversity.

As the Human Genome Project and related efforts identify and determine the DNA sequences of human genes, it is important that highly reliable and efficient mechanisms are found to access individual genetic variation. It is only through a greater understanding of genetic diversity that the true benefit of the Human Genome Project will be realized. One approach, hybridization to high-density arrays of oligonucleotides, is a fast and effective means of accessing this genetic variation. Light-directed chemical synthesis has been used to generate miniaturized, high-density arrays of oligonucleotide probes. Application-specific oligonucleotide probe array designs have been developed for the rapid screening of characterized genes. Dedicated instrumentation and software have been developed for array hybridization, fluorescence detection and data acquisition and analysis. In a specific and challenging application, oligonucleotide probe arrays have been used to screen the reverse transcriptase and protease genes of the highly polymorphic HIV-1 genome to explore genetic diversity and detect mutations conferring resistance to antiviral drugs. Results from this application strongly suggest that oligonucleotide probe arrays will be a powerful tool for rapid investigations in sequence checking, pathogen detection, expression monitoring and DNA molecular recognition.

Base Sequence↗

PubMed1994

DNA sequence confidence estimation.

A significant bottleneck in the current DNA sequencing process is the manual editing of trace data generated by automated DNA sequencers. This step is used to correct base calls and to associate to each base call a confidence level. The confidence levels are used in the assembly process to determine overlaps and to resolve discrepancies in determining the consensus sequence. This single step may cost as much as 4 to 8 cents per finished base. We report an approach to automated trace editing using classification trees to detect and exploit context-based patterns in trace peak heights. Local base composition and nearby peak heights account for 80% of the variations in peak heights. Classification algorithms were developed to identify 37% of automated base calls that differ from the consensus sequence. With these algorithms, 12% of the base calls had confidence levels less than 90%.

Algorithms↗

PubMed1993

Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP.

We have used affinity panning of libraries of bacteriophages that display random octapeptide or dodecapeptide sequences at the N-terminus of the adsorption protein (pIII) to characterize peptides that bind to the endoplasmic reticulum chaperone BiP and to develop a scoring system that predicts potential BiP-binding sequences in naturally occurring polypeptides. BiP preferentially binds peptides containing a subset of aromatic and hydrophobic amino acids in alternating positions, suggesting that peptides bind in an extended conformation, with the side chains of alternating residues pointing into a cleft on the BiP molecule. Synthetic peptides with sequences corresponding to those displayed by BiP-binding bacteriophages bind to BiP and stimulate its ATPase activity, with a half-maximal concentration in the range 10-60 microM.

Adenosine Triphosphatases↗

PubMed1993

Likelihood DNA sequencing by hybridization.

Sequencing by hybridization (SBH) extracts local sequence information from a DNA fragment using hybridization with oligonucleotides and then reconstructs the sequence using the derived information. We describe an improvement to the SBH methodology which will allow it to work efficiently in the presence of hybridization errors. In particular, given a set of hybridizing probes, and the empirically derived rates of false positive and false negative hybridization, we can estimate the most likely DNA fragment to have produced the set of probes, and then estimate the probability that it generated the hybridization data. This methodology extends earlier results by identifying the most probable fragment to have generated the actual hybridization data. The methodology described will also generate longer unambiguous sequence fragments without the use of overlapping fragments.

Algorithms↗