DNA sequencing directly from a mixture using terminal-base-selective primers.

H Furuyama; K Okano; H Kambara

doi:10.1093/dnares/1.5.231

Home/Archive/PubMed

PubMed · 7584045

DNA sequencing directly from a mixture using terminal-base-selective primers.

H Furuyama·K Okano·H Kambara

View original source ↗Export metadata ↓

Abstract

DNA cloning is often used to select and amplify one DNA species from a mixture. However, the cloning process is complex and labor-intensive. We have developed a new two-step method for DNA sequencing directly from a mixture. The first is the introduction of a known oligonucleotide (common part) into the terminus of unknown DNA by ligation. The second is selective DNA sequencing using primers with two additional nucleotides at the 3' terminus in addition to the common part (terminal-base-selective primers). The primers work only for templates on which the primers perfectly hybridized. This method was found to be effective for the HindIII digestion products of lambda phage.

Explore related subjects

Bacteriophage lambda Base Sequence DNA Primers DNA, Single-Stranded DNA, Viral Deoxyribonuclease HindIII Molecular Sequence Data Sequence Analysis, DNA Journal Article

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

H Furuyama, K Okano, H Kambara. 1994. DNA sequencing directly from a mixture using terminal-base-selective primers.. https://doi.org/10.1093/dnares%2F1.5.231

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

Propagation of fluorescent viruses in growing plaques.

To study virus propagation, we have developed a method by which the propagation of the Lambda bacteriophage can be observed and quantified. This is done by creating a fusion protein of the capsid protein gpD and the enhanced yellow fluorescent protein (EYFP). We show that this fusion allows capsid formation and that the modified viruses propagate on a surface covered with host bacteria thus forming fluorescent plaques. The intensity of fluorescence in a growing plaque determines the distribution of phages. This provides a new tool to study the propagation of infection at the microscopic level.

Bacteriophage lambda↗

PubMed2007

Combining optical tweezers and scanning probe microscopy to study DNA-protein interactions.

We present the first results obtained with a new instrument designed and built to study DNA-protein interactions at the single molecule level. This microscope combines optical tweezers with scanning probe microscopy and allows us to locate DNA-binding proteins on a single suspended DNA molecule. A single DNA molecule is stretched taut using the optical tweezers, while a probe is scanned along the molecule. Interaction forces between the probe and the sample are measured with the optical tweezers. The instrument thus enables us to correlate mechanical and functional properties of bound proteins with the tension within the DNA molecule. The typical friction force between a micropipette used as probe and a naked DNA molecule was found to be <1 pN. A 16 micro m DNA molecule with approximately 10-15 digoxygenin (DIG) molecules located over a 90 nm range in the middle of the DNA was used as a model system. By scanning with an antidigoxygenin (alpha-DIG) antibody-coated pipette we were able to localize these sites by exploiting the high binding affinity between this antibody-antigen pair. The estimated experimental resolution assuming an infinitesimally thin and rigid probe and a single alpha-DIG/DIG bond was 15 nm.

Bacteriophage lambda↗

PubMed2007

A rapid DNA digestion system.

This paper presents a novel microfluidic DNA digestion system incorporating a high performance micro-mixer. Through the appropriate control of fixed and periodic switching DC electric fields, electrokinetic forces are established to mix the DNA and restriction enzyme samples and to drive them through the reaction column of the device. The experimental and numerical results show that a mixing performance of 98% can be achieved within a mixing channel of length 1.6 mm when a 150 V/cm driving voltage and a 5 Hz switching frequency are applied. The relationship between the mixing performance, switching frequency, and main applied electric field is derived. It is found that the optimal switching frequency depends upon the magnitude of the main applied electric field. The successful digestion of lambda-DNA using Eco RI restriction enzyme is demonstrated. The DNA-enzyme reaction is completed within 15 min in the proposed microfluidic system, compared to 50 min in a conventional large-scale system. Hence, the current device provides a valuable tool for rapid lambda-DNA digestion, while its mixer system delivers a simple yet effective solution for mixing problems in the micro-total-analysis-systems field.

Bacteriophage lambda↗