University of Nebraska

Li-cor, Inc.

National Institutes of Health, a Component of the US Dept. of Health & Human Services

John A Brumbaugh

Lyle R Middendorf

Gi Young Jang

Gabor Patonay

Narasimhachari Narayanan

Stephen H Hughes

Ramesh Kumar

A universal terminator includes a heterocycle other than naturally occuring DNA heterocycles such as adenine, cytosine, guanine and thymine. In terminating strand synthesis, the hybridized primer-template is split into four aliquots. In the &ldquo;A&rdquo; vial is added DNA polymerase and normal amounts of &ldquo;C&rdquo;, &ldquo;G&rdquo;, and &ldquo;T&rdquo; deoxynucleotides, along with a reduced amount of &ldquo;A&rdquo; deoxynucleotide and an amount of the universal terminator such that statistically the universal terminator has less than a one percent chance of being incorporated at sites where a &ldquo;C&rdquo;, &ldquo;G&rdquo;, or &ldquo;T&rdquo; deoxynucleotide should be incorporated and about a one percent chance of being incorporated at sites where an &ldquo;A&rdquo; deoxynucleotide should be incorporated. Similar strategies are followed for the &ldquo;C&rdquo;, &ldquo;G&rdquo;, and &ldquo;T&rdquo; vials, except that the amount of &ldquo;C&rdquo;, &ldquo;G&rdquo; and &ldquo;T&rdquo; deoxynucleotides are reduced for their respective vial.

DNA sequencing and DNA terminators

The present invention relates to a method of detecting a DNA sequence by means of a DNA:DNA hybrid in real time using fluorescence. The present invention eliminates the need to use radioactive probes to detect the DNA and eliminates the delay needed for autoradiographic exposure of the X-ray to the radioactive label.

Method for the fluorescent detection of a DNA sequence in real time

To sequence long strands of DNA, clone strands having lengths longer than 100 bases are, in one embodiment, marked on one end with biotin. These strands are divided into 4 aliquots and each aliquot: (1) is uniquely chemically treated to randomly terminate the strands at the non-biotinylated end at a selected type of base; and (2) is moved continuously by electrophoresis through a different one of four identical channels. In the one embodiment, the strands are randomly terminated at a selected base type and they are moved into avidin, which due to high affinity, combines with the biotin marked ends of shorter strands before the longer strands are fully resolved in the gel. The avidin is marked with fluorescein, the strands are scanned and the signals are decoded. In another embodiment, the strands are synthesized, with termination at a selected base type and marked either by the above method of by ethidium bromide.

DNA sequencing

To sequence long strands of DNA, clone strands having lengths longer than 100 bases are, in one embodiment, marked on one end with biotin. These strands are divided into 4 aliquots and each aliquot: (1) is uniquely chemically treated to randomly terminate the strands at the nonbiotinylated end at a selected type of base; and (2) is moved continuously by electrophoresis through a different one of four identical channels. In the one embodiment, the strands are randomly terminated at a selected base type and they are moved into avidin, which due to high affinity, combines with the biotin marked ends of shorter strands before the longer strands are fully resolved in the gel. The avidin is marked with fluorescein, the strands are scanned and the signals are decoded. In another embodiment, the strands are synthesized, with termination at a selected base type and marked either by the above method of by ethidium bromide.

To sequence DNA automatically, DNA marked with far infrared, near infrared, or infrared fluorescent dyes are electrophoresed in a plurality of channels through a gel electrophoresis slab or capillary tubes wherein the DNA samples are resolved in accordance with the size of DNA fragments in the gel electrophoresis slab or capillary tubes into fluorescently marked DNA bands. The separated samples are scanned photoelectrically with a laser diode and a sensor, wherein the laser scans with scanning light at a wavelength within the absorbance spectrum of said fluorescently marked DNA samples and light is sensed at the emission wavelength of the marked DNA.

Sequencing near infrared and infrared fluorescence labeled DNA for

To sequence DNA automatically, fluorescently marked DNA are electrophoresed in a plurality of channels through a gel electrophoresis slab; wherein the DNA samples are resolved in accordance with the size of DNA fragments in the gel electrophoresis slab into fluorescently marked DNA bands. The separated samples are scanned photoelectrically with a laser and a sensor, wherein the laser scans with scanning light at a scanning light frequency within the absorbance spectrum of said fluorescently marked DNA samples and light is sensed at the emission frequency of the marked DNA. The light is modulated from said laser at a predetermined modulation frequency and fluorescent light emitted by said DNA bands at said modulation frequency is detected, whereby background noise from the medium through which the light is transmitted is discriminated against.

Digital DNA typing

To sequence long strands of DNA, cloned strands having lengths longer than 100 bases are, in one embodiment, marked on one end with biotih. These strands are divided into 4 aliquots and each aliquot: (1) is uniquely chemically treated to randomly terminate the strands at the non-biotinylated end at a selected type of base; and (2) is moved continuously by electrophoresis through a different one of four identical channels. In the one embodiment, the strands are randomly terminated at a selected base type and they are moved into avidin, which due to high affinity, combines with the biotin marked ends of shorter strands before the longer strands are fully resolved in the gel. The avidin is marked with fluorescein, the strands are scanned and the signals are decoded. In another embodiment, the strands are synthesized, with termination at a selected base type and marked either by the above method or by ethidium bromide.

To sequence DNA automatically, flourescently marked DNA are electrophoresed in a plurality of channels through a gel electrophoresis slab; wherein the DNA samples are resolved in accordance with the size of DNA fragments in the gel electrophoresis slab into fluorescently marked DNA bands. The separated samples are scanned photoelectrically with a laser and a sensor, wherein the laser scans with scanning light at a scanning light frequency within the absorbance spectrum of said fluorescently marked DNA samples and light is sensed at the emission frequency of the marked DNA. The light is modulated from said laser at a predetermined modulation frequency and fluorescent light emitted by said DNA bands at said modulation frequency is detected, whereby background noise from the medium through which the light is transmitted is discriminated against.

To sequence long strands of DNA, cloned strands having lengths longer than 100 bases are, in one embodiment, marked on one end with biotin. These strands are divided into 4 aliquots and each aliquot: (1) is uniquely chemically treated to randomly terminate the strands at the non-biotinylated end at a selected type of base; and (2) is moved continuously by electrophoresis through a different one of four identical channels. In the one embodiment, the strands are randomly terminated at a selected base type and they are moved into avidin, which due to high affinity, combines with the biotin marked ends of shorter strands before the longer strands are fully resolved in the gel. The avidin is marked with fluorescein, the strands are scanned and the signals are decoded. In another embodiment, the strands are synthesized, with termination at a selected base type and marked either by the above method or by ethidium bromide.

Growing community of inventors

Lincoln, NE, United States of America

John A Brumbaugh