DNA sequencing

Claim 1

A method for sequencing DNA comprising the steps of: preparing a multiplicity of identical DNA strands; marking the DNA strands on one end with biotin; dividing the DNA strands into at least four batches; preparing at least one additional batch of DNA strands with known lengths to be electrophoresed as a time base; randomly cleaving at least some of the DNA strands in a first batch at adenine bases by a chemical treatment to form an adenine-strand batch; randomly cleaving at least some of the DNA strands in a second batch at guanine bases by a chemical treatment to form a guanine-strand batch; randomly cleaving at least some of the DNA strands in a third batch at cytosine bases by a chemical treatment to form a cytosine-strand batch; randomly cleaving at least some of the DNA strands in a fourth batch at thymine bases by a chemical treatment to form a thymine-strand batch; applying samples from each of the four batches after cleaving at their respective bases to four identical channels of gel electrophoresis apparatus; applying at least one time base source of DNA of known length strands to at least one additional channel positioned among the aforementioned four channels whereby a reference time of electrophoresing may be obtained from said channel; electrophoresing the DNA strands through a gel slab so that bands of more mobile strands, each of which has substantially uniform length strands, are fully resolved while the less mobile strands to be later formed into bands are unresolved in a continuous process such that at least ten percent of the bands are fully resolved and electrophoresed through the gel while the less mobile strands are yet unresolved into bands in the gel; whereby a plurality of adenine-strand bands is formed in a first channel in an order corresponding to the length of the strands; a plurality of guanine-strand bands is formed in a second channel in an order corresponding to the length of the strands; a plurality of cytosine-strand bands is formed in a third channel in an order corresponding to the length of the strands; and a plurality of thymine-strand bands is formed in a fourth channel in an order corresponding to the length of the strands; attaching fluorescent markers to avidin whereby the fluorescently labeled avidin is combined with the biotin markers at the end of the DNA strands; moving the fluorescently-marked bands in sequence through a medium; scanning said bands with laser having a narrow bandwidth substantially comforming to the optimum absorption spectrum of the fluorescent markers; pulsing the laser light with pulses of shorter duration than three nanoseconds during a first period of time; detecting the fluorescent emission from the markers across a narrow selective band of wavelengths conforming substantially to the optimum emission spectrum of the markers during a second period of time; said second period of time beginning at least fifty nanoseconds from the start of its corresponding pulse of laser light and terminating at a time no greater than one hundred fifty nanoseconds from the start of the pulse of the laser light; and identifying and recording the sequence of the bands in each of the channels so as to indicate the DNA sequence.

Claim 2

A method for sequencing DNA comprising the steps of: preparing a multiplicity of identical DNA strands; marking the DNA strands on one end with biotin; dividing the DNA strands into at least four batches; preparing at least one additional batch of DNA strands with known lengths to be electrophoresed as a time base; randomly cleaving at least some of the DNA strands in a first batch at adenine bases by a chemical treatment to form an adenine-strand batch; randomly cleaving at least some of the DNA strands in a second batch at guanine bases by a chemical treatment to form a guanine-strand batch; randomly cleaving at least some of the DNA strands in a third batch at cytosine bases by a chemical treatment to form a cytosine-strand batch; randomly cleaving at least some of the DNA strands in a fourth batch at thymine bases by a chemical treatment to form a thymine-strand batch; applying samples from each of the four batches after cleaving at their respective bases to four identical channels of gel electrophoresis apparatus; applying at least one time base source of DNA of known length strands to at least one additional channel positioned among the aforementioned four channels whereby a reference time of electrophoresing may be obtained from said channel; electrophoresing the DNA strands through a gel slab so that bands of more mobile strands, each of which has substantially uniform length strands, are fully resolved while the less mobile strands to be later formed into bands are unresolved in a continuous process such that at least ten percent of the bands are fully resolved and electrophoresed through the gel while the less mobile strands are yet unresolved into bands in the gel; whereby a plurality of adenine-strand bands is formed in a first channel in an order corresponding to the length of the strands; a plurality of guanine-strand bands is formed in a second channel in an order corresponding to the length of the strands; a plurality of cytosine-strand bands is formed in a third channel in an order corresponding to the length of the strands; and a plurality of thymine-strand bands is formed in a fourth channel in an order corresponding to the length of the strands; attaching fluorescent markers to avidin whereby the fluorescently labeled avidin is combined with the biotin markers at the end of the DNA strands; moving the fluorescently-marked bands in sequence through a medium; scanning said bands with laser having a narrow bandwidth substantially comforming to the optimum absorption spectrum of the fluorescent markers; pulsing the laser light with pulses of shorter duration than three nanoseconds during a first period of time; detecting the fluorescent emission from the markers across a narrow selective band of wavelengths conforming substantially to the optimum emission spectrum of the markers during a second period of time; said second period of time beginning at least fifty nanoseconds from the start of its corresponding pulse of laser light and terminating at a time no greater than one hundred fifty nanoseconds from the start of the pulse of the laser light; and identifying and recording the sequence of the bands in each of the channels so as to indicate the DNA sequence.

Claim 3

A method for sequencing DNA comprising the steps of: preparing a multiplicity of identical DNA strands; creating DNA strands with random terminations at adenine bases and with random lengths to form an adenine-strand batch; creating DNA strands with random terminations at guanine bases and with random lengths to form a guanine-strand batch; creating DNA strands with random terminations at cytosine bases and with random lengths to form a cytosine-strand batch; creating DNA strands with random terminations at thymine bases and with random lengths to form a thymine-strand batch; applying a sample from a different one of each of the four batches to a corresponding one of four identical channels of separating apparatus; separating the DNA strands into bands within the channels so that the bands of more mobile strands in the channels are fully resolved while some of the DNA strands to be later formed into bands are unresolved in a continuous process such that at least ten percent of the bands are resolved while the less mobile DNA strands are yet unresolved into bands in the channel; fluorescently marking the DNA strands; and applying light to the resolved bands while the bands are still moving with respect to each other in the continuous process and identifying and recording the sequence of the bands in each of the channels from a response of the fluorescently marked DNA strands to the applied light, so as to indicate the DNA sequence.

Claim 4

A method for sequencing DNA comprising the steps of: preparing a multiplicity of identical DNA strands; creating DNA strands with random terminations at adenine bases and with random lengths to form an adenine-strand batch; creating DNA strands with random terminations at guanine bases and with random lengths to form a guanine-strand batch; creating DNA strands with random terminations at cytosine bases and with random lengths to form a cytosine-strand batch; creating DNA strands with random terminations at thymine bases and with random lengths to form a thymine-strand batch; applying a sample from a different one of each of the four batches to a corresponding one of four identical channels of separating apparatus; separating the DNA strands into bands within the channels so that the bands of more mobile strands in the channels are fully resolved while some of the DNA strands to be later formed into bands are unresolved in a continuous process such that at least ten percent of the bands are resolved while the less mobile DNA strands are yet unresolved into bands in the channel; fluorescently marking the DNA strands; and applying light to the resolved bands while the bands are still moving with respect to each other in the continuous process and identifying and recording the sequence of the bands in each of the channels from a response of the fluorescently marked DNA strands to the applied light, so as to indicate the DNA sequence.

Claim 14

A method for sequencing DNA comprising the steps of: preparing a multiplicity of identical DNA strands; preparing, from the multiplicity of identical DNA strands, fluorescently marked DNA strands with random lengths terminated at least at one of different ones of the adenine base, guanine base, cytosine base and thymine base, wherein at least one batch of DNA strands is formed, which batch is terminated at one of the adenine base, guanine base, cytosine base and thymine base; applying samples of the fluorescently marked DNA strands to at least one channel of a separating apparatus; separating the strands within at least one channel so that the bands of more mobile strands in the channels are fully resolved while some of the less mobile strands to be later formed into bands are unresolved in a continuous process such that at least ten percent of the bands are fully resolved while the less mobile strands are yet unresolved into bands in the channel; applying light from a source of light to the resolved bands; and identifying and recording the sequence of the bands in the channel from the reaction of the fluorescently marked strands with the light so as to indicate the DNA sequence.

Claim 15

A method for sequencing DNA comprising the steps of: preparing a multiplicity of identical DNA strands; preparing, from the multiplicity of identical DNA strands, fluorescently marked DNA strands with random lengths terminated at least at one of different ones of the adenine base, guanine base, cytosine base and thymine base, wherein at least one batch of DNA strands is formed, which batch is terminated at one of the adenine base, guanine base, cytosine base and thymine base; applying samples of the fluorescently marked DNA strands to at least one channel of a separating apparatus; separating the strands within at least one channel so that the bands of more mobile strands in the channels are fully resolved while some of the less mobile strands to be later formed into bands are unresolved in a continuous process such that at least ten percent of the bands are fully resolved while the less mobile strands are yet unresolved into bands in the channel; applying light from a source of light to the resolved bands; and identifying and recording the sequence of the bands in the channel from the reaction of the fluorescently marked strands with the light so as to indicate the DNA sequence.