DNA SEQUENCING

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

Infrared Fluorescent Detection of D1S80 Alleles from Blood and Body Fluid Collected on IsoCode™ Devices

A genetic locus D1S80 (pMCT 118) containing variable number of tandem repeats (VNTR) has been used extensively in forensic analysis and paternity testing. In the current research, DNA was isolated from blood, saliva and nasal secretions collected on two types of IsoCode™ paper-based devices. The D1S80 locus was amplified using PCR technology, and the alleles were separated by gel electrophoresis and then detected using an infrared (IR) fluorescence automated DNA sequencer. IR-labeled amplification products were generated from human genomic DNA using oligonucleotide primers, which were covalently linked to an infrared fluorescent dye (IRD41) at the 5′ end. This system combines IR fluorescence chemistry and laser technology, thus eliminating the need for post-electrophoretic gel handling for the detection of the alleles. Real-time detection after separation of the alleles is valuable for visualization of the data. The VNTR alleles are displayed as familiar autoradiogram-like images, which can also be analyzed by computer. Since DNA is eluted from the IsoCode devices only with sterile distilled water and without time-consuming methods of extraction, amplification can be performed from numerous samples within a short period of time

DNA SEQUENCING

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

Continuous, on-line DNA sequencing using oligodeoxynucleotide primers with multiple fluorophores

A method for sequencing DNA by using a difluoresceinated primer and laser excitation is described. Dideoxy protocols have been determined that provide sequences for 600 bases starting with base 1 with \u3c1% error in a single load. Electrophoresis is at 20 W and the bands are detected 24 cm from the bottom of the loading well with a scanning fluorescence detector. Bands are imaged on a TV screen in two dimensions. The sequences can be read from the TV screen manually or semiautomatically by using a simple software program. The system allows more bases to be read with a lower error rate than any other reported automated sequencing method