Molecular diagnostics system and methods
The present invention relates to methods for the extraction of nucleic acids from cells, the amplification of segments of nucleic acid and the detection of nucleic acids, all in a convenient and portable manner. In one embodiment, a sample comprising cells containing nucleic acid is exposed to an aqueous mixture comprising a lytic reagent and one or more beads capable of binding the nucleic acid released from said cells to form a nucleic acid-bead complex. The nucleic acid-bead complex is passed through an immiscible liquid layer to separate the nucleic acid from the aqueous mixture. The one or more beads are magnetic, and the nucleic acid-bead complex is passed through and separated from the immiscible liquid layer with an applied magnetic field. The invention is particularly suited for use in point-of-care medical diagnostics testing.
1. A method for performing a nucleic acid amplification assay comprising:
(a) combining reagents, DNA polymerase, a target nucleic acid and an amount of a first modified primer comprising a sequence of bases complementary to a first region of the target nucleic acid, a polymerase blocking region attached to the first modified primer, a first single stranded hybridization region attached to the polymerase blocking region, and a detectable label,
(b) cycling a mixture of (a) to provide multiple copies of an amplicon incorporating the first modified primer;
(c) substantially eliminating excess unincorporated first modified primer from the mixture;
(d) exposing the mixture to a capture oligonucleotide complementary to the first single stranded hybridization region;
(e) hybridizing the first single stranded hybridization region of the amplicon incorporating the first modified primer, with the capture oligonucleotide; and
(f) detecting the label associated with the hybridization.
2. The method of claim 1 , wherein:
the combining further comprises combining an amount of a second modified primer comprising a sequence of bases complementary to a second region of the target nucleic acid, a polymerase blocking region attached to the second modified primer, a second single stranded hybridization region attached to the polymerase blocking region, and a detectable label;
the cycling further comprises cycling a mixture of (a) to provide multiple copies of an amplicon incorporating the first modified primer and the second modified primer;
the substantially eliminating further comprises the substantially eliminating excess unincorporated second modified primer from the mixture;
exposing the mixture further comprises exposing the mixture to a capture oligonucleotide complementary to both of the first single stranded hybridization region and the second single stranded hybridization region; and
hybridizing further comprises hybridizing both of the first single stranded hybridization region and the second single stranded hybridization region of the amplicon incorporating the first modified primer and the second modified primer, with the capture oligonucleotide.
3. The method of claim 1 , wherein the polymerase blocking region is phosphoraramidite 18-O-dimethoxyltritylhexaethyleneglycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.
4. The method of claim 1 , wherein the delectable label is biotin, FAM, or fluorocein.