The present methods are exemplified by a process in which maternal blood containing fetal DNA is diluted to a nominal value of approximately 0.5 genome equivalent of DNA per reaction sample. Digital analysis is then be used to detect aneuploidy, such as the trisomy that causes Down Syndrome. Since aneuploidies do not present a mutational change in sequence, and are merely a change in the number of chromosomes, it has not been possible to detect them in a fetus without resorting to invasive techniques such as amniocentesis or chorionic villi sampling. Digital amplification allows the detection of aneuploidy using massively parallel amplification and detection methods, examining, e.g., 10,000 genome equivalents.

The present methods are exemplified by a process in which maternal blood containing fetal DNA is diluted to a nominal value of approximately 0.5 genome equivalent of DNA per reaction sample. Digital analysis is then be used to detect aneuploidy, such as the trisomy that causes Down Syndrome. Since aneuploidies do not present a mutational change in sequence, and are merely a change in the number of chromosomes, it has not been possible to detect them in a fetus without resorting to invasive techniques such as amniocentesis or chorionic villi sampling. Digital amplification allows the detection of aneuploidy using massively parallel amplification and detection methods, examining, e.g., 10,000 genome equivalents.

The present invention is directed to methods and kits for template-free enzymatic synthesis of polynucleotides employing hybridization stringency and/or nuclease digestion for removing failure sequences.

The present invention is directed to methods and kits for template-free enzymatic synthesis of polynucleotides employing hybridization stringency and/or nuclease digestion for removing failure sequences.

The present disclosure provides methods of sequencing polynucleotides and compounds, compositions for sequencing of polynucleotides, and synthesis of such compositions. The chemical compounds include nucleotides and their analogs which possess a sugar moiety comprising a cleavable chemical group capping the 3′-OH group and a base, but without covalently bounded dye. The cleavable chemical group is reactive to form covalent bond(s) with a dye used to confirm the presence of the expected base-pairing. The cleavable chemical group capping the 3′OH group can be removed together with the covalently bounded dye. Furthermore, after the cleavable chemical group is cleaved, the free 3′-OH group can be active in continued elongation. Example chemical compounds according to the present disclosure are shown as Formulas (II) and (V):

##STR00001##

The present disclosure provides methods of sequencing polynucleotides and compounds, compositions for sequencing of polynucleotides, and synthesis of such compositions. The chemical compounds include nucleotides and their analogs which possess a sugar moiety comprising a cleavable chemical group capping the 3′-OH group and a base, but without covalently bounded dye. The cleavable chemical group is reactive to form covalent bond(s) with a dye used to confirm the presence of the expected base-pairing. The cleavable chemical group capping the 3′OH group can be removed together with the covalently bounded dye. Furthermore, after the cleavable chemical group is cleaved, the free 3′-OH group can be active in continued elongation. Example chemical compounds according to the present disclosure are shown as Formulas (II) and (V):

##STR00001##

The present disclosure provides improved nucleic acid sequencing-by-synthesis (SBS) methods, related kits and reagents, and systems for performing such methods using such kits and reagents.

The present disclosure provides improved nucleic acid sequencing-by-synthesis (SBS) methods, related kits and reagents, and systems for performing such methods using such kits and reagents.

This disclosure provides chips, systems and methods for sequencing a nucleic acid sample. Tagged nucleotides are provided into a reaction chamber comprising a nanopore in a membrane. An individual tagged nucleotide of the tagged nucleotides can contain a tag coupled to a nucleotide, which tag is detectable with the aid of the nanopore. Next, an individual tagged nucleotide of the tagged nucleotides can be incorporated into a growing strand complementary to a single stranded nucleic acid molecule derived from the nucleic acid sample. With the aid of the nanopore, a tag associated with the individual tagged nucleotide can be detected upon incorporation of the individual tagged nucleotide. The tag can be detected with the aid of the nanopore when the tag is released from the nucleotide.

This disclosure provides chips, systems and methods for sequencing a nucleic acid sample. Tagged nucleotides are provided into a reaction chamber comprising a nanopore in a membrane. An individual tagged nucleotide of the tagged nucleotides can contain a tag coupled to a nucleotide, which tag is detectable with the aid of the nanopore. Next, an individual tagged nucleotide of the tagged nucleotides can be incorporated into a growing strand complementary to a single stranded nucleic acid molecule derived from the nucleic acid sample. With the aid of the nanopore, a tag associated with the individual tagged nucleotide can be detected upon incorporation of the individual tagged nucleotide. The tag can be detected with the aid of the nanopore when the tag is released from the nucleotide.