Methods of assaying cells of a cellular sample for the presence of a target nucleic acid are provided. Aspects of the methods include evaluating a cellular sample that has been contacted with a nuclease inhibitor for the presence of a target nucleic acid. Also provided are devices and kits that find use in practicing the methods described herein.

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 PCR 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 PCR 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 invention provides methods of detecting a nucleic acid present in a biological sample, comprising combining the biological sample with a lysis buffer to form a lysis mixture comprising nucleic acid released from cells in said biological sample; and subjecting a volume of the lysis mixture to size-exclusion chromatography in a column comprising a volume of size-exclusion medium. In certain embodiments, the lysis buffer separates double-stranded nucleic acid into single-stranded nucleic acid. In certain embodiments, the elution can have a flow rate of separation of less than 10 minutes to produce an eluted solution comprising isolated nucleic acid. The invention provides for a method of accurately and rapidly detecting products of nucleic acid amplification.

The invention provides methods of detecting a nucleic acid present in a biological sample, comprising combining the biological sample with a lysis buffer to form a lysis mixture comprising nucleic acid released from cells in said biological sample; and subjecting a volume of the lysis mixture to size-exclusion chromatography in a column comprising a volume of size-exclusion medium. In certain embodiments, the lysis buffer separates double-stranded nucleic acid into single-stranded nucleic acid. In certain embodiments, the elution can have a flow rate of separation of less than 10 minutes to produce an eluted solution comprising isolated nucleic acid. The invention provides for a method of accurately and rapidly detecting products of nucleic acid amplification.

Methods, compositions, kits and apparatuses that include a fluid, the fluid containing a ternary complex and Li.sup.+, wherein the ternary complex includes a primed template nucleic acid, a polymerase, and a nucleotide cognate for the next correct base for the primed template nucleic acid molecule. As an alternative or addition to Li.sup.+, the fluid can contain betaine or a metal ion that inhibits polymerase catalysis such as Ca.sup.2+. In addition to Li.sup.+, the fluid can contain polyethylenimine (PEI) with or without betaine.

Methods, compositions, kits and apparatuses that include a fluid, the fluid containing a ternary complex and Li.sup.+, wherein the ternary complex includes a primed template nucleic acid, a polymerase, and a nucleotide cognate for the next correct base for the primed template nucleic acid molecule. As an alternative or addition to Li.sup.+, the fluid can contain betaine or a metal ion that inhibits polymerase catalysis such as Ca.sup.2+. In addition to Li.sup.+, the fluid can contain polyethylenimine (PEI) with or without betaine.

The invention provides methods of amplification from a single primer or a pair of forward and reverse primers of limited nucleotide composition. Limited nucleotide composition means that the primers are underrepresented in at least one nucleotide type. Such primers have much reduced capacity to prime from each other or to extend initiated by mispriming from other than at their intended primer binding sites in a target nucleic acid.

The present disclosure provides compositions and methods that employ the compositions for conducting pairwise sequencing and for generating concatemer template molecules for pairwise sequencing. The concatemers can be generated using a rolling circle amplification reaction which is conducted either on-support, or conducted in-solution and then distributed onto a support. The rolling circle amplification reaction generates concatemers containing tandem copies of a sequence of interest and at least one universal adaptor sequence. An increase in the number of tandem copies in a given concatemer increases the number of sites along the concatemer for hybridizing to multiple sequencing primers which serve as multiple initiation sites for polymerase-catalyzed sequencing reactions. When the sequencing reaction employs detectably labeled nucleotides and/or detectably labeled multivalent molecules (e.g., having nucleotide units), the signals emitted by the nucleotides or nucleotide units that participate in the parallel sequencing reactions along the concatemer yields an increased signal intensity for each concatemer.

The present disclosure provides compositions and methods that employ the compositions for conducting pairwise sequencing and for generating concatemer template molecules for pairwise sequencing. The concatemers can be generated using a rolling circle amplification reaction which is conducted either on-support, or conducted in-solution and then distributed onto a support. The rolling circle amplification reaction generates concatemers containing tandem copies of a sequence of interest and at least one universal adaptor sequence. An increase in the number of tandem copies in a given concatemer increases the number of sites along the concatemer for hybridizing to multiple sequencing primers which serve as multiple initiation sites for polymerase-catalyzed sequencing reactions. When the sequencing reaction employs detectably labeled nucleotides and/or detectably labeled multivalent molecules (e.g., having nucleotide units), the signals emitted by the nucleotides or nucleotide units that participate in the parallel sequencing reactions along the concatemer yields an increased signal intensity for each concatemer.