The present disclosure provides methods for determining the ploidy status of a chromosome in a gestating fetus from genotypic data measured from a mixed sample of DNA comprising DNA from both the mother of the fetus and from the fetus, and optionally from genotypic data from the mother and father. The ploidy state is determined by using a joint distribution model to create a plurality of expected allele distributions for different possible fetal ploidy states given the parental genotypic data, and comparing the expected allelic distributions to the pattern of measured allelic distributions measured in the mixed sample, and choosing the ploidy state whose expected allelic distribution pattern most closely matches the observed allelic distribution pattern. The mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias, for example using massively multiplexed targeted PCR.

A method and system to decode information stored on a polymer sequence, such as a DNA strand, is described herein. The method and system use molecular probes to label sections of the polymer sequence. Each molecular probe includes a fluorophore and a quencher. The fluorophore produces light with a color and wavelength corresponding to the information stored on the section of the polymer sequence the molecular probe labels. The quencher inhibits the production of light by an adjacent fluorophore. When adjacent sections of the polymer sequence are labeled with molecular probes, the fluorophore of the leading molecular probe produces light while the trailing molecular probe's light is quenched. The method and system then sequentially unbind the molecular probes from the sections of the polymer sequence within a waveguide, producing a sequence of observable fluorescence signals. The sequence can be used to determine the information stored on a polymer sequence.

A method and system to decode information stored on a polymer sequence, such as a DNA strand, is described herein. The method and system use molecular probes to label sections of the polymer sequence. Each molecular probe includes a fluorophore and a quencher. The fluorophore produces light with a color and wavelength corresponding to the information stored on the section of the polymer sequence the molecular probe labels. The quencher inhibits the production of light by an adjacent fluorophore. When adjacent sections of the polymer sequence are labeled with molecular probes, the fluorophore of the leading molecular probe produces light while the trailing molecular probe's light is quenched. The method and system then sequentially unbind the molecular probes from the sections of the polymer sequence within a waveguide, producing a sequence of observable fluorescence signals. The sequence can be used to determine the information stored on a polymer sequence.

The invention is directed to methods for synthesizing a plurality of oligonucleotides in the same reaction vessel, and in some embodiments, using the synthesized oligonucleotides in an oligonucleotide-based assay in such reaction vessel. In some embodiments, methods of the invention are implemented by steps of (a) providing a plurality of different initiators attached to one or more supports, each different initiator having a terminal nucleotide with a different 3-O-blocking group; (b) for each different initiator, synthesizing a polynucleotide by repeated cycles of template-free enzymatic additions of 3′-O-blocked nucleoside triphosphates, wherein the blocking group of the 3-O-blocked nucleoside triphosphate is removable under deblocking conditions orthogonal to the deblocking conditions for removing blocking groups of the other initiators; and (c) releasing the oligonucleotides from the polynucleotides and the one or more solid supports.

The invention is directed to methods for synthesizing a plurality of oligonucleotides in the same reaction vessel, and in some embodiments, using the synthesized oligonucleotides in an oligonucleotide-based assay in such reaction vessel. In some embodiments, methods of the invention are implemented by steps of (a) providing a plurality of different initiators attached to one or more supports, each different initiator having a terminal nucleotide with a different 3-O-blocking group; (b) for each different initiator, synthesizing a polynucleotide by repeated cycles of template-free enzymatic additions of 3′-O-blocked nucleoside triphosphates, wherein the blocking group of the 3-O-blocked nucleoside triphosphate is removable under deblocking conditions orthogonal to the deblocking conditions for removing blocking groups of the other initiators; and (c) releasing the oligonucleotides from the polynucleotides and the one or more solid supports.

Disclosed are methods for detecting a target nucleic acid in a sample. The methods include contacting the sample, in the presence of a polymerase and an endonuclease, with a first oligonucleotide comprising, in the 5′ to 3′ direction, a first signal DNA generation sequence, an endonuclease recognition site, and a sequence complementary to the 3′ end of a target nucleic acid; a second oligonucleotide comprising, in the 5′ to 3′ direction, a second signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the first signal DNA generation sequence of the first oligonucleotide; a third oligonucleotide comprising, in the 5′ to 3′ direction, a third signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the second signal DNA generation sequence of the second oligonucleotide.

Disclosed are methods for detecting a target nucleic acid in a sample. The methods include contacting the sample, in the presence of a polymerase and an endonuclease, with a first oligonucleotide comprising, in the 5′ to 3′ direction, a first signal DNA generation sequence, an endonuclease recognition site, and a sequence complementary to the 3′ end of a target nucleic acid; a second oligonucleotide comprising, in the 5′ to 3′ direction, a second signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the first signal DNA generation sequence of the first oligonucleotide; a third oligonucleotide comprising, in the 5′ to 3′ direction, a third signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the second signal DNA generation sequence of the second oligonucleotide.

A method for generating an image of a region of interest in a biological sample comprising the steps of: generating a first image including the region of interest of the biological sample having undergone a first protocol but not a second protocol; and generating a second image including the region of interest of the biological sample after having undergone a second protocol; wherein the region of interest is smaller than said sample. Also provided is a method of analyzing a biological sample, comprising providing an image of the biological sample according to the method for generating an image of a region of interest in a biological sample, and analyzing the biological sample from the image. Further provided are system and kit that comprise the means for executing the novel methods.

A method for generating an image of a region of interest in a biological sample comprising the steps of: generating a first image including the region of interest of the biological sample having undergone a first protocol but not a second protocol; and generating a second image including the region of interest of the biological sample after having undergone a second protocol; wherein the region of interest is smaller than said sample. Also provided is a method of analyzing a biological sample, comprising providing an image of the biological sample according to the method for generating an image of a region of interest in a biological sample, and analyzing the biological sample from the image. Further provided are system and kit that comprise the means for executing the novel methods.

The invention is directed to a method for detecting RNA, DNA or protein target sequences by a) Hybridizing a library of probes having the general formula (I)

P—(CL-D).sub.x  (I)  With P: probes having at least 10 nucleotides or amino acids CL: cleavable linker D: fluorescent dye X: integer between 1 and 5  to RNA, DNA or protein target sequences wherein the library comprises probes P having different sequences of nucleotides or amino acids and cleavable linkers CL of different groups which are cleavable with different means b) Removing unhybridized probes and detecting the hybridized probes via the fluorophores D by a first image c) Cleaving sequentially by different means each group of chemical linkers CL from the hybridized probes; removing the thus cleaved fluorophores D and detecting the remaining hybridized probes via their fluorophores D by a second image d) Detecting the removed fluorophores D by comparing the first and second image. e) Obtaining a part of the sequence information of the target sequences via the sequence information of the probes P associated with the removed fluorophores D f) Repeating step c) until all groups of chemical linkers CL are cleaved.