A method for detecting one, two, three or four copies of a fetal chromosome 13, chromosome 18 and chromosome 21 or portions thereof in a sample, comprising: (a) contacting a sample comprising circulating cell-free nucleic acid from a human pregnant female bearing a fetus with a methylation sensitive restriction enzyme, thereby generating cleaved nucleic acid and non-cleaved nucleic acid; (b) determining in a single multiplex reaction amounts of each target polynucleotide in chromosome 13, chromosome 18 and chromosome 21 in the non-cleaved nucleic acid of (a), wherein the target polynucleotides are: chromosome 13 polynucleotides of SEQ ID NOs: 193-198, 200-204, 206, 208-210, and 212-215; chromosome 18 polynucleotides of SEQ ID NOs: 216-218, 220-230, and 232; and chromosome 21 polynucleotides of SEQ ID NOs: 234, 236, 238-240, 242-246, 248-253, 255, and 256, which single multiplex reaction comprises contacting the nucleic acid of (a) with (i) a collection of primer pairs specifically hybridizing to the target polynucleotides; and (ii) known amounts of competitor polynucleotides, wherein the competitor oligonucleotides are polynucleotides that comprise a nucleic acid sequence that is identical to its corresponding target polynucleotide apart from a single nucleotide base substitution within the competitor oligonucleotides that differentiates the competitor oligonucleotides from its corresponding target polynucleotide, under amplification conditions, thereby generating amplicons, which amplicons are further processed by contacting them with extension oligonucleotides under conditions in which the extension oligonucleotides anneal to the amplicons and are extended by one or more nucleotides, thereby generating extension products; and (c) quantifying, from the amounts of extension products the amounts of target polynucleotides, one, two, three or four copies of chromosome 13, chromosome 18, chromosome 21, or portions thereof, in the fetus. (a) contacting a sample comprising circulating cell-free nucleic acid from a human pregnant female bearing a fetus with a methylation sensitive restriction enzyme, thereby generating cleaved nucleic acid and non-cleaved nucleic acid; (b) determining in a single multiplex reaction amounts of each target polynucleotide in chromosome 13, chromosome 18 and chromosome 21 in the non-cleaved nucleic acid of (a), wherein the target polynucleotides are: chromosome 13 polynucleotides of SEQ ID NOs: 193-198, 200-204, 206, 208-210, and 212-215; chromosome 18 polynucleotides of SEQ ID NOs: 216-218, 220-230, and 232; and chromosome 21 polynucleotides of SEQ ID NOs: 234, 236, 238-240, 242-246, 248-253, 255, and 256, which single multiplex reaction comprises contacting the nucleic acid of (a) with (i) a collection of primer pairs specifically hybridizing to the target polynucleotides; and (ii) known amounts of competitor polynucleotides, wherein the competitor oligonucleotides are polynucleotides that comprise a nucleic acid sequence that is identical to its corresponding target polynucleotide apart from a single nucleotide base substitution within the competitor oligonucleotides that differentiates the competitor oligonucleotides from its corresponding target polynucleotide, under amplification conditions, thereby generating amplicons, which amplicons are further processed by contacting them with extension oligonucleotides under conditions in which the extension oligonucleotides anneal to the amplicons and are extended by one or more nucleotides, thereby generating extension products; and (i) a collection of primer pairs specifically hybridizing to the target polynucleotides; and (ii) known amounts of competitor polynucleotides, wherein the competitor oligonucleotides are polynucleotides that comprise a nucleic acid sequence that is identical to its corresponding target polynucleotide apart from a single nucleotide base substitution within the competitor oligonucleotides that differentiates the competitor oligonucleotides from its corresponding target polynucleotide, (c) quantifying, from the amounts of extension products the amounts of target polynucleotides, one, two, three or four copies of chromosome 13, chromosome 18, chromosome 21, or portions thereof, in the fetus.