Methods and processes for non-invasive assessment of genetic variations

A method of for amplifying target polynucleotides in a sample, wherein the target polynucleotides are located in one or more genomic loci, wherein the method comprises:

• a) selecting one or more genomic loci wherein each locus comprises three or more features selected from:
• (i) a locus length of about 5000 contiguous base pairs, or less,
• (ii) at least 5 CpG methylation sites,
• (iii) a plurality of restriction endonuclease recognition sites wherein the mean, median or absolute distance between each restriction endonuclease recognition site on the locus is about 20 to about 125 base pairs, and each of the restriction endonuclease recognition sites is recognized by one or more methylation-sensitive restriction endonucleases,
• (iv) at least 1 restriction endonuclease recognition site per 1000 base pairs, wherein the at least one restriction endonuclease recognition site can be specifically digested by a methylation-sensitive restriction endonuclease,
• (v) a locus comprising a methylation status of 60% or more in a minority nucleic acid species,
• (vi) a locus comprising a methylation status of 40% or less in a majority nucleic acid species, and
• (vii) a locus comprising a difference in methylation status of 5% or more between a minority nucleic acid species and a majority nucleic acid species,
• wherein the one or more genomic loci are on chromosome 13, 18, or 21;
• and
• (b) contacting the sample with a plurality of oligonucleotide primer pairs, wherein each primer of each primer pair hybridizes to a portion of a strand of the locus selected in (a) for which the primer pair is specific, thereby amplifying the target polynucleotides.

A method for analyzing a minority nucleic acid in a sample, wherein the sample comprises the minority nucleic acid and the majority nucleic acid, wherein the minority nucleic acid is fetal DNA or cancer DNA,

• the method comprising:
• (a) contacting the sample with one or more methylation sensitive cleavage agents that specifically digests the nucleic acid at non-methylated recognition sites, thereby generating digested nucleic acid fragments;
• and
• (b) analyzing the digested nucleic acid fragments.