Method for determining genetic condition of fetus

A method for determining a genetic condition of a fetus, the method comprising:

• an objective region selection step of selecting an objective region for determining the genetic condition, from regions on a human genome;
• a single cell isolation step of isolating a single cell from a maternal blood sample;
• a genomic DNA extraction step of extracting genomic DNA from the single cell;
• an amplification step performed through a polymerase chain reaction in which the objective region is amplified through the polymerase chain reaction using a primer set designed so as to amplify the objective region through the polymerase chain reaction using the genomic DNA extracted from the single cell as a template;
• and
• a DNA sequencing step of decoding a DNA base sequence of an amplification product of the objective region obtained through the polymerase chain reaction,
• wherein the objective region selection step is performed before or after both steps of the single cell isolation step and the genomic DNA extraction step, or performed in parallel with both steps of the single cell isolation step and the genomic DNA extraction step,
• wherein the primer set designed so as to amplify the objective region through the polymerase chain reaction is designed through a method for designing a primer set used for the polymerase chain reaction, the method for designing a primer set including
• a target region selection step of selecting a target region in which the primer set used for the polymerase chain reaction is designed, from the objective region,
• a primer candidate base sequence generation step of generating at least one base sequence of a primer candidate for amplifying the target region based on each base sequence in vicinity regions at both ends of the target region on the genome,
• a local alignment step of obtaining a local alignment score by performing pairwise local alignment on the base sequence of the primer candidate under a condition that a partial sequence to be subjected to comparison includes the 3′ terminal of the base sequence of the primer candidate,
• a first stage selection step of performing first stage selection of the base sequence of the primer candidate based on the local alignment score obtained in the local alignment step,
• a global alignment step of obtaining a global alignment score by performing pairwise global alignment on a base sequence which is of three base length and includes the 3′ terminal of the base sequence of the primer candidate,
• a second stage selection step of performing second stage selection of a base sequence of the primer candidate based on the global alignment score obtained in the global alignment step, and
• a primer employment step of employing the base sequence of the primer candidate which has been selected in both of the first stage selection step and the second stage selection step as a base sequence of a primer for amplifying the target region, and
• wherein both steps of the local alignment step and the first stage selection step are performed before or after both steps of the global alignment step and the second stage selection step, or performed in parallel with both steps of the global alignment step and the second stage selection step, and
• wherein the genetic condition is a numerical abnormality of a chromosome of the fetus,
• an objective region for determining the genetic condition on at least one of chromosome 13, chromosome 18, and chromosome 21 is selected from regions on a human genome in the objective region selection step, and
• the DNA base sequence of the amplification product of the objective region obtained through the polymerase chain reaction is decoded, a ratio of number of times of sequence reading of mother-derived amplification product to number of times of sequence reading of fetus-derived amplification product is obtained, and it is determined that the fetus has the numerical abnormality of the chromosome in a case where the ratio is greater than or equal to a predetermined cutoff value, in the DNA sequencing step.