REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “2017-11-16 1110-0574PUS1 ST25.txt” created on Nov. 16, 2017 and is 12,908 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for determining a genetic condition of a fetus.

2. Description of the Related Art

In the related art, an amniotic fluid test that investigates chromosomes of fetal cells in the amniotic fluid through amniocentesis has been performed as a prenatal diagnosis. However, for this method, a significant problem of having a possibility of miscarriage has been mentioned.

A prenatal diagnosis has recently been performed as a safe prenatal diagnosis through a method for analyzing fetus-derived cell-free deoxyribonucleic acid (DNA) which exists in maternal blood while being mixed with maternal cell-free DNA. This method has been attracting attention as a method which is noninvasive and can acquire fetal genetic information with high probability. A method for determining a polyploidy status of a chromosome in a fetus during pregnancy using genotype data measured from a mixed sample of mother-derived DNA of a fetus and fetus-derived DNA, and genotype data pieces from a mother or a father as necessary is disclosed in WO2003/074723A.

On the other hand, it has been known that fetal cells migrate into maternal blood and circulate the maternal body with the blood. In a case where it is possible to reliably analyze genomic DNA in such fetal cells with good reproducibility, it is possible to realize a prenatal diagnosis in which there is no possibility of miscarriage and which is safe and directly analyzes fetus-derived DNA.

However, it is understood that there is only about one fetal cell (for example, fetal nucleated red blood cell) existing in maternal blood, per several mL of maternal blood, and therefore, various methods for reliably obtaining fetal cells have been studied. In some cases, the number of fetal cells that can be obtained in a case where rare fetal cells are reliably sorted out using such well-known techniques is large or is limited to one.

Regarding an amplification technique for amplifying DNA in cells, a method for amplifying a plurality of target positions of interest through a multiplex polymerase chain reaction (PCR) using a plurality of primer sets, and detecting genetic diseases such as mutation and chromosomal abnormality is disclosed in WO2003/074723A. In addition, a method for amplifying a specific sequence of fetus-derived genomic DNA through a highly multi-targeted polymerase chain reaction and determining an allele frequency at each target genetic position using a sequencer is disclosed in WO2012/108920A.

The multiplex PCR is known as a reaction for simultaneously amplifying two or more amplification target regions in one reaction system. The multiplex PCR is known as a technique useful for efficiently amplifying amplification target regions from a small amount of DNA extracted from a trace amount of blood and typing a large number of single nucleotide polymorphisms (SNPs). A method for designing a primer for multiplex PCR which can efficiently amplify a plurality of amplification sites (targets) is disclosed in WO2008/004691A.

SUMMARY OF THE INVENTION

In order to determine a genetic condition of a fetus by isolating fetus-derived nucleated red blood cells and analyzing DNA in the obtained cells using a well-known technique, it is necessary to extract DNA from a single cell and amplify DNA to an amount required for analysis. Therefore, in many cases, whole genome amplification is performed.

However, although the whole genome amplification has an advantage of remarkably increasing the trace amount of genomic DNA which has been extracted from one cell, it is difficult to constantly maintain the amplification factor using an amplification region. Therefore, there is a disadvantage that a PCR product becomes biased through amplification.

For this reason, even if the whole genome amplification is not performed, a method for determining a genetic condition of a fetus is required which uniformly performs PCR amplification of an objective region accurately using the genomic DNA extracted from a single cell as a template.

An object of the present invention is to provide a method for determining a genetic condition of a fetus which uniformly performs amplification of an objective region accurately through a polymerase chain reaction (PCR).

The present inventors have conducted extensive studies to solve the above-described problems. As a result, they have found that a primer set can be obtained which is used for a polymerase chain reaction and can selectively amplify an objective gene region efficiently in a case where primers selected in both a first stage and a second stage are employed by performing: first stage selection based on a local alignment score obtained by evaluating formability of a primer dimer and obtaining a local alignment score through performing pairwise local alignment on a base sequence of a primer candidate under the condition that a partial sequence to be subjected to comparison includes the 3′ terminal of a base sequence of a primer; and a second stage selection based on a global alignment score obtained by performing pairwise global alignment on a base sequence which has a predetermined sequence length and includes the 3′ terminal of the base sequence of the primer candidate, and have completed the present invention.

That is, the present invention is the following (1) to (6).

(1) 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, in which 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, 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 has a predetermined sequence 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 in which 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.
(2) The method for determining a genetic condition of a fetus according to (1), in which 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 first target region selection step of selecting a first target region in which the primer set used for the polymerase chain reaction is designed, from the objective region, a first primer candidate base sequence generation step of generating at least one base sequence of a primer candidate for amplifying the first target region based on each base sequence in vicinity regions at both ends of the first target region on the genome, a first 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 step of first stage selection of performing first stage selection of the base sequence of the primer candidate based on the local alignment score obtained in the first local alignment step, a first global alignment step of obtaining a global alignment score by performing pairwise global alignment on a base sequence which has a predetermined sequence length and includes the 3′ terminal of the base sequence of the primer candidate, a first step of second stage selection of performing second stage selection of the base sequence of the primer candidate based on the global alignment score obtained in the first global alignment step, a first primer employment step of employing the base sequence of the primer candidate which has been selected in both of the first step of first stage selection and the first step of second stage selection as a base sequence of a primer for amplifying the first target region, a second target region selection step of selecting a second target region in which the primer set used for the polymerase chain reaction is designed, from the objective region, a second primer candidate base sequence generation step of generating at least one base sequence of each primer candidate for amplifying the second target region based on each base sequence in vicinity regions at both ends of the second target region on the genome, a second local alignment step of obtaining a local alignment score by performing pairwise local alignment on the base sequence of the primer candidate for amplifying the second target region and the base sequence of the primer which has already been employed, under a condition that partial sequences to be subjected to comparison include the 3′ terminal of the base sequence of the primer candidate and the 3′ terminal of the base sequence of the primer which has already been employed, a second step of first stage selection of performing first stage selection of the base sequence of the primer candidate for amplifying the second target region based on the local alignment score obtained in the second local alignment step, a second global alignment step of obtaining a global alignment score by performing pairwise global alignment on base sequences which have a predetermined sequence length and include the 3′ terminal of the base sequence of the primer candidate for amplifying the second target region and the 3′ terminal of the base sequence of the primer which has already been employed, a second step of second stage selection of performing second stage selection of the base sequence of the primer candidate for amplifying the second target region based on the global alignment score obtained in the second global alignment step, and a second primer employment step of employing the base sequence of the primer candidate which has been selected in both of the second step of first stage selection and the second step of second stage selection as a base sequence of a primer for amplifying the second target region, in which both steps of the first local alignment step and the first step of first stage selection are performed before or after both steps of the first global alignment step and the first step of second stage selection, or performed in parallel with both steps of the first global alignment step and the first step of second stage selection, both steps of the second local alignment step and the second step of first stage selection are performed before or after both steps of the second global alignment step and the second step of second stage selection, or performed in parallel with both steps of the second global alignment step and the second step of second stage selection, and in a case where there are three or more of the target regions, the steps from the second target region selection step to the second primer employment step are repeated with respect to all of the target regions until a base sequence of a primer for amplifying each target region is employed.
(3) The method for determining a genetic condition of a fetus according to (1) or (2), in which the steps at least from the genomic DNA extraction step to the DNA sequencing step are repeated until a fetus-derived single cell can be isolated in the single cell isolation step.
(4) The method for determining a genetic condition of a fetus according to any one of (1) to (3), in which the genetic condition is a numerical abnormality of a chromosome.
(5) The method for determining a genetic condition of a fetus according to any one of (1) to (4), the method further comprising: a magnetic bead purification step of purifying the amplification product of the objective region obtained through the polymerase chain reaction, using magnetic beads, between the amplification step performed through the polymerase chain reaction and the DNA sequencing step.
(6) The method for determining a genetic condition of a fetus according to any one of (1) to (5), in which the number of times of sequence reading is further measured in the DNA sequencing step.

According to the present invention, it is possible to provide a method for determining a genetic condition of a fetus which uniformly performs amplification of an objective region accurately through a polymerase chain reaction.

In addition, according to the present invention, the determination of a gene sequence in the objective region is reliably performed efficiently in a short process.

