JP5177712B2 - Method and apparatus for correcting gene data in competitive hybridization - Google Patents

Description

  The present invention relates to a method and apparatus for correcting gene data in competitive hybridization.

  Nucleic acids are substances that are used to store and transmit genetic information of living organisms, but artificial nucleic acids with improved stability and functionality, such as peptide nucleic acids, have been created, and their application fields are expanding. is there. In particular, the utilization of the basic physical properties of nucleic acids (hereinafter referred to as hybridization phenomena) in which a nucleic acid fragment pair whose base sequence is completely specific (complementary) or partially specific to form a duplex is used in medicine / Not only biotechnology such as biology, but also information technology has begun to be applied.

Currently, the field of biotechnology uses the hybridization phenomenon most actively. In this field, techniques such as microarray (see Patent Document 1) and quantitative in situ hybridization are used as powerful measuring means for comprehensively analyzing genes or their activity states in organisms and cells. These technologies measure the amount of specific binding of nucleotide sequences using several to several million probes for nucleic acid detection, thereby simultaneously quantifying all genes or gene activities on a genome scale, It is possible to measure the localization and quantitative distribution in the tissue.

Such a high-precision measurement technique for the amount of nucleic acid is indispensable for the development and clinical application of highly effective and safe nucleic acid medicine and gene therapy, and technological development aimed at improving quantitativeness is underway.
Also, in the field of information engineering, by replacing the problem to be calculated with a base sequence and performing an in vitro reaction, an attempt to solve a large number of problems simultaneously and at high speed (hybrid parallel computer or DNA computer) Application is seen. When such an apparatus is put to practical use, it is necessary to further improve the accuracy of nucleic acid amount measurement.

JP-A-11-512293

  In general, in a measurement apparatus using a hybridization phenomenon, it is assumed that only a target nucleic acid fragment (target) binds to a specific probe, and the target concentration is estimated from the label intensity of each probe. . However, a phenomenon (cross-hybridization) in which an unintended nucleic acid fragment binds to each probe is known, and there is a problem that it is difficult to accurately measure the target concentration from the label intensity.

  Accordingly, the present invention has been made in view of the above problems, and provides a method and apparatus for correcting genetic data in competitive hybridization that can improve the quantitativeness in measuring the abundance of nucleic acid fragments. This is the issue.

<Single-stranded nucleic acid molecule number estimation apparatus 20 in FIG. 3>
In order to solve the above-described problem, the single-stranded nucleic acid molecule number estimation apparatus of the present invention (single-stranded nucleic acid molecule number apparatus 20 in FIG. 3)
(Component 1) (Refer to Formula (6))
A number of molecules _C ^{i t} of the first single stranded nucleic acid,
The number of molecules C _j ^{t of} the second single-stranded nucleic acid that can bind to the first single-stranded nucleic acid to form a double-stranded nucleic acid;
A _release rate coefficient β _ij , which is a rate at which a double-stranded nucleic acid bound to the first single-stranded nucleic acid and the second single-stranded nucleic acid is released,
Based on the number of molecules M _ij ^t of the double-stranded nucleic acid in which the first single-stranded nucleic acid and the second single-stranded nucleic acid are bound to each other, based on the first single-stranded nucleic acid and the second single-stranded nucleic acid. A double-stranded nucleic acid molecule number updating unit (double-stranded nucleic acid molecule number updating unit 21 in FIG. 3) to be updated for each combination;
(Component 2) (Refer to Formula (7))
The number of molecules M _ij ^{t + 1 of} each double-stranded nucleic acid updated by the double-stranded nucleic acid molecule number updating unit (double-stranded nucleic acid molecule number updating unit 21 in FIG. 3);
The total number of molecules S _{i of} nucleic acid fragments given from the outside of its own device (single-stranded nucleic acid molecule device 20 in FIG. 3),
Based on the single-stranded nucleic acid molecule number updating unit (single-stranded nucleic acid molecule number updating unit 22 in FIG. 3) for updating the single-stranded nucleic acid molecule number C _i ^{t + 1} for each type i of the single-stranded nucleic acid,
(Component 3)
A number of molecules C _i ^t single-stranded nucleic acid before being updated by the single-stranded nucleic acid molecule number updating unit (single-stranded nucleic acid molecule number updating unit 22 of FIG. 3),
The difference | C _i ^{t + 1} −C _i ^t | from the molecular number C _i ^{t + 1} of the single-stranded nucleic acid after being updated by the single-stranded nucleic acid molecule number updating unit (single-stranded nucleic acid molecule number updating unit 22 in FIG. 3) Calculate for each type i of single-stranded nucleic acid,
Each difference ^{_{| C i t + 1 -C i}} t | based on, each single strand single-stranded nucleic convergence determining unit determines whether the estimation result of the number of molecules C _i ^t converges nucleic acid (single-stranded nucleic acid of Figure 3 Convergence determination unit 23);
It is characterized by providing.

<Single-stranded nucleic acid molecule number estimation apparatus 20 in FIG. 3>
In addition, the single-stranded nucleic acid molecule number estimating device of the present invention (single-stranded nucleic acid molecule number estimating device 20 in FIG. 3)
(Component 1) (Refer to Formula (8))
When it is determined that the single-stranded nucleic acid convergence determination unit (single-stranded nucleic acid convergence determination unit 23 in FIG. 3) has not converged,
Based on the number of molecules C _i ^{t + 1} of each single-stranded nucleic acid updated by the single-stranded nucleic acid molecule number updating unit (single-stranded nucleic acid molecule number updating unit 22 in FIG. 3),
An acceleration coefficient calculator (acceleration coefficient calculator 24 in FIG. 3) that calculates an acceleration coefficient s _ij ^{t + 1} for each type of the double-stranded nucleic acid;
(Component 2) (See Equation (6))
The double-stranded nucleic acid molecule number updating unit (double-stranded nucleic acid molecule number updating unit 21 in FIG. 3)
The number of molecules C _i ^{t + 1 of} the first single-stranded nucleic acid updated by the single-stranded nucleic acid molecule number updating unit (single-stranded nucleic acid molecule number updating unit 22 in FIG. 3);
The number of molecules C _j ^{t + 1 of} the second single-stranded nucleic acid that can be combined with the first single-stranded nucleic acid to form a double-stranded nucleic acid;
An acceleration coefficient s _ij ^{t + 1} for each combination of the first single-stranded nucleic acid and the second single-stranded nucleic acid calculated by the acceleration coefficient calculating section (acceleration coefficient calculating section 24 in FIG. 3);
A _release rate coefficient β _ij , which is a rate at which a double-stranded nucleic acid bound to the first single-stranded nucleic acid and the second single-stranded nucleic acid is released,
The molecular number M _ij ^{t + 1} of the double-stranded nucleic acid is updated for each combination of the first single-stranded nucleic acid and the second single-stranded nucleic acid.

<Total Molecular Number Estimator in FIG. 2>
Further, the total molecular number estimation device of the present invention (total molecular number estimation device 10 in FIG. 2)
(Component 1)
A separation rate coefficient storage unit (detachment rate coefficient storage unit 12 in FIG. 2) in which information indicating a separation rate coefficient β _ij from which the single-stranded nucleic acid is detached from the double-stranded nucleic acid is stored for each type of the double-stranded nucleic acid; ,
(Component 2)
The number of molecules C _i of a single-stranded nucleic acid in which the nucleic acid fragment is in a single-stranded state, the number of molecules of a double-stranded nucleic acid containing at least one single-stranded nucleic acid (ΣM _ij (i = 1 to n)) and M _ii A total molecular number storage unit (total molecular number storage unit 11 in FIG. 2) in which information indicating the total molecular number S _i of the nucleic acid fragment that is the sum of the single-stranded nucleic acid is stored,
(Component 3) (See Equation (6) and Equation (7))
Read the information indicating the separation speed coefficient β _ij from the separation speed coefficient storage unit,
Read information indicating the total number of molecules S _i ^k from the total number of molecules storage unit,
The read out on the basis of the read the the dissociation rate coefficient beta _ij on the total molecular number S _i ^k,
A single-stranded nucleic acid molecule number acquisition unit (single-stranded nucleic acid molecule of FIG. 2) that causes the single-stranded nucleic acid molecule number estimation device to estimate the number of molecules C _i ^k of the single-stranded nucleic acid existing in the solution for each type i of the single-stranded nucleic acid Number acquisition unit 13),
(Component 4)
The estimated number of molecules C _i ^k of the single-stranded nucleic acid,
Differences C _i ^k a -Cg _i is calculated for each type i of the single stranded nucleic acid single-stranded nucleic acid difference calculator with a known number of molecules Cg _i of the single-stranded nucleic acid that is input from an external host device (in FIG. 2 A single-stranded nucleic acid difference calculating unit 14),
(Component 5)
Based on the difference C _i ^k -Cg _i for each type i of the single-stranded nucleic acid, the total number of molecules convergence determining unit determines whether the estimated result is converged total molecular number S _i of the nucleic acid fragment (FIG. 2 A total molecular number convergence determination unit 15) of
(Component 6) (Refer to Formula (5))
When it is determined that the total molecular number convergence determination unit has not converged,
Read out the information indicating each separation speed coefficient β _ij from the separation speed coefficient storage unit,
Based on each read out rate coefficient β _ij and the difference C _j ^k -Cg _{j of} the second single-stranded nucleic acid that can bind to the first single-stranded nucleic acid to form a double-stranded nucleic acid, in the total number of molecules S _i ^k of the first single stranded nucleic acid having at least one nucleic acid fragment is corrected for each type i of the single-stranded nucleic acid, the information indicating the total number of molecules S _{i k} ^{+ 1} obtained by correcting the total molecular A total molecular number correction unit (total molecular number correction unit 16 in FIG. 2) that updates information indicating the total molecular number S _i stored in the number storage unit,
The single-stranded nucleic acid molecule number acquisition unit reads information indicating the total molecule number S _i updated by the total molecule number correction unit from the total molecule number storage unit, and based on the read total molecule number S _i ^k , The number C _i ^k of the single-stranded nucleic acid is estimated by the single-stranded nucleic acid molecule number estimation apparatus for each type i of the single-stranded nucleic acid.

In addition, the single-stranded nucleic acid molecule number acquisition unit in the total molecular number estimation apparatus (total molecular number estimation apparatus 10 in FIG. 2) of the present invention provides the total number of molecules of the nucleic acid fragment to be output to the single-stranded nucleic acid molecule number estimation apparatus. The initial value S _i ⁰ is a known molecular number Cg _{i of} a single-stranded nucleic acid inputted from the outside.

<Separation speed coefficient calculation device in FIG. 1>
In addition, the separation speed coefficient calculation device of the present invention (the separation speed coefficient calculation device 1 in FIG. 1)
(Component 1)
Based on the separation rate coefficient β _ij , which is the rate at which the single-stranded nucleic acid is released from the double-stranded nucleic acid, and the known number of molecules Cg _{i of} each single-stranded nucleic acid, the number of molecules S _i of the nucleic acid fragment is calculated from the single-stranded nucleic acid. A total number-of-molecules estimation device (total number-of-molecules estimation device 10 in FIG. 1) for each type,
(Component 2)
Wherein the respective dissociation rate coefficient beta _ij, wherein based on the number of molecules S _i of the estimated each nucleic acid fragments, single chain molecules number C _i number single stranded nucleic acid molecule is estimated for each type of single-chain nucleic estimated nucleic acid An apparatus (single-stranded nucleic acid molecule number estimation apparatus 20 in FIG. 1);
(Component 3)
Based on the estimated number of molecules C _i of each single-stranded nucleic acid, a control unit (control unit 30 in FIG. 1) that calculates the separation rate coefficient β _ij for each type of the double-stranded nucleic acid;
It is characterized by providing.

<Control part of FIG. 4>
Further, the control unit (the control unit 30 in FIG. 4) in the separation speed coefficient calculation device (the separation speed coefficient calculation device in FIG. 1) of the present invention includes:
(Component 1)
In the total molecular number estimation device (total molecular number estimation device 10 in FIG. 4), based on the separation rate coefficient β _ij and the known molecular number Cg _i of the single-stranded nucleic acid, the total molecular number S _i is calculated as the single chain. Obtain single-stranded nucleic acid by estimating for each type of nucleic acid i, and causing the single-stranded nucleic acid estimation apparatus to estimate the number of molecules C _i of single-stranded nucleic acid corresponding to the total number of molecules S _i for each type of single-stranded nucleic acid i Part (single-stranded nucleic acid obtaining unit 31 in FIG. 4);
(Component 2)
For each type i of the single-stranded nucleic acid,
The number of molecules C _i ^l of the single-stranded nucleic acid obtained by the single-stranded nucleic acid obtaining unit (single-stranded nucleic acid obtaining unit 31 in FIG. 4),
A known molecule number Cg _i of the single-stranded nucleic acid input from the outside of the device is compared for each type i of the single-stranded nucleic acid,
If known molecular smaller number Cg _i of the single strand nucleic acid molecule number C _i of the single-stranded nucleic acid has been input the acquired, increasing the dissociation rate coefficient beta _ij ^l of the single-stranded nucleic acid,
When the number of molecules C _{i of the} obtained single-stranded nucleic acid is equal to or larger than the known number of molecules Cg _{i of} the inputted single-stranded nucleic acid, the separation rate coefficient for reducing the separation rate coefficient β _ij ^l of the single-stranded nucleic acid A correction unit (withdrawal speed coefficient correction unit 32 in FIG. 4);
With
The single-stranded nucleic acid acquisition unit (single-stranded nucleic acid acquisition unit 31 in FIG. 4) is based on the separation rate coefficient β ′ _ij ^l corrected by the separation rate coefficient correction unit (see FIG. 4). The total molecular number estimation device 10) estimates the total molecular number S ′ _i for each type i of the single-stranded nucleic acid, and the single-stranded nucleic acid estimation device uses the single-stranded nucleic acid corresponding to the total molecular number S ′ _i . The number of molecules C ′ _i ¹ is estimated for each type i of the single-stranded nucleic acid,
The control unit (control unit 30 in FIG. 4)
(Component 3)
For each kind of single-stranded nucleic acid _i , the difference between the number of molecules C _i ^l of the single-stranded nucleic acid before correction and the known number of molecules Cg _i of the inputted single-stranded nucleic acid is the difference before correction | C _i ^l −Cg _i |
For each type i of single-stranded nucleic acid, the difference after correcting for the difference between the corrected number of molecules C ′ _i ¹ of the single-stranded nucleic acid and the known number of molecules Cg _i of the input single-stranded nucleic acid | C a before-and-after-correction difference calculating unit ('before-and-after-correction calculating unit 34 in FIG. 4) that is calculated as' _i ¹ -Cg _i |;
(Component 4)
The difference | C _i ¹ −Cg _i | before correction and the difference | C ′ _i ¹ −Cg _i | after correction are compared for each type i of the single-stranded nucleic acid, and the difference before correction | C _i ^l -Cg _i | difference of the corrected | C _'i ^l -Cg _i | is greater than, select dissociation rate coefficient .beta.' _ij ^l after being corrected by the dissociation rate coefficient correction unit, the correction When the previous difference | C _i ¹ −Cg _i | is equal to or smaller than the corrected difference | C ′ _i ¹ −Cg _i |, the separation speed coefficient β _ij ^l before being corrected by the separation speed coefficient correction unit is selected. A separation speed coefficient selection unit (a separation speed coefficient selection unit 35 in FIG. 4),
Is further provided.

<Separation Speed Coefficient Convergence Determination Unit 36 in FIG. 4>
Further, the control unit (the control unit 30 in FIG. 4) in the separation speed coefficient calculation device (the separation speed coefficient calculation device in FIG. 1) of the present invention includes:
For each separation speed coefficient β _ij , the separation speed coefficient β _ij ^l before being corrected by the separation speed coefficient correction section and the separation speed coefficient selection section (the separation speed coefficient selection section 35 in FIG. 4) are selected. A separation speed coefficient convergence determination unit (the separation speed in FIG. 4) that determines whether or not the estimation result of the separation speed coefficient β _ij has converged based on the difference from the separation speed coefficient (β _ij ^l or β ′ _ij ^l ). A coefficient convergence determination unit 36),
When the single-stranded nucleic acid obtaining unit determines that the separation rate coefficient convergence determination unit has not converged, the single-stranded nucleic acid acquisition unit determines each separation rate coefficient (β _ij ^l or β ′ _ij ^l ) selected by the separation rate coefficient selection unit. based on, and acquires the number of molecules C _i of each single-stranded nucleic acid from said single-stranded nucleic acid molecule number estimating apparatus.

  The separation speed coefficient correction unit corrects the separation speed coefficient using a random number.

The initial value β _ij ⁰ of the _release rate coefficient is a release rate coefficient that has been obtained so far in its own device when a new known number of molecules of the single-stranded nucleic acid is obtained. .

The initial value β _ij ⁰ of the _release rate coefficient is the same as the sequence of the first single-stranded nucleic acid and the sequence of the second single-stranded nucleic acid when there is no release rate coefficient obtained so far in the device. The value is dependent on the length of the part.

<Method for estimating the number of single-stranded nucleic acid molecules>
The single-stranded nucleic acid molecule number estimation method of the present invention is a single-stranded nucleic acid molecule number estimation method executed by a single-stranded nucleic acid molecule number estimation device (single-stranded nucleic acid molecule number estimation device 20 in FIG. 3),
(Component 1) (Refer to Formula (6))
A number of molecules _C ^{i t} of the first single stranded nucleic acid,
The number of molecules C _j ^{t of} the second single-stranded nucleic acid that can bind to the first single-stranded nucleic acid to form a double-stranded nucleic acid;
A _release rate coefficient β _ij , which is a rate at which a double-stranded nucleic acid bound to the first single-stranded nucleic acid and the second single-stranded nucleic acid is released,
Based on the number of molecules M _ij ^t of the double-stranded nucleic acid in which the first single-stranded nucleic acid and the second single-stranded nucleic acid are bound to each other, based on the first single-stranded nucleic acid and the second single-stranded nucleic acid. A procedure for updating the number of double-stranded nucleic acid molecules to be updated for each combination;
(Component 2) (Refer to Formula (7))
The number of molecules M _ij ^{t + 1 of} each double-stranded nucleic acid updated by the double-stranded nucleic acid molecule number updating procedure;
The total number of molecules S _{i of} nucleic acid fragments given from outside the single-stranded nucleic acid molecule number estimating device (single-stranded nucleic acid molecule device 20 in FIG. 3);
A single-stranded nucleic acid molecule number updating procedure for updating the single-stranded nucleic acid molecule number C _i ^{t + 1} for each type i of the single-stranded nucleic acid,
(Component 3)
A number of molecules C _i ^t single-stranded nucleic acid before being updated by the single-stranded nucleic acid molecule number updating procedure,
The difference | C _i ^{t + 1} −C _i ^t | from the number of molecules C _i ^{t + 1} of the single-stranded nucleic acid after being updated by the single-stranded nucleic acid molecule number updating procedure is calculated for each type i of the single-stranded nucleic acid,
Each difference ^{_{| C i t + 1 -C i}} t | based on a single-stranded nucleic convergence determination procedure for determining whether the converged estimation result of the number of molecules C _i ^t of each single-stranded nucleic acid,
It is characterized by having.

<Total number of molecules estimation method>
In addition, the method for estimating the total number of molecules of the present invention includes a separation rate coefficient storage unit in which information indicating a separation rate coefficient β _{ij at} which a single-stranded nucleic acid is detached from a double-stranded nucleic acid is stored for each type of the double-stranded nucleic acid. and (dissociation rate coefficient storage unit 12 of FIG. 2), nucleic acid fragment is a sum of the number of molecules of double-stranded nucleic acid comprising at least one molecular number C _i and the single stranded nucleic acids single-stranded nucleic acid which is a single-stranded state A total molecular number estimation device comprising: a total molecular number storage unit (total molecular number storage unit 11 in FIG. 2) in which information indicating the total molecular number S _i of a fragment is stored for each type of single-stranded nucleic acid A method for estimating the total number of molecules,
(Component 1) (See Equation (6) and Equation (7))
Read the information indicating the separation speed coefficient β _ij from the separation speed coefficient storage unit,
Read information indicating the total number of molecules S _i ^k from the total number of molecules storage unit,
The read out on the basis of the read the the dissociation rate coefficient beta _ij on the total molecular number S _i ^k,
A single chain number nucleic acid molecule acquisition procedure for estimating the number of molecules C _i ^k single-stranded nucleic acid present in the solution for each type i of the single stranded nucleic acid to said single-stranded nucleic acid molecule number estimating apparatus,
(Component 2)
The estimated number of molecules C _i ^k of the single-stranded nucleic acid,
A single-stranded nucleic acid difference computation steps a difference C _i ^k -Cg _i with known molecular number Cg _i is calculated for each type i of the single-stranded nucleic acid of the single-stranded nucleic acid that is input from an external of the apparatus,
(Component 3)
Based on the difference C _i ^k -Cg _i for each type i of the single-stranded nucleic acid, the total number of molecules convergence determination procedure for determining whether estimation result or not convergence of the total number of molecules S _i of the nucleic acid fragments,
(Component 4) (Refer to Formula (5))
When it is determined that the total molecular number convergence determination unit procedure has not converged,
Read out the information indicating each separation speed coefficient β _ij from the separation speed coefficient storage unit,
Based on each read out rate coefficient β _ij and the difference C _j ^k -Cg _{j of} the second single-stranded nucleic acid that can bind to the first single-stranded nucleic acid to form a double-stranded nucleic acid, in the total number of molecules S _i ^k of the first single stranded nucleic acid having at least one nucleic acid fragment is corrected for each type i of the single-stranded nucleic acid, the information indicating the total number of molecules S _{i k} ^{+ 1} obtained by correcting the total molecular A total molecular number correction procedure for updating the information indicating the total molecular number S _i stored in the number storage unit;
Have
The single-stranded nucleic acid molecule number acquisition procedure reads information indicating the total molecular number S _i updated by the total molecular number correction procedure from the total molecular number storage unit, and based on the read total molecular number S _i ^k , The number C _i ^k of the single-stranded nucleic acid is estimated by the single-stranded nucleic acid molecule number estimation apparatus for each type i of the single-stranded nucleic acid.

<How to calculate the separation speed coefficient>
The separation speed coefficient calculation method executed by the separation speed coefficient calculation apparatus of the present invention (the separation speed coefficient calculation apparatus 1 in FIG. 1)
(Component 1)
Based on the separation rate coefficient β _ij , which is the rate at which the single-stranded nucleic acid is released from the double-stranded nucleic acid, and the known number of molecules Cg _{i of} each single-stranded nucleic acid, the number of molecules S _i of the nucleic acid fragment is calculated from the single-stranded nucleic acid. The total number-of-molecules estimation method for estimating each type;
(Component 2)
The number of single-stranded nucleic acid molecules for estimating the number of molecules C _i of single-stranded nucleic acids for each type of single-stranded nucleic acid based on each of the separation rate coefficients β _ij and the estimated number of molecules S _i of each nucleic acid fragment An estimation method;
(Component 3)
A control procedure for calculating the separation rate coefficient β _ij for each type of the double-stranded nucleic acid based on the estimated number C _i of each single-stranded nucleic acid;
It is characterized by having.

<Single-stranded nucleic acid molecule number estimation program>
Moreover, the single-stranded nucleic acid molecule number estimation program of the present invention is stored in a computer which is a single-stranded nucleic acid molecule number estimation device (detachment rate coefficient calculation device 13 in FIG.
(Component 1) (Refer to Formula (6))
A number of molecules _C ^{i t} of the first single stranded nucleic acid,
The number of molecules C _j ^{t of} the second single-stranded nucleic acid that can bind to the first single-stranded nucleic acid to form a double-stranded nucleic acid;
A _release rate coefficient β _ij , which is a rate at which a double-stranded nucleic acid bound to the first single-stranded nucleic acid and the second single-stranded nucleic acid is released,
Based on the number of molecules M _ij ^t of the double-stranded nucleic acid in which the first single-stranded nucleic acid and the second single-stranded nucleic acid are bound to each other, based on the first single-stranded nucleic acid and the second single-stranded nucleic acid. A step of updating the number of double-stranded nucleic acid molecules to be updated for each combination;
(Component 2) (Refer to Formula (7))
The number of molecules M _ij ^{t + 1 of} each double-stranded nucleic acid updated by the double-stranded nucleic acid molecule number updating step;
The total number of molecules S _{i of} nucleic acid fragments given from outside the single-stranded nucleic acid molecule number estimating device (single-stranded nucleic acid molecule device 20 in FIG. 3);
A single-stranded nucleic acid molecule number updating step for updating the single-stranded nucleic acid molecule number C _i ^{t + 1} for each type i of the single-stranded nucleic acid,
(Component 3)
A number of molecules C _i ^t single-stranded nucleic acid before being updated by the single-stranded nucleic acid molecule number updating step,
The difference | C _i ^{t + 1} −C _i ^t | from the number of molecules C _i ^{t + 1} of the single-stranded nucleic acid after updated by the step of updating the number of single-stranded nucleic acid molecules is calculated for each type i of the single-stranded nucleic acid,
Each difference ^{_{| C i t + 1 -C i}} t | based on a single-stranded nucleic convergence determination step of determining whether the estimated result is converged molecular number C _i ^t of each single-stranded nucleic acid,
Is a program for estimating the number of single-stranded nucleic acid molecules.

<Total number of molecules estimation program>
Further, the total molecular number estimation program of the present invention includes a separation rate coefficient storage unit in which information indicating a separation rate coefficient β _{ij at} which a single-stranded nucleic acid is detached from a double-stranded nucleic acid is stored for each type of the double-stranded nucleic acid. and (dissociation rate coefficient storage unit 12 of FIG. 2), nucleic acid fragment is a sum of the number of molecules of double-stranded nucleic acid comprising at least one molecular number C _i and the single stranded nucleic acids single-stranded nucleic acid which is a single-stranded state A computer as a total number-of-molecules estimation device comprising: a total number-of-molecules storage unit (total number-of-molecules storage unit 11 in FIG. 2) in which information indicating the total number of molecules S _i of fragments is stored for each type of single-stranded nucleic acid In addition,
(Component 1) (See Equation (6) and Equation (7))
Read the information indicating the separation speed coefficient β _ij from the separation speed coefficient storage unit,
Read information indicating the total number of molecules S _i ^k from the total number of molecules storage unit,
The read out on the basis of the read the the dissociation rate coefficient beta _ij on the total molecular number S _i ^k,
A single chain number nucleic acid molecule acquisition procedure for estimating the number of molecules C _i ^k single-stranded nucleic acid present in the solution for each type i of the single stranded nucleic acid to said single-stranded nucleic acid molecule number estimating apparatus,
(Component 2)
The estimated number of molecules C _i ^k of the single-stranded nucleic acid,
A single-stranded nucleic acid difference computation steps a difference C _i ^k -Cg _i with known molecular number Cg _i is calculated for each type i of the single-stranded nucleic acid of the single-stranded nucleic acid that is input from an external of the apparatus,
(Component 3)
Based on the difference C _i ^k -Cg _i for each type i of the single-stranded nucleic acid, the total number of molecules convergence determination procedure for determining whether estimation result or not convergence of the total number of molecules S _i of the nucleic acid fragments,
(Component 4) (Refer to Formula (5))
When it is determined that the total molecular number convergence determination unit procedure has not converged,
Read out the information indicating each separation speed coefficient β _ij from the separation speed coefficient storage unit,
Based on each read out rate coefficient β _ij and the difference C _j ^k -Cg _{j of} the second single-stranded nucleic acid that can bind to the first single-stranded nucleic acid to form a double-stranded nucleic acid, in the total number of molecules S _i ^k of the first single stranded nucleic acid having at least one nucleic acid fragment is corrected for each type i of the single-stranded nucleic acid, the information indicating the total number of molecules S _{i k} ^{+ 1} obtained by correcting the total molecular A total molecular number correction procedure for updating the information indicating the total molecular number S _i stored in the number storage unit;
A total molecular number estimation program for executing
The single-stranded nucleic acid molecule number acquisition procedure reads information indicating the total molecular number S _i updated by the total molecular number correction procedure from the total molecular number storage unit, and based on the read total molecular number S _i ^k , A total molecular number estimation program for causing the single-stranded nucleic acid molecule number estimation apparatus to estimate the number of molecules C _i ^k of the single-stranded nucleic acid for each type i of the single-stranded nucleic acid.

<Separation speed coefficient calculation program>
Further, the separation speed coefficient calculation program of the present invention is applied to a computer which is a separation speed coefficient calculation apparatus (the separation speed coefficient calculation apparatus 1 in FIG. 1).
(Component 1)
Based on the separation rate coefficient β _ij , which is the rate at which the single-stranded nucleic acid is released from the double-stranded nucleic acid, and the known number of molecules Cg _{i of} each single-stranded nucleic acid, the number of molecules S _i of the nucleic acid fragment is determined as The total number-of-molecules estimation program for estimating each type;
(Component 2)
The number of single-stranded nucleic acid molecules for estimating the number of molecules C _i of single-stranded nucleic acids for each type of single-stranded nucleic acid based on each of the separation rate coefficients β _ij and the estimated number of molecules S _i of each nucleic acid fragment An estimation program;
(Component 3)
A control step of calculating the separation rate coefficient β _ij for each type of the double-stranded nucleic acid based on the estimated number C _i of each single-stranded nucleic acid;
Is a separation speed coefficient calculation program.

  According to the present invention, the number of molecules of each single-stranded nucleic acid can be estimated with high accuracy. Further, since the total number of molecules of each estimated nucleic acid fragment can be estimated, the number of molecules of single-stranded nucleic acid and the total number of molecules of nucleic acid fragments can be clearly distinguished and estimated.

It is a block block diagram of the separation speed coefficient calculation apparatus in embodiment of this invention. It is a block block diagram of a total molecule number estimation apparatus. It is a block block diagram of a single strand nucleic acid molecule number estimation apparatus. It is a block block diagram of a control part. It is the flowchart which showed the flow of the process of estimation of the separation speed coefficient by the separation speed coefficient calculation apparatus. 6 is a flowchart showing a flow of processing for obtaining the number of molecules of each single-stranded nucleic acid in step S102 or step S105 of FIG. It is the flowchart which showed the flow of the process of estimation of the total molecule number of each nucleic acid fragment by the total molecule number estimation apparatus in FIG. FIG. 8 is a flowchart showing the flow of processing for estimating the number of molecules of each single-stranded nucleic acid by the single-stranded nucleic acid molecule number estimating apparatus in step S202 of FIG. 6 or step S303 of FIG.

  One embodiment of the present invention (method and apparatus for correcting gene data in competitive hybridization), single-stranded nucleic acid molecule number estimating apparatus, total molecular number estimating apparatus, withdrawal rate coefficient calculating apparatus, single-stranded nucleic acid molecule number estimating A method, a total molecule number estimation method, a withdrawal rate coefficient calculation method, a single-stranded nucleic acid molecule number estimation program, a total molecule number estimation program, and a withdrawal rate coefficient calculation program will be described.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. First, an outline of the present invention will be described. The nucleic acid fragment exists mainly in a single-stranded state and a double-stranded state in which a nucleic acid strand having a complementary base sequence is bound. The single chain and the double chain are in physicochemical equilibrium, and their ratios are determined depending on the temperature and salt concentration of the solution and the binding constant (detachment rate constant) based on the base sequence.

  Single-stranded bonds can form a binding state not only when the nucleotide sequences of the paired nucleic acid fragments are completely complementary, but also with partial complementarity. Since there are only four types of bases, and the sequence pattern becomes shorter as the length of the nucleic acid fragment is shorter, partial complementary strands are present by chance, and partial nucleic acid fragment binding is very likely to occur. Therefore, in a system in which a large number of nucleic acid fragments having various base sequences are present simultaneously, the number of completely specific nucleic acid fragment bonds is rather small, and a large amount of partially specific nucleic acid fragment bonds are generated.

  When measuring the total number of molecules of a specific nucleic acid fragment (target), the amount of single-stranded nucleic acid by a method such as a pair of nucleic acid fragments that have specific (complementary) base sequences bind to form a duplex Using a device that measures the total number of molecules in an existing target, a double-stranded nucleic acid is formed in solution due to the formation of double-stranded nucleic acids due to complete and partial specific binding. In the measurement method that equates with the amount of strand nucleic acid, the quantitativeness of the measurement result is lowered.

  In the present invention, the abundance of the nucleic acid fragment to be measured is measured with high accuracy by correcting the influence of double strand formation by completely specific and partial specific binding of the nucleic acid fragment in solution. Single-stranded nucleic acid molecule number estimation device, total molecular number estimation device, separation rate coefficient calculation device, single-stranded nucleic acid molecule number estimation method, total molecule number estimation method, separation rate coefficient calculation method, single-stranded nucleic acid molecule number An estimation program, a total molecular number estimation program, and a withdrawal rate coefficient calculation program are provided.

<Theory>
Next, the theory that forms the basis of the present invention will be described. In a solution containing a plurality of types of single-stranded nucleic acids, it is considered that single-stranded nucleic acids are bound to each other. The amount of double-stranded nucleic acid that increases due to binding depends on the probability that two single-stranded nucleic acids are encountered and is thought to be proportional to the concentration of each single-stranded nucleic acid.

Here, M _ij is the number of molecules of the double-stranded nucleic acid in which the i (i is an integer from 1 to n) -th single-stranded nucleic acid and the j (j is an integer from 1 to n) -th single-stranded nucleic acid are combined. C _i and C _j represent the number of molecules of the i-th single-stranded nucleic acid and the number of molecules of the j-th single-stranded nucleic acid, respectively. Here, K is a constant representing the rate of binding, and V is the volume of the solution. The amount of double-stranded nucleic acid that decreases by dissociating into single-stranded nucleic acid is considered to depend on the binding force between the nucleic acid fragments, and the amount of double-stranded nucleic acid that decreases is expressed by the following equation.

Here, β _ij is a detachment rate coefficient indicating the rate at which the double-stranded nucleic acid in which the i-th type single-stranded nucleic acid and the j-th type single-stranded nucleic acid are combined is dissociated as described above. When the number of molecules of the i-th single-stranded nucleic acid before mixing is S _i , the number of molecules C _i of the i-th type single-stranded nucleic acid is expressed by the following equation.

Here, the third term on the right side is a term that is required when the double-stranded nucleic acid is a bond of the same single-stranded nucleic acid. That is, when the same single-stranded nucleic acid is combined to produce a double-stranded nucleic acid, at the same time, the number of the single-stranded nucleic acid is reduced by two, so the third term needs to be subtracted on the right side.
After mixing, after a sufficient time, the state is considered to be in an equilibrium state, and the right side of equation (1) is equal to the right side of equation (2), so the following equation is established.

<Separation speed coefficient calculation device>
Next, the separation speed coefficient calculation device 1 according to the embodiment of the present invention will be described with reference to FIG. FIG. 1 is a block diagram of a separation speed coefficient calculation device 1 according to an embodiment of the present invention. The withdrawal rate coefficient calculation device 1 includes a total molecular number estimation device 10, a single-stranded nucleic acid molecule number estimation device 20, and a control unit 30.

The control unit 30 indicates the known number of molecules Cg _{i of} each single-stranded nucleic acid (i is an index indicating the type of single-stranded nucleic acid) and is an integer from 1 to n input from the outside of the separation rate coefficient calculating device 1 Based on the information and the information indicating the initial value β _ij ⁰ of each separation rate coefficient, the total molecular number estimation device 10 indicates information indicating the known number of molecules Cg _i of each single-stranded nucleic acid and each separation rate coefficient β _ij. The information indicating is output. The control unit 30 causes the total number-of-molecules estimation apparatus 10 to calculate the number of molecules C _i of the single-stranded nucleic acid for each type of single-stranded nucleic acid based on the output information.

Control unit 30, based on the information acquired information indicating the number of molecules C _i of each single-stranded nucleic acid from single-stranded nucleic acid molecule number estimating device 20 indicates the number of molecules C _i of each single-stranded nucleic acid obtained, each withdrawal The speed coefficient β _ij is calculated, and information indicating each calculated separation speed coefficient β _ij is output to the outside of the separation speed coefficient calculation apparatus 1.

Total molecular number estimation device 10 receives the information indicating the known number of molecules Cg _i single-stranded nucleic acid that is input from the control unit 30, based on the information indicating the dissociation rate coefficient beta _ij, the total number of molecules of the nucleic acid fragment S _i is calculated for each type of single-stranded nucleic acid.

The total number-of-molecules estimation apparatus 10 outputs information indicating the separation rate coefficient β _ij and information indicating the calculated total number of molecules S _i of nucleic acid fragments to the single-stranded nucleic acid molecule number estimation apparatus 20, and the number of single-stranded nucleic acid molecules based on the output information to the estimation device 20, the number of molecules C _i of the single-stranded nucleic acid is calculated for each type of single-chain nucleic acids.
Single-stranded nucleic acid molecule number estimating device 20 outputs information indicating the number of molecules C _i of each single-stranded nucleic acid that is calculated in the control unit 30.

The single-stranded nucleic acid molecule number estimation device 20 is based on the information indicating the withdrawal rate coefficient β _ij and the information indicating the total number of molecules S _i of the nucleic acid fragments input from the total molecule number estimation device 10. the number C _i is calculated for each type of single-chain nucleic acids. Single-stranded nucleic acid molecule number estimating device 20 outputs to the calculated respective single chain molecular number C _i total molecular number estimating apparatus information indicating a 10 nucleic acid control unit 30.

Summarizing the above processing, the total number-of-molecules estimation device 10 of the separation rate coefficient calculation device 1 has a separation rate coefficient β _ij , which is a rate at which a single-stranded nucleic acid is detached from a double-stranded nucleic acid, and each single-stranded nucleic acid. Based on the known number of molecules Cg _i , the number of molecules S _i of each nucleic acid fragment is estimated for each type of single-stranded nucleic acid. The single-stranded nucleic acid molecule number estimating device 20 calculates the molecular number C _i of the single-stranded nucleic acid based on each of the above-mentioned withdrawal rate coefficient β _ij and the estimated molecular number S _i of each nucleic acid fragment, and the kind of single-stranded nucleic acid. Estimate every time. Based on the estimated number C _i of each single-stranded nucleic acid, the control unit 30 calculates the separation rate coefficient β _ij for each type of the double-stranded nucleic acid.

<Total number of molecules estimation apparatus 10>
Next, the total molecular number estimation apparatus 10 will be described with reference to FIG. FIG. 2 is a block configuration diagram of the total number-of-molecules estimation apparatus 10. The total molecular number estimation device 10 includes a total molecular number storage unit 11, a withdrawal rate coefficient storage unit 12, a single-stranded nucleic acid molecule number acquisition unit 13, a single-stranded nucleic acid difference calculation unit 14, and a total molecular number convergence determination unit 15. And a total molecular number correction unit 16.

The total molecular number storage unit 11, the second and subsequent step k (k ≧ 1), by being allowed to store the total molecular number correction section 16, the information S _i indicating the total number of molecules of each nucleic acid fragment of the corrected ^k is stored.

In other words, the total number-of-molecules storage unit 11 includes the number of molecules C _i of a single-stranded nucleic acid whose nucleic acid fragment is in a single-stranded state and the number of molecules of a double-stranded nucleic acid containing at least one single-stranded nucleic acid (M _ij and M _ii information indicating the total number of molecules S _i of the nucleic acid fragments are stored for each type of single-stranded nucleic acid which is the sum of the sum) between.
The separation rate coefficient storage unit 12 stores information for each type of double-stranded nucleic acid that is stored by the control unit 30 and indicates the separation rate coefficient β _ij from which the single-stranded nucleic acid is detached from the double-stranded nucleic acid. ing.

The single-stranded nucleic acid molecule number acquisition unit 13 reads information indicating each separation rate coefficient β _ij from the separation rate coefficient storage unit 12.
Single-stranded nucleic acid molecule number obtaining unit 13, the first step (k = 0, k is the number of steps), the obtaining information indicating a known number of molecules Cg _i of each single-stranded nucleic acid that is input from the control unit 30. Single-stranded nucleic acid molecule number obtaining unit 13, the known number of molecules Cg _i of each single-stranded nucleic acid, the information (i indicating the total number of molecules of each nucleic acid fragments from 1 an index indicating the type of single-stranded nucleic acids to n Integer).

Single-stranded nucleic acid molecule number obtaining unit 13, in step the second and subsequent (k ≧ 1), the total number of molecules of the total molecular number of S _i each nucleic acid fragment information indicating for each nucleic acid fragment from the total molecular number storage unit 11 S ^{Read out} as information indicating _i ^k .

The single-stranded nucleic acid molecule number acquisition unit 13 outputs information indicating each withdrawal rate coefficient β _ij and information indicating information S _i indicating the total number of molecules of each nucleic acid fragment to the single-stranded nucleic acid molecule number estimation device 20. it allows to calculate the number of molecules C _i of each single-stranded nucleic acid to single stranded nucleic acid molecule number estimation device 20.
Further, the single-stranded nucleic acid molecule number acquisition unit 13 in the second and subsequent steps k (k ≧ 1), each total molecule number S _i updated from the total molecule number storage unit 11 by the total molecule number correction unit 16 described later. And the single-stranded nucleic acid molecule number estimation device 20 is made to estimate the number of single-stranded nucleic acid molecules C _i ^k for each type of single-stranded nucleic acid _i based on the read total number of molecules S _i ^k .

Single-stranded nucleic acid molecule number obtaining unit 13 obtains information indicating the number of molecules C _i of each single-stranded nucleic acid that is calculated by the single-stranded nucleic acid molecule number estimating device 20, the number of molecules C _i ^k of each single-stranded nucleic acid obtained Is output to the single-stranded nucleic acid difference calculation unit 14.

Single-stranded nucleic acid difference calculation unit 14, the control unit 30 receives information indicating a known number of molecules Cg _i of each single-stranded nucleic acid that is input from an external of the apparatus. The single-stranded nucleic acid difference calculating unit 14 calculates the number of molecules C _i ^k of the single-stranded nucleic acid estimated by the single-stranded nucleic acid molecule number estimation device 20 and the known number of molecules Cg of the single-stranded nucleic acid input from the outside of the device. the difference _C ⁱ k -Cg _i and _i is calculated for each type of single-chain nucleic acid i. Single-stranded nucleic acid difference calculating unit 14 outputs information indicating the calculated difference C _i ^k -Cg _i the total number of molecules convergence determination unit 15.

Total molecular number of the convergence determination unit 15, based on the difference C _i ^k -Cg _i for each type of single-stranded nucleic acid, S _i determines whether the estimation result of the total number of molecules S _i of the nucleic acid fragment has converged It is determined whether or not the estimation result has converged.

Specifically, for example, the total number of molecules convergence determination unit 15, when the absolute value of the difference C _i ^k -Cg _i for each type of single-stranded nucleic acid are all less than a predetermined threshold value, the total number of molecules of the nucleic acid fragments It is determined that the estimation result has converged. On the other hand, the total number of molecules convergence determination unit 15, if it exceeds a predetermined threshold value, even one of the absolute value of the difference C _i ^k -Cg _i for each type of single-stranded nucleic acid, the estimation result of the total number of molecules of the nucleic acid fragment Judge that it has not converged.

Total molecular number of the convergence determination unit 15, when the estimated result the total number of molecules of the nucleic acid fragment is determined to have converged, reads information indicating the total number of molecules S _i of each nucleic acid fragments from the total molecular number storage unit 11, the read and outputs as the information indicating the total number of molecules S _i of each nucleic acid fragment.
On the other hand, when the total molecular number convergence determination unit 15 determines that the estimation result of the total number of molecules of the nucleic acid fragment has not converged, information indicating that the estimation result of the total number of molecules has not converged is corrected for the total number of molecules. To the unit 16.

The total number of molecules convergence determination unit 15, when it reaches a predetermined number the number predetermined step may determine that the estimation result of the total number of molecules S _i ^k of each nucleic acid fragment has converged. Thus, the total number of molecules estimating apparatus 10, since it is determined that all differences C _i ^k -Cg _i is the number of steps without fall below a predetermined threshold value has converged if reaches a predetermined number, the computation time It can be within a predetermined range.

The total molecular number correction unit 16 receives information indicating that the estimation result of the total molecular number has not converged from the total molecular number convergence determination unit 15, that is, the total molecular number convergence determination unit 15 has not converged. If it is determined, information indicating each separation speed coefficient β _ij is read from the separation speed coefficient storage unit.
The total number-of-molecules correction unit 16 determines the difference C _j between each read-out rate coefficient β _ij and the second single-stranded nucleic acid that can be combined with the first single-stranded nucleic acid to form a double-stranded nucleic acid. Based on ^k− Cg _j , the total number of molecules S _i ^k of the nucleic acid fragment having at least one first single-stranded nucleic acid is corrected for each type i of the single-stranded nucleic acid, and the corrected total number of molecules S _i ^{k + 1} The information indicating the total number of molecules S _i stored in the total number-of-molecules storage unit 11 is updated.

Specifically, for example, the total molecular number correction unit 16 calculates the correction amount ΔS _i ^k of the total number of molecules of the nucleic acid fragment including the single-stranded nucleic acid of the single-stranded nucleic acid type i in the k-th step according to the following formula. The total number of molecules S _i ^k of the single-stranded nucleic acid type i nucleic acid fragment is corrected with the calculated correction amount ΔS _i ^k .

Here, s is a predetermined value, C _j ^k is the number of molecules of the j-th single-stranded nucleic acid estimated in the k-th step, and Cg _j is the known number of molecules of the j-th single-stranded nucleic acid.

<Single-stranded nucleic acid molecule number estimation device>
Next, the single-stranded nucleic acid molecule number estimation apparatus 20 will be described with reference to FIG. FIG. 3 is a block configuration diagram of the single-stranded nucleic acid molecule number estimation apparatus 20. The single-stranded nucleic acid molecule number estimation device 20 includes a double-stranded nucleic acid molecule number update unit 21, a single-stranded nucleic acid molecule number update unit 22, a single-stranded nucleic acid convergence determination unit 23, an acceleration coefficient calculation unit 24, a single-stranded nucleic acid A molecular number storage unit 25 and a double-stranded nucleic acid molecule number storage unit 26 are provided.

In the first step (t = 0, t is an integer representing the number of steps), the double-stranded nucleic acid molecule number updating unit 21 receives the initial number of total molecules for each type of single-stranded nucleic acid from the single-stranded nucleic acid molecule number acquiring unit 13. Information indicating the value S _i and information indicating each separation speed coefficient are acquired. In the first step (t = 0), each double-stranded nucleic acid does not exist in the solution, and only each single-stranded nucleic acid exists. That is, the initial value S _i for numbers of total molecular is the number of molecules of each single-stranded nucleic acid. Therefore, in the first step (t = 0), the double-stranded nucleic acid molecule number updating unit 21 sets the initial value C _i ⁰ of the number of molecules of the single-stranded nucleic acid to S _i . The double-stranded nucleic acid molecule number updating unit 21 sets the initial value M _ij ⁰ of the number of molecules of each double-stranded nucleic acid to 0.

In the second and subsequent steps, the double-stranded nucleic acid molecule number updating unit 21 reads information indicating the number of molecules M _ij ^t of each double-stranded nucleic acid from the double-stranded nucleic acid molecule number storage unit 26. Further, the double-stranded nucleic acid molecule number updating unit 21 acquires information indicating the acceleration coefficient s _ij ^t for accelerating the convergence calculation calculated by the acceleration coefficient calculating unit 24.

In addition, the double-stranded nucleic acid molecule number updating unit 21 obtains information indicating the molecular number C _i of the first type i single-stranded nucleic acid updated by the single-stranded nucleic acid convergence determining unit 23 described later, in the t-th step. as information indicating the number of molecules C _i ^t single-stranded nucleic acid of the first type i, read from a single-stranded nucleic acid molecule number storage unit 25. The double-stranded nucleic acid molecule number updating unit 21 _obtains information indicating the molecular number C _j of the second-type j single-stranded nucleic acid updated by the single-stranded nucleic acid convergence determining unit 23, which will be described later, in the second t-th step. Is read from the single-stranded nucleic acid molecule number storage unit 25 as information indicating the number of molecules C _j ^t of the single-stranded nucleic acid of type j.

The double-stranded nucleic acid molecule number updating unit 21 calculates the number of molecules M _ij ^{t + 1} of each double-stranded nucleic acid in the t + 1-th step according to the following formula.

Here, s _ij ^t is an acceleration coefficient for accelerating the convergence calculation, and a calculation method of s _ij ^t will be described later.
The double-stranded nucleic acid molecule number updating unit 21 calculates the number of molecules M _ij ^{t + 1} of each double-stranded nucleic acid according to the formula (6) for all i and j combinations.

In the first step (t = 0), in the above formula (6), M _ij ^{t in} the first term on the right side is 0, and C _j ^t and C _j ^t are S _i. The number updating unit 21 calculates the number of molecules of each double-stranded nucleic acid according to M _ij ¹ = s _ij ^t × K × (S _i × S _i ) / β _ij .

To summarize the above process, double-stranded nucleic acid molecule number updating unit 21, and the number of molecules C _i ^t of the first single stranded nucleic acid to form a duplex nucleic acid binding to a single stranded nucleic acid of the first The number of molecules C _j ^{t of} the second single-stranded nucleic acid, and a removal rate coefficient β _ij , which is the rate at which the double-stranded nucleic acid bound to the first single-stranded nucleic acid and the second single-stranded nucleic acid is released; Based on the number of molecules M _ij ^t of the double-stranded nucleic acid in which the first single-stranded nucleic acid and the second single-stranded nucleic acid are bound to each other, based on the first single-stranded nucleic acid and the second single-stranded nucleic acid. Update for each combination.

The double-stranded nucleic acid molecule number updating unit 21 combines the first single-stranded nucleic acid molecule number C _i ^{t + 1} updated by the single-stranded nucleic acid molecule number updating unit 22 with the first single-stranded nucleic acid. The number of molecules C _j ^{t + 1 of} the second single-stranded nucleic acid that can form the heavy chain nucleic acid, and the acceleration coefficient s for each combination of the first single-stranded nucleic acid and the second single-stranded nucleic acid calculated by the acceleration coefficient calculating unit 24 _{Based on ij} ^{t + 1} and the _release rate coefficient β _ij , which is the rate at which the double-stranded nucleic acid bound to the first single-stranded nucleic acid and the second single-stranded nucleic acid is released, the number of molecules of the double-stranded nucleic acid M _ij ^{t + 1} is updated for each combination of the first single-stranded nucleic acid and the second single-stranded nucleic acid.

The double-stranded nucleic acid molecule number updating unit 21 outputs information indicating the updated molecular number M _ij ^{t + 1} of each double-stranded nucleic acid to the single-stranded nucleic acid molecule number updating unit 22. In addition, the double-stranded nucleic acid molecule number updating unit 21 causes the double-stranded nucleic acid molecule number storage unit 26 to store information indicating the updated molecule number M _ij ^{t + 1} of each double-stranded nucleic acid.

The single-stranded nucleic acid molecule number updating unit 22 includes the number of molecules M _ij ^{t + 1 of} each double-stranded nucleic acid updated by the double-stranded nucleic acid molecule number updating unit 21 and a nucleic acid fragment given from the outside of the single-stranded nucleic acid molecule device 20. Based on the total number of molecules S _i , the number of molecules C _i ^{t + 1} of the single-stranded nucleic acid is updated for each type i of the single-stranded nucleic acid.

Specifically, for example, single chain number nucleic acid molecule updating unit 22 reads information indicating the total number of molecules S _i of each nucleic acid fragments from a single chain nucleic acid molecule number obtaining unit 13. Single-stranded nucleic acid molecule number updating unit 22 according to the following equation based on the equation (3), and updates the number of molecules C _i ^t single-stranded nucleic acid for each type of single-chain nucleic acid i.

That is, a single-stranded nucleic acid molecule number updating unit 22, from the total number of molecules S _i of the nucleic acid fragments of type i in the single-stranded nucleic acids, double stranded nucleic acid comprising at least one single-stranded nucleic acid of the type i of the single-stranded nucleic acid molecule By subtracting the number (ΣM _ij (sum of i = 1 to n) and M _ii ), the number of molecules C _i ^{t + 1} of the single-stranded nucleic acid of type i of the single-stranded nucleic acid is calculated. The single-stranded nucleic acid molecule number updating unit 22 calculates the number of molecules C _i ^{t + 1} of each single-stranded nucleic acid by performing the above calculation for all types i of single-stranded nucleic acids.

The single-stranded nucleic acid molecule number updating unit 22 outputs information indicating the updated molecular number C _i ^{t + 1} of each single-stranded nucleic acid to the single-stranded nucleic acid convergence determining unit 23.

Single stranded nucleic convergence determination unit 23, the number of molecules C _i ^t single-stranded nucleic acid before being updated by the single-stranded nucleic acid molecule number updating section 22, a single chain after being updated by the single-stranded nucleic acid molecule number updating section 22 The difference | C _i ^{t + 1} −C _i ^t | from the number of nucleic acid molecules C _i ^{t + 1} is calculated for each type i of single-stranded nucleic acid.
The single-stranded nucleic acid convergence determination unit 23 determines whether or not the estimation result of the number of molecules C _i ^t of each single-stranded nucleic acid has converged based on each difference | C _i ^{t + 1} −C _i ^t |.

Specifically, for example, a single-stranded nucleic acid convergence determination unit 23, all differences | estimation results in the case is less than a predetermined threshold value, the number of molecules C _i ^t of each single-stranded nucleic acid ^{_{| C i t + 1 -C i}} t Determined to have converged. Then, the single-stranded nucleic acid convergence determining unit 23 outputs information indicating the updated number of molecules C _i ^{t + 1} of each single-stranded nucleic acid to the single-stranded nucleic acid molecule number acquiring unit 13.

On the other hand, the difference | determines that exceed the predetermined threshold value even any one of the estimation result of the number of molecules C _i ^t of each single-stranded nucleic acid is not converged ^{_{| C i t + 1 -C i}} t. Then, the single-stranded nucleic acid convergence determination unit 23 outputs information indicating the updated number of molecules C _i ^{t + 1} of each single-stranded nucleic acid to the acceleration coefficient calculation unit 24. In addition, the single-stranded nucleic acid convergence determining unit 23 stores the updated information indicating the number of molecules C _i ^{t + 1} of each single-stranded nucleic acid in the single-stranded nucleic acid molecule number storage unit 25 as the number of molecules C _i of each single-stranded nucleic acid.

Incidentally, single-stranded nucleic convergence determination unit 23, when it reaches a predetermined number the number predetermined step may determine that the estimation result of the number of molecules C _i ^t of each single-stranded nucleic acid has converged. Thereby, the single-stranded nucleic acid molecule number estimation device 20 determines that the convergence has been achieved if the number of steps reaches a predetermined number even if all the differences | C _i ^{t + 1} −C _i ^t | do not fall below a predetermined threshold. Therefore, the calculation time can be kept within a predetermined range.

Subsequently, the acceleration coefficient s _ij ^t will be described. Since the binding force between the nucleic acid fragments is weak, the number of molecules C _i ^{t + 1} of each single-stranded nucleic acid converges quickly when the concentration of the single-stranded nucleic acid is small from the initial concentration even after equilibration. However, the process of estimating the number of molecules of single-stranded nucleic acid is difficult when the difference between the concentration of single-stranded nucleic acid after equilibration is high and the percentage of double-stranded nucleic acid is high due to the strong binding force between nucleic acid fragments. Thus, the phenomenon that the number of molecules of single-stranded nucleic acid oscillates around a certain value is likely to occur. In order to suppress the vibration, the acceleration coefficient calculation unit 24 decreases the acceleration coefficient s _ij ^t in Equation (6) as the number of molecules of each single-stranded nucleic acid decreases.

If the single-stranded nucleic acid convergence determination unit 23 determines that the single-stranded nucleic acid convergence determination unit 23 has not converged, the acceleration coefficient calculation unit 24 calculates the number of molecules C _i ^{t + 1} of each single-stranded nucleic acid updated by the single-stranded nucleic acid molecule number update unit 22 based on two An acceleration coefficient s _ij ^{t + 1} is calculated for each type of heavy chain nucleic acid.
Specifically, the acceleration coefficient calculation unit 24 receives the information indicating the number of molecules C _{i t} ^{+ 1} of each single-stranded nucleic acid that is updated from a single-stranded nucleic acid convergence determination unit 23, the number of molecules C _{i t} ^{+ 1} of each single-stranded nucleic acid Based on the above, each acceleration coefficient s _ij ^{t + 1} is calculated. For example, the acceleration coefficient calculation unit 24 calculates each acceleration coefficient s _ij ^t according to the following equation.

Here, S _i is the total number of molecules of single-stranded nucleic acid of type i of single-stranded nucleic acid, and S _j is the total number of molecules of single-stranded nucleic acid of type j of single-stranded nucleic acid. The acceleration coefficient s _ij ^t is 1 in the first step (t = 0).
Acceleration coefficient calculation unit 24, in accordance with the equation (8), as the number of molecules C _j of a single-stranded nucleic acid molecule number C _i or type j of single-stranded nucleic acid of type i is reduced to reduce the acceleration coefficient s _ij ^t.

Thereby, as the acceleration coefficient s _ij ^t becomes smaller, the number of the second term on the right side of the equation (6) becomes smaller. Therefore, the double-stranded nucleic acid molecule number updating unit 21 changes the molecular number M _ij of the double-stranded nucleic acid. The amount decreases. As a result, when the amount of change in the number of molecules M _ij of the double-stranded nucleic acid is reduced, the amount of change in the number of molecules of the single-stranded nucleic acid by the single-stranded nucleic acid molecule number updating unit 22 is also reduced. In the process, it can be suppressed that the number of single-stranded nucleic acid molecules vibrates around a certain value.

The acceleration coefficient calculation unit 24 may adjust the acceleration coefficient s _ij ^t based on the relationship between the number C _i of each single-stranded nucleic acid and the binding rate constant K indicating the binding rate between the single-stranded nucleic acids.
The acceleration coefficient calculation unit 24 outputs information indicating each calculated acceleration coefficient s _ij ^t to the double nucleic acid molecule number updating unit 21.

<Control unit>
Subsequently, processing of the control unit 30 will be described. FIG. 4 is a block configuration diagram of the control unit 30. The control unit 30 includes a single-stranded nucleic acid acquisition unit 31, a detachment rate coefficient correction unit 32, a detachment coefficient storage unit 33, a before / after correction difference calculation unit 34, a detachment rate coefficient selection unit 35, and a detachment rate coefficient convergence determination unit. 36.

The single-stranded nucleic acid obtaining unit 31 uses the total number-of-molecules estimation apparatus 10 to calculate the total number of molecules S _i of the nucleic acid fragments based on the separation rate coefficient β _ij and the known number of molecules Cg _{i of the} single-stranded nucleic acid. For each i, the single-stranded nucleic acid estimation apparatus 20 is made to estimate the number of molecules C _i of the single-stranded nucleic acid corresponding to the total number of molecules S _i of the nucleic acid fragments for each type i of the single-stranded nucleic acid.

Specifically, for example, in the first step (l = 0), the single-stranded nucleic acid acquisition unit 31 receives information indicating the initial value β _ij ⁰ of each separation rate coefficient from the outside of the separation rate coefficient calculation device 1, and each obtaining a known number of molecules Cg _i single-stranded nucleic acid. Then, the single-stranded nucleic acid acquisition unit 31 uses the initial value β _ij ⁰ of each separation rate coefficient as information indicating each separation rate coefficient β _ij , together with the known number of molecules Cg _{i of} each single-stranded nucleic acid, the total number-of-molecules estimation device 10 Output to.

Single-stranded nucleic acid acquiring unit 31, based on the known number of molecules Cg _i of the respective dissociation rate coefficient beta _ij and each single strand nucleic acid, the total number of molecules estimating apparatus 10, the nucleic acid fragments of each type of single-stranded nucleic acid The total number of molecules S _i is calculated.
In addition, when the single-stranded nucleic acid acquisition unit 31 determines that the separation rate coefficient convergence determination unit 36 has not converged, the single-stranded nucleic acid acquisition unit 31 is based on each separation rate coefficient β _ij ^{l + 1} selected by the separation rate coefficient selection unit 35. from the nucleic acid molecule number estimating device 20 obtains the number of molecules C _i of each single-stranded nucleic acid.

Specifically, for example, when it is determined that the separation rate coefficient convergence determination unit 36 has not converged, the single-stranded nucleic acid acquisition unit 31 sets each separation rate coefficient β _ij ^{l + 1} selected by the separation rate coefficient selection unit 35. When the information indicating the separation rate coefficient β _ij ^{1 + 1} is received from the separation rate coefficient convergence unit 26, the information indicating the separation rate coefficient β _ij is used as information indicating each separation rate coefficient β _{ij. i} is output to the total number-of-molecules estimation device 10 together with _i , and the total number-of-molecules estimation device 10 is caused to calculate the total number of molecules S _i of nucleic acid fragments for each type of single-stranded nucleic acid.

In the second and subsequent steps (l ≧ 1), the single-stranded nucleic acid acquisition unit 31 indicates information indicating each separation speed coefficient β _ij ^{l + 1} input from the separation speed coefficient convergence determination unit, and indicates each separation speed coefficient β _ij . information as outputs with known molecular number Cg _i the total number of molecules estimating apparatus 10 of each single-stranded nucleic acid.

The single-stranded nucleic acid acquisition unit 31 causes the single-stranded nucleic acid molecule number estimation device 20 to calculate the number of molecules of each single-stranded nucleic acid based on the calculated total number of molecules S _i of each nucleic acid fragment and each separation rate coefficient β _ij. C _i is calculated.
Single-stranded nucleic acid acquiring unit 31 acquires information indicating the number of molecules C _i of each single-stranded nucleic acid that is calculated from the single-stranded nucleic acid molecule number estimation device 20.

The single-stranded nucleic acid acquisition unit 31 uses the information indicating the number of molecules C _i of each single-stranded nucleic acid acquired from the single-stranded nucleic acid molecule number estimation device 20 as information indicating the number of molecules C _i ^l of the single-stranded nucleic acid before correction. It outputs to the coefficient correction part 32 and the difference calculation part 34 before and behind correction | amendment. In addition, the single-stranded nucleic acid acquisition unit 31 outputs information indicating each separation rate coefficient β _ij ^l to the correction rate coefficient correction unit 32.

The single-stranded nucleic acid acquisition unit 31 also sets the total number of molecules S ′ _i to the total number-of-molecules estimation device 10 based on the separation rate coefficient β ′ _ij ^l corrected by the separation rate coefficient correction unit 32. For each type i, the single-stranded nucleic acid estimation apparatus 20 is made to estimate the number of single-stranded nucleic acids C ′ _i ¹ corresponding to the total number of molecules S ′ _i for each type i of single-stranded nucleic acids.

Specifically, for example, the single-stranded nucleic acid acquisition unit 31 acquires each separation rate coefficient β ′ _ij corrected by the separation rate coefficient correction unit 32 from the separation rate coefficient correction unit 32. Single-stranded nucleic acid acquisition unit 31, information indicating each dissociation rate coefficient .beta. _'Ij corrected by dissociation rate coefficient correcting unit 32, as information indicating the respective dissociation rate coefficient beta _ij, known number of molecules of each single-stranded nucleic acid outputs with cg _i the total number of molecules estimating device 10. Single-stranded nucleic acid acquiring unit 31, based on the known number of molecules Cg _i of the respective dissociation rate coefficient beta _ij and each single strand nucleic acid, the total number of molecules estimating apparatus 10, the nucleic acid fragments of each type of single-stranded nucleic acid The total number of molecules S _i is calculated.

The single-stranded nucleic acid acquisition unit 31 causes the single-stranded nucleic acid molecule number estimation device 20 to calculate the number of molecules of each single-stranded nucleic acid based on the calculated total number of molecules S _i of each nucleic acid fragment and each separation rate coefficient β _ij. C _i is calculated. Single-stranded nucleic acid acquiring unit 31 acquires information indicating the number of molecules C _i of each single-stranded nucleic acid that is calculated from the single-stranded nucleic acid molecule number estimation device 20.

The single-stranded nucleic acid acquisition unit 31 uses the information indicating the molecular number C _i of each single-stranded nucleic acid acquired from the single-stranded nucleic acid molecule number estimation apparatus 20 as information indicating the corrected molecular number C ′ _i ^l of the single-stranded nucleic acid. The result is output to the difference calculation unit 34 before and after correction.

The withdrawal rate coefficient correction unit 32 receives information indicating the number of molecules C _i ^l of the single-stranded nucleic acid acquired by the single-stranded nucleic acid acquisition unit 31. For each type i of single-stranded nucleic acid, the detachment rate coefficient correction unit 32 determines the number of molecules C _i ^l of the single-stranded nucleic acid acquired by the single-stranded nucleic acid acquisition unit 31 and the single-stranded nucleic acid input from the outside of the device. The known molecular number Cg _i is compared for each type i of the single-stranded nucleic acid.

When the number of molecules C _i of the obtained single-stranded nucleic acid is smaller than the known number of molecules Cg _{i of the} input single-stranded nucleic acid, the separation rate coefficient correcting unit 32 determines the above-mentioned separation rate coefficient β _ij ^{l of} the single-stranded nucleic acid. When the number of molecules C _{i of} the obtained single-stranded nucleic acid is equal to or greater than the known number of molecules Cg _{i of} the inputted single-stranded nucleic acid, the separation rate coefficient β _ij ^l of the single-stranded nucleic acid is reduced.

Note that the separation speed coefficient correction unit 32 may correct the separation speed coefficient using a random number. In that case, for example, dissociation rate coefficient correcting unit 32, if known molecular smaller number Cg _i of the single strand nucleic acid molecule number C _i of the single-stranded nucleic acid has been input is acquired, the withdrawal of the single-stranded nucleic acid A predetermined value and a random number are added to the speed coefficient β _ij ^l . On the other hand, when the molecular number C _{i of} the obtained single-stranded nucleic acid is equal to or larger than the known molecular number Cg _{i of} the inputted single-stranded nucleic acid, the separation rate coefficient correction unit 32 determines the separation rate coefficient β of the single-stranded nucleic acid. _ij ^l is subtracted by a predetermined value and a random number is added.
Thus, dissociation rate coefficient correcting unit 32, by adding a further random number to dissociation rate coefficient beta _ij ^l, dissociation rate coefficient beta _ij ^l can be prevented from falling into a local optimum value.

The separation speed coefficient correction unit 32 outputs information indicating the corrected separation speed coefficient β ′ _ij ^l to the single-stranded nucleic acid acquisition unit 31. As a result, the single-stranded nucleic acid acquisition unit 31 uses the corrected withdrawal rate coefficient β ′ _ij ^l to give the single-stranded nucleic acid molecule number estimation device 20 the corrected molecular number C ′ _i ^l of each single-stranded nucleic acid. The information shown can be estimated.

Also, the separation speed coefficient correction unit 32 includes information indicating the separation speed coefficient β ′ _ij ^l before correction and information indicating the correction speed separation coefficient β ′ _ij ^l after correction. Remember me.

The separation speed coefficient storage unit 33 stores information indicating the initial value β _ij ⁰ of the separation speed coefficient input from the outside of the separation speed coefficient calculation device 1. The initial value β _ij ⁰ of the separation rate coefficient is a separation rate coefficient that has been obtained so far by the device itself when a new known number of molecules of the single-stranded nucleic acid is obtained. The initial value β _ij ⁰ of the _release rate coefficient is the same as the sequence of the first single-stranded nucleic acid and the sequence of the second single-stranded nucleic acid when there is no release rate coefficient obtained so far in the device. The value depends on the length of the part.

The before-and-after-difference difference calculation unit 34 for each type i of the single-stranded nucleic acid, the number of molecules C _i ^l of the single-stranded nucleic acid before correction and the single-stranded nucleic acid input from the outside of the separation rate coefficient calculating device 1. The difference from the known number of molecules Cg _i is calculated as a difference | C _i ¹ −Cg _i | before correction. Further, the difference calculation unit 34 before and after correction, for each type i of single-stranded nucleic acid, the number of molecules C ′ _i ¹ of the single-stranded nucleic acid after correction and the single-stranded nucleic acid input from the outside of the separation rate coefficient calculating device 1. is calculated as | of the difference between the known number of molecules Cg _i differences corrected | C _'i ^l -Cg _i.

The before-and-after-correction difference calculating unit 34 selects the information indicating the calculated difference | C _i ¹ −Cg _i | before correction and the information indicating the corrected difference | C ′ _i ¹ −Cg _i | To the unit 35.

The separation rate coefficient selection unit 35 compares the difference | C _i ¹ −Cg _i | before correction with the difference after correction | C ′ _i ¹ −Cg _i | for each type i of single-stranded nucleic acid. When the difference | C _i ¹ −Cg _i | before correction is larger than the difference after correction | C ′ _i ¹ −Cg _i | dissociation rate coefficient beta _i1 to leave from the heavy chain nucleic _acid, ..., as beta _in, the dissociation rate coefficient beta after being corrected by the respective dissociation rate coefficient correcting unit ^{_{32 'i1 l, ..., β}} ' single chain nucleic acids _in ^l For each type i.

On the other hand, when the difference | C _i ¹ −Cg _i | before correction is equal to or less than the difference after correction | C ′ _i ¹ −Cg _i |, the separation rate coefficient selection unit 35 selects the single-stranded nucleic acid of type i of the single-stranded nucleic acid dissociation rate coefficient beta _i1 but to leave the double-stranded nucleic _acid, ..., as beta _in, dissociation rate coefficient beta _{i1 l} before being corrected by the respective dissociation rate coefficient correcting unit _32, ^..., beta _in ^l a single-stranded nucleic acid Select for each type i.

Dissociation rate coefficient selecting unit 35, dissociation rate coefficient after correction has been selected dissociation rate coefficient beta _ij ^l before being corrected .beta. _'Ij ^l leaving the selection information indicating the selected rate coefficient convergence determination unit 36 Output to.

The separation speed coefficient convergence determination unit 36 indicates the separation speed coefficient (β _ij ^l or β ′ _ij ^l ) selected by the separation speed coefficient selection unit 35 based on the selection information input from the separation speed coefficient selection unit 35. Read information.
The separation speed coefficient convergence determination unit 36 for each separation speed coefficient β _ij , the separation speed coefficient β _ij ^l before being corrected by the separation speed coefficient correction unit 32, and the separation speed coefficient selected by the separation speed coefficient selection unit 35. Based on the difference from (β _ij ^l or β ′ _ij ^l ), it is determined whether or not the estimation result of the separation speed coefficient β _ij has converged.

Specifically, for example, the separation speed coefficient convergence determination unit 36 determines the separation speed coefficient β _ij ^l before being corrected by the separation speed coefficient correction unit 32 and the separation speed coefficient selected by the separation speed coefficient selection unit 35 ( When all of the differences from β _ij ^l or β ′ _ij ^l ) are smaller than a predetermined threshold, the departure speed coefficient convergence determination unit 36 determines that the estimation result of the separation speed coefficient β _ij has converged. Then, the separation speed coefficient convergence determination unit 36 calculates the separation speed coefficient using the separation speed coefficient (β _ij ^l or β ′ _ij ^l ) selected by the separation speed coefficient selection unit 35 as information indicating the separation speed coefficient β _ij. Output to the outside of the device 1.

On the other hand, the separation speed coefficient convergence determination unit 36 has a separation speed coefficient β _ij ^l before being corrected by the separation speed coefficient correction unit 32 and a separation speed coefficient (β _ij ^l or β β selected by the separation speed coefficient selection unit 35. If any one of the differences from ' _ij ^l ) is equal to or greater than the predetermined threshold, the departure speed coefficient convergence determination unit 36 determines that the estimation result of the separation speed coefficient β _ij has not converged. The dissociation rate coefficient convergence determination unit 36, dissociation rate coefficient selected by the dissociation rate coefficient selecting section 35 (beta _ij ^l or β _'ij ^l), the dissociation rate coefficient β _ij ^{l + 1} in the next step (l + 1) Information to be output is output to the single-stranded nucleic acid acquisition unit 31.

FIG. 5 is a flowchart showing the flow of the estimation process of the separation speed coefficient by the separation speed coefficient calculation apparatus 1. First, the control unit 30 of the separation rate coefficient calculation device 1 uses information indicating the initial value β _ij ⁰ of each separation rate coefficient and information indicating the known number of molecules Cg _i of each single-stranded nucleic acid of the separation rate coefficient calculation device 1. Obtained from the outside (step S101).
Next, the single-stranded nucleic acid acquisition unit 31 acquires the number of molecules C _i ¹ of each single-stranded nucleic acid before correction (step S102). Next, the difference calculator 34 before and after correction calculates a difference | C _i ¹ −Cg _i | before each correction (step S103).

Next, the separation speed coefficient correction unit 32 corrects the separation speed coefficient (step S104). Next, the single-stranded nucleic acid acquisition unit 31 acquires the corrected molecular number C ′ _i ¹ of each single-stranded nucleic acid (step S105). Next, the difference calculator 34 before and after correction calculates a difference | C ′ _i ¹ −Cg _i | after each correction (step S106).

Next, the separation speed coefficient selection unit 35 determines whether or not the difference after correction is smaller than the difference before correction (step S107). When the difference after correction becomes smaller than the difference before correction (YES in step S107), the separation rate coefficient selecting unit 35 causes the separation rate coefficient β at which the single-stranded nucleic acid of type i of the single-stranded nucleic acid is detached from the double-stranded nucleic acid. _i1, ..., as beta _in, dissociation rate coefficient after each correction ^{_{β 'i1 l, ..., β}} ' select _in ^l, the process proceeds to step S110 (step S108).

On the other hand, if the difference after correction is equal to or greater than the difference before correction (NO in step S107), the separation rate coefficient selection unit 35 separates the single-stranded nucleic acid of type i from single-stranded nucleic acid from the double-stranded nucleic acid β. _i1, ..., as beta _in, dissociation rate coefficient before each correction β _i1 ^l, ..., select beta _in ^l, the process proceeds to step S110 (step S109).

Next, the separation rate coefficient selection unit 35 determines whether or not the difference before correction and the difference after correction have been compared for all types of single-stranded nucleic acids i (step S110). When the difference before correction and the difference after correction are not compared for all types of single-stranded nucleic acids i (NO in step S110), the separation rate coefficient selection unit 35 increments i by one and performs the process of step S107. Return.
On the other hand, when the difference | C _i ¹ −Cg _i | and the difference after correction | C ′ _i ¹ −Cg _i | are compared for all types of single-stranded nucleic acid i (YES in step S110), the withdrawal speed The coefficient convergence determination unit 36 determines that the difference between the difference before correction | C _i ¹ −Cg _i | and the difference after correction | C ′ _i ¹ −Cg _i | is a predetermined threshold value for all types of single-stranded nucleic acids i. It is determined whether it is below (step S112).

The separation rate coefficient convergence determination unit 36 determines that the difference between the difference before correction | C _i ^l −Cg _i | and the difference after correction | C ′ _i ^l −Cg _i | is predetermined for all types of single-stranded nucleic acids i. If it is not less than the threshold value (NO in step S112), the separation rate coefficient convergence determination unit 36 updates the separation rate coefficient β _ij held by the single-stranded nucleic acid acquisition unit 31 with the selected separation rate coefficient β _ij ^{l + 1} (step S113). The process returns to step S102.

The separation rate coefficient convergence determination unit 36 determines that the difference between the difference before correction | C _i ^l −Cg _i | and the difference after correction | C ′ _i ^l −Cg _i | Is equal to or less than the threshold value (YES in step S112), the separation speed coefficient convergence determination unit 36 outputs each separation speed coefficient _βij to the outside (step S114). Above, the process of this flowchart is complete | finished.

FIG. 6 is a flowchart showing the flow of processing for obtaining the number of molecules of each single-stranded nucleic acid in step S102 or step S105 of FIG. First, the total number of molecules estimating device 10 estimates the total number of molecules _{S i} of each nucleic acid fragment (step S201). Next, based on the estimated total number of molecules S _i of each nucleic acid fragment, the single-stranded nucleic acid molecule number estimation device 20 estimates the number of molecules C _i of each single-stranded nucleic acid, and outputs information indicating the number of molecules _{C i} of the single-stranded nucleic acid (step S202). Above, the process of this flowchart is complete | finished.

By the above, dissociation rate coefficient calculating apparatus 1 of this embodiment, the total molecular number estimating apparatus 10, by a configuration that uses a single-stranded nucleic acid molecule number estimating device 20, the number of molecules C _i of each single-stranded nucleic acid Since each separation rate coefficient β _ij can be estimated using the number of molecules C _i of each single-stranded nucleic acid, genomics analysis can be remarkably improved in efficiency and precision. .

In addition, since the total number-of-molecules estimation device 10 can use the calculated separation rate coefficients β _ij when estimating the total number of molecules S _i of each nucleic acid fragment, the total number-of-molecules estimation device 10 can accurately it is possible to estimate the total number of molecules S _i of nucleic acid fragments.
Similarly, each calculated rate-of-detachment coefficient β _ij can be used when the single-stranded nucleic acid molecule number estimating device 20 estimates the number of molecules C _i of each single-stranded nucleic acid, so that the single-stranded nucleic acid molecule number estimating device 20 it can be estimated accurately molecular number C _i of each single-stranded nucleic acid.

FIG. 7 is a flowchart showing the flow of processing for estimating the total number of molecules of each nucleic acid fragment by the total number-of-molecules estimation apparatus 10 in step S201 of FIG. First, the single-stranded nucleic acid molecule number acquisition unit 13 reads each separation rate coefficient β _ij from the separation rate coefficient storage unit 12 (step S301). Next, single-stranded nucleic acid molecule number obtaining unit 13 obtains the known number of molecules Cg _i of each single-stranded nucleic acid that is input from the control unit 30 as an example as the total number of molecules S _i of each nucleic acid fragments (Step S302) .

Next, single-stranded nucleic acid molecule number estimating device 20 estimates the number of molecules _{C i} of each single-stranded nucleic acid (step S303). Next, the single-stranded nucleic acid difference calculation unit 14 calculates the difference between the number of molecules C _i of each single-stranded nucleic acid estimated by the single-stranded nucleic acid molecule number estimation device 20 and the known number of molecules Cg _{i of} each single-stranded nucleic acid. Calculate (step S304).

The total molecular number convergence determination unit 15 determines whether all of the calculated differences are equal to or less than a predetermined threshold (step S305). If all of the calculated differences are not less than or equal to the predetermined threshold (NO in step S305), the total molecular number correction unit 16 corrects the total number of molecules of each nucleic acid fragment, and returns to the process of step S303.
On the other hand, when all the calculated differences are equal to or smaller than the predetermined threshold (YES in step S305), the total molecular number convergence determination unit 15 outputs information indicating the total number of molecules of each nucleic acid fragment to the outside (step S307). Above, the process of this flowchart is complete | finished.

As described above, the total number-of-molecules estimation apparatus 10 in the present embodiment acquires the number of molecules of each single-stranded nucleic acid by using the single-stranded nucleic acid molecule number estimation apparatus 20, and uses the acquired number of molecules of each single-stranded nucleic acid. , to estimate the total number of molecules S _i of each nucleic acid fragment. By adopting such a configuration, the single-stranded nucleic acid molecule number estimating device 20 is configured to calculate the number of molecules of each single-stranded nucleic acid estimated by the single-stranded nucleic acid molecule number estimating device 20 and the number of known single-stranded nucleic acid molecules. by correcting the total number of molecules S _i of each nucleic acid fragment so as to reduce the difference, since the total number of molecules S _i of each nucleic acid fragment can be accurately estimated, dramatically streamline genomics analysis, and Can be refined.

  FIG. 8 is a flowchart showing the flow of the process of estimating the number of molecules of each single-stranded nucleic acid by the single-stranded nucleic acid molecule number estimating apparatus 20 in step S202 of FIG. 6 or step S303 of FIG. First, the double-stranded nucleic acid molecule number updating unit 21 updates the number of each double-stranded nucleic acid molecule (step S401). Next, the single-stranded nucleic acid molecule number updating unit 22 updates the number of each single-stranded nucleic acid molecule (step S402). Next, the single-stranded nucleic acid convergence determining unit 23 determines whether or not all the amount of change in the number of molecules of each single-stranded nucleic acid in the single-stranded nucleic acid molecule number updating unit 22 is equal to or less than a predetermined threshold (step S403).

When even one change amount of the number of molecules of each single-stranded nucleic acid exceeds a predetermined threshold (NO in step S403), the acceleration coefficient calculation unit 24 updates the acceleration coefficient (step S404), and the single-stranded nucleic acid molecule number estimation device 20 returns to the process of step S401.
On the other hand, when all of the amount of change in the number of molecules of each single-stranded nucleic acid in the single-stranded nucleic acid molecule number updating unit 22 is equal to or less than a predetermined threshold (YES in step S403), the single-stranded nucleic acid molecule number estimating device 20 The information indicating the number of molecules is output to the outside (step S405). Above, the process of this flowchart is complete | finished.

  As described above, the single-stranded nucleic acid molecule number estimation device 20 in the present embodiment updates the number of molecules of each double-stranded nucleic acid, and updates the number of molecules of each single-stranded nucleic acid based on the updated number of molecules of double-stranded nucleic acid. And estimating the number of molecules of each single-stranded nucleic acid by determining whether the estimation of the number of molecules of each single-stranded nucleic acid has converged based on the amount of change in the number of single-stranded nucleic acid molecules at the time of the update. Can do. By adopting such a configuration, the number of molecules of each double-stranded nucleic acid is updated and the number of molecules of each single-stranded nucleic acid is updated until the estimation of the number of molecules of each single-stranded nucleic acid converges. It is possible to accurately estimate the number of molecules. Thereby, genomics analysis can be remarkably improved in efficiency and refined.

As described above, according to the present invention, by using the polysolute binding equilibrium equation shown in the present embodiment, the existing measurement data is generated due to the non-target nucleic acid fragment binding included in the original data. By removing the error, the number of molecules of each single-stranded nucleic acid and the number of molecules of each nucleic acid fragment can be obtained with high accuracy.
In addition, error correction due to saturation phenomenon in complex systems, which could not be dealt with by the prior art, and the quantitative performance of nucleic acid fragments that are present only in small amounts that were greatly affected by errors due to cross-hybridization can be significantly improved. .

  This makes it possible to perform highly accurate quantification that could not be achieved with the prior art, and in the fields of medicine and biology, genomics analysis is dramatically improved and refined. In addition to strongly promoting the development and clinical application of highly effective and safe nucleic acid drugs and gene therapy methods, it can also contribute to the development of next-generation massively parallel computers in the field of information engineering.

  Moreover, you may make it implement | achieve the function of the withdrawal rate coefficient calculation apparatus 1, the total number-of-molecules estimation apparatus 10, or the single-stranded nucleic acid molecule number estimation apparatus 20 in the embodiment of the present invention or a part of the functions by a computer. In this case, the computer program for realizing the function may be recorded on a computer-readable recording medium, and the computer program recorded on the recording medium may be read by the computer system and executed. Here, the “computer system” includes an OS (Operating System) and hardware of peripheral devices. The “computer-readable recording medium” refers to a portable recording medium such as a flexible disk, a magneto-optical disk, an optical disk, and a memory card, and a storage device such as a hard disk built in the computer system. Furthermore, the “computer-readable recording medium” dynamically holds a program for a short time like a communication line when transmitting a program via a network such as the Internet or a communication line such as a telephone line. In this case, a volatile memory in the computer system that becomes a server or a client in that case may be included and a program that holds a program for a certain period. Further, the above computer program may be for realizing a part of the above-described functions, and further realizing the above functions by a combination with a computer program already recorded in the computer system. Also good.

  As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the concrete structure is not restricted to this embodiment, The design etc. of the range which does not deviate from the summary of this invention are included.

DESCRIPTION OF SYMBOLS 1 Separation rate coefficient calculation device 10 Total molecule number estimation device 11 Total molecule number storage unit 12 Separation rate coefficient storage unit 13 Single strand nucleic acid molecule number acquisition unit 14 Single strand nucleic acid difference calculation unit 15 Total molecule number convergence determination unit 16 Total number of molecules Correction unit 20 Single-stranded nucleic acid molecule number estimation device 21 Double-stranded nucleic acid molecule number update unit 22 Single-stranded nucleic acid molecule number update unit 23 Single-stranded nucleic acid convergence determination unit 24 Acceleration coefficient calculation unit 25 Single-stranded nucleic acid molecule number storage unit 30 Control unit 31 Single-stranded nucleic acid acquisition unit 32 Separation rate coefficient correction unit 33 Separation rate coefficient storage unit 34 Pre-correction difference calculation unit 35 Separation rate coefficient selection unit 36 Separation rate coefficient convergence determination unit

Claims (16)

The number of molecules of the first single-stranded nucleic acid, the number of molecules of the second single-stranded nucleic acid that can bind to the first single-stranded nucleic acid to form a double-stranded nucleic acid, the first single-stranded nucleic acid, and the A double strand in which the first single-stranded nucleic acid and the second single-stranded nucleic acid are bound to each other based on a separation rate coefficient that is a rate at which the double-stranded nucleic acid bound to the second single-stranded nucleic acid is detached. A double-stranded nucleic acid molecule number updating unit for updating the number of nucleic acid molecules for each combination of the first single-stranded nucleic acid and the second single-stranded nucleic acid;
The number of molecules of the single-stranded nucleic acid based on the number of molecules of each double-stranded nucleic acid updated by the double-stranded nucleic acid molecule number updating unit and the total number of molecules of nucleic acid fragments given from the outside of the device A single-stranded nucleic acid molecule number updating unit for updating each single-stranded nucleic acid type,
The number of single-stranded nucleic acid molecules before being updated by the single-stranded nucleic acid molecule number updating unit,
The difference between the number of single-stranded nucleic acid molecules updated by the single-stranded nucleic acid molecule number updating unit is calculated for each type of single-stranded nucleic acid, and the number of molecules of each single-stranded nucleic acid is estimated based on each difference. A single-stranded nucleic acid convergence determination unit for determining whether the result has converged, and
An apparatus for estimating the number of single-stranded nucleic acid molecules. When it is determined that the single-stranded nucleic acid convergence determination unit has not converged, based on the number of molecules of each single-stranded nucleic acid updated by the single-stranded nucleic acid molecule number update unit, an acceleration factor for each type of double-stranded nucleic acid An acceleration coefficient calculation unit for calculating
The double-stranded nucleic acid molecule number updating unit binds the first single-stranded nucleic acid and the number of molecules of the first single-stranded nucleic acid updated by the single-stranded nucleic acid molecule number updating unit. The number of molecules of the second single-stranded nucleic acid that can be formed, the acceleration coefficient for each combination of the first single-stranded nucleic acid and the second single-stranded nucleic acid calculated by the acceleration coefficient calculating unit, and the first Based on the separation rate coefficient, which is the rate at which the double-stranded nucleic acid bound to the single-stranded nucleic acid and the second single-stranded nucleic acid is separated, the number of molecules of the double-stranded nucleic acid is determined from the first single-stranded nucleic acid and the above-mentioned The apparatus for estimating the number of single-stranded nucleic acid molecules according to claim 1, wherein the number of single-stranded nucleic acid molecules is updated for each combination of second single-stranded nucleic acids. Information indicating a separation rate coefficient at which the single-stranded nucleic acid is detached from the double-stranded nucleic acid is stored for each type of the double-stranded nucleic acid,
Information indicating the total number of nucleic acid fragments, which is the sum of the number of molecules of a single-stranded nucleic acid in which the nucleic acid fragment is in a single-stranded state and the number of molecules of a double-stranded nucleic acid containing at least one single-stranded nucleic acid is the type of single-stranded nucleic acid A total molecular number storage unit stored for each;
The information indicating the separation rate coefficient is read from the separation rate coefficient storage unit, the information indicating the total number of molecules is read from the total molecule number storage unit, the read separation rate coefficient and the total number of molecules read out Based on the above, the number of single-stranded nucleic acid molecules that cause the single-stranded nucleic acid molecule number estimation device according to claim 1 to estimate the number of molecules of the single-stranded nucleic acid in the solution for each type of the single-stranded nucleic acid An acquisition unit;
A single-stranded nucleic acid difference calculating unit that calculates the difference between the estimated number of molecules of the single-stranded nucleic acid and the number of known molecules of the single-stranded nucleic acid input from the outside of the device for each type of the single-stranded nucleic acid; ,
Based on the difference for each type of the single-stranded nucleic acid, a total molecular number convergence determination unit that determines whether or not the estimation result of the total number of molecules of the nucleic acid fragment has converged,
When it is determined that the total molecular number convergence determination unit has not converged, information indicating the respective separation rate coefficients is read from the separation rate coefficient storage unit, and each of the read separation rate coefficients and the first unit Based on the difference of the second single-stranded nucleic acid that can form a double-stranded nucleic acid by binding to a strand nucleic acid, the total number of nucleic acid fragments having at least one first single-stranded nucleic acid is calculated as the single-stranded nucleic acid. A total molecular number correction unit that updates the information indicating the total number of molecules stored in the total molecular number storage unit with information indicating the corrected total number of molecules.
With
The single-stranded nucleic acid molecule number acquisition unit reads information indicating the total number of molecules updated by the total molecular number correction unit from the total molecular number storage unit, and based on the read total number of molecules, An apparatus for estimating the total number of molecules, wherein the number of molecules is estimated for each kind of the single-stranded nucleic acid by the single-stranded nucleic acid molecule number estimating apparatus.   The single-stranded nucleic acid molecule number acquisition unit sets an initial value of the total number of molecules of the nucleic acid fragment to be output to the single-stranded nucleic acid molecule number estimation device as a known number of molecules of single-stranded nucleic acid input from the outside. The total number-of-molecules estimation apparatus according to claim 3, A request to estimate the number of molecules of a nucleic acid fragment for each type of single-stranded nucleic acid based on each of the separation rate coefficients, which are the rates at which single-stranded nucleic acids are released from double-stranded nucleic acids, and the known number of molecules of each single-stranded nucleic acid The total number-of-molecules estimation apparatus according to Item 3 or Claim 4,
The single strand according to claim 1 or 2, wherein the number of molecules of single-stranded nucleic acid is estimated for each kind of single-stranded nucleic acid based on each of the separation rate coefficients and the estimated number of molecules of each nucleic acid fragment. An apparatus for estimating the number of nucleic acid molecules;
Based on the estimated number of molecules of each single-stranded nucleic acid, a controller that calculates the separation rate coefficient for each type of double-stranded nucleic acid,
A separation speed coefficient calculating device comprising: The controller is
The total molecular number estimation apparatus according to claim 3 or 4, wherein the total number of molecules is estimated for each type of the single-stranded nucleic acid based on the separation rate coefficient and the known number of molecules of the single-stranded nucleic acid, A single-stranded nucleic acid obtaining unit that causes the single-stranded nucleic acid estimation apparatus according to claim 1 or 2 to estimate the number of molecules of a single-stranded nucleic acid corresponding to the total number of molecules for each type of the single-stranded nucleic acid,
For each type of single-stranded nucleic acid, the number of molecules of the single-stranded nucleic acid obtained by the single-stranded nucleic acid obtaining unit and the known number of molecules of the single-stranded nucleic acid input from the outside of the device are the single strand. When the number of molecules of the obtained single-stranded nucleic acid is smaller than the known number of molecules of the inputted single-stranded nucleic acid, compared with each type of nucleic acid, the separation rate coefficient of the single-stranded nucleic acid is increased and the obtained When the number of molecules of the single-stranded nucleic acid is equal to or greater than the known number of molecules of the input single-stranded nucleic acid, a separation rate coefficient correction unit that reduces the separation rate coefficient of the single-stranded nucleic acid,
With
5. The single-stranded nucleic acid acquisition unit calculates the total number of molecules in the total number-of-molecules estimation apparatus according to claim 3 or 4 based on the separation rate coefficient corrected by the separation rate coefficient correction unit. Estimating for each type of nucleic acid, causing the single-stranded nucleic acid estimation apparatus according to claim 1 or claim 2 to estimate the number of molecules of single-stranded nucleic acid corresponding to the total number of molecules for each type of single-stranded nucleic acid,
The controller is
For each type of single-stranded nucleic acid, the difference between the number of molecules of the single-stranded nucleic acid before correction and the number of known molecules of the input single-stranded nucleic acid is calculated as a difference before correction, For each type, a difference calculation unit before and after correction that calculates the difference between the number of molecules of the corrected single-stranded nucleic acid and the number of known molecules of the input single-stranded nucleic acid as a corrected difference;
The difference before correction and the difference after correction are compared for each type i of the single-stranded nucleic acid, and when the difference before correction is larger than the difference after correction, the difference is corrected by the detachment speed coefficient correction unit. A separation speed coefficient selection unit that selects a separation speed coefficient before being corrected by the separation speed coefficient correction unit when the difference before correction is equal to or less than the difference after correction;
The separation speed coefficient calculating device according to claim 5, further comprising: The controller is
For each separation speed coefficient, the separation speed coefficient is estimated based on the difference between the separation speed coefficient before being corrected by the separation speed coefficient correction section and the separation speed coefficient selected by the separation speed coefficient selection section. A separation speed coefficient convergence determination unit that determines whether or not the result has converged,
When the single-stranded nucleic acid acquisition unit determines that the separation rate coefficient convergence determination unit has not converged, the number of single-stranded nucleic acid molecules is estimated based on each separation rate coefficient selected by the separation rate coefficient selection unit. The separation rate coefficient calculation apparatus according to claim 6, wherein the number of molecules of each single-stranded nucleic acid is obtained from the apparatus.   The separation speed coefficient calculation device according to claim 6 or 7, wherein the separation speed coefficient correction unit corrects the separation speed coefficient using a random number.   6. The initial value of the separation rate coefficient is a separation rate coefficient that has been obtained so far in the device itself when a new known number of molecules of the single-stranded nucleic acid is obtained. The separation speed coefficient calculation device according to any one of claims 1 to 8.   The initial value of the separation rate coefficient is the length of the coincidence between the sequence of the first single-stranded nucleic acid and the sequence of the second single-stranded nucleic acid when there is no separation rate coefficient obtained so far in the apparatus. The separation speed coefficient calculation device according to any one of claims 5 to 8, wherein the value is a value depending on the height. A single-stranded nucleic acid molecule number estimation method executed by a single-stranded nucleic acid molecule number estimation apparatus,
The number of molecules of the first single-stranded nucleic acid, the number of molecules of the second single-stranded nucleic acid that can bind to the first single-stranded nucleic acid to form a double-stranded nucleic acid, the first single-stranded nucleic acid, and the A double strand in which the first single-stranded nucleic acid and the second single-stranded nucleic acid are bound to each other based on a separation rate coefficient that is a rate at which the double-stranded nucleic acid bound to the second single-stranded nucleic acid is detached. A double-stranded nucleic acid molecule number updating procedure for updating the number of molecules of nucleic acid for each combination of the first single-stranded nucleic acid and the second single-stranded nucleic acid;
Based on the number of molecules of each double-stranded nucleic acid updated by the update procedure of the number of double-stranded nucleic acid molecules and the total number of molecules of nucleic acid fragments given from the outside of the single-stranded nucleic acid molecule estimation device, Updating the number of single-stranded nucleic acid molecules for each type of single-stranded nucleic acid,
The number of single-stranded nucleic acid molecules before being updated by the single-stranded nucleic acid molecule number updating procedure;
The difference from the number of molecules of the single-stranded nucleic acid after being updated by the procedure for updating the number of single-stranded nucleic acid molecules is calculated for each type of the single-stranded nucleic acid, and the number of molecules of each single-stranded nucleic acid is estimated based on each difference. A single-stranded nucleic acid convergence determination procedure for determining whether the result has converged, and
A method for estimating the number of single-stranded nucleic acid molecules. Information indicating a separation rate coefficient, which is a rate at which a single-stranded nucleic acid is separated from a double-stranded nucleic acid, is stored for each type of the double-stranded nucleic acid, the number of molecules of the single-stranded nucleic acid, The nucleic acid that is the sum of the number of molecules of a single-stranded nucleic acid that can be combined with a single-stranded nucleic acid to form a double-stranded nucleic acid and the number of molecules of a double-stranded nucleic acid in which the single-stranded nucleic acid and the single-stranded nucleic acid are combined A total molecular number estimation method that is executed by a total molecular number estimation device including a total molecular number storage unit in which information indicating the total number of molecules is stored for each type of single-stranded nucleic acid,
The information indicating the separation rate coefficient is read from the separation rate coefficient storage unit, the information indicating the total number of molecules is read from the total molecule number storage unit, the read separation rate coefficient and the total number of molecules read out Based on the above, a single-stranded nucleic acid molecule number obtaining procedure for estimating the number of single-stranded nucleic acid molecules present in a solution for each kind of single-stranded nucleic acid by the single-stranded nucleic acid molecule number estimating method according to claim 11;
A single-stranded nucleic acid difference calculation procedure for calculating, for each type of single-stranded nucleic acid, a difference between the estimated number of molecules of the single-stranded nucleic acid and the known number of molecules of the single-stranded nucleic acid input from the outside of the device; ,
Based on the difference for each type of the single-stranded nucleic acid, a total molecular number convergence determination procedure for determining whether or not the estimation result of the total number of molecules of the nucleic acid fragment has converged,
When it is determined that the total number-of-molecules convergence determination procedure has not converged, information indicating each of the separation speed coefficients is read from the separation speed coefficient storage unit, and each of the read separation speed coefficients and the first unit Based on the difference of the second single-stranded nucleic acid that can form a double-stranded nucleic acid by binding to a strand nucleic acid, the total number of nucleic acid fragments having at least one first single-stranded nucleic acid is calculated as the single-stranded nucleic acid. And a total molecule number correction procedure for updating the information indicating the total number of molecules stored in the total molecule number storage unit with information indicating the corrected total number of molecules.
Have
The single-stranded nucleic acid molecule number acquisition procedure reads information indicating the total number of molecules updated by the total molecular number correction procedure from the total molecular number storage unit, and based on the read total number of molecules, A method for estimating the total number of molecules, comprising causing the single-stranded nucleic acid molecule number estimating apparatus to estimate the number of molecules for each type of the single-stranded nucleic acid. A separation speed coefficient calculation method executed by the separation speed coefficient calculation device,
A request to estimate the number of molecules of a nucleic acid fragment for each type of single-stranded nucleic acid based on each of the separation rate coefficients, which are the rates at which single-stranded nucleic acids are released from double-stranded nucleic acids, and the known number of molecules of each single-stranded nucleic acid Item 12. The total molecular number estimation method according to Item 12,
12. The number of single-stranded nucleic acid molecules according to claim 11, wherein the number of molecules of single-stranded nucleic acid is estimated for each type of single-stranded nucleic acid based on each of the separation rate coefficients and the estimated number of molecules of each nucleic acid fragment. Method and
Based on the estimated number of molecules of each single-stranded nucleic acid, a control procedure for calculating the separation rate coefficient for each type of the double-stranded nucleic acid,
A method of calculating a separation speed coefficient, comprising: In a computer that is an apparatus for estimating the number of single-stranded nucleic acid molecules,
The number of molecules of the first single-stranded nucleic acid, the number of molecules of the second single-stranded nucleic acid that can bind to the first single-stranded nucleic acid to form a double-stranded nucleic acid, the first single-stranded nucleic acid, and the A double strand in which the first single-stranded nucleic acid and the second single-stranded nucleic acid are bound to each other based on a separation rate coefficient that is a rate at which the double-stranded nucleic acid bound to the second single-stranded nucleic acid is detached. A double-stranded nucleic acid molecule number updating step of updating the number of molecules of nucleic acid for each combination of the first single-stranded nucleic acid and the second single-stranded nucleic acid;
Based on the number of molecules of each double-stranded nucleic acid updated in the double-stranded nucleic acid molecule number updating step and the total number of molecules of nucleic acid fragments given from the outside of the single-stranded nucleic acid molecule estimation device, Updating the number of single-stranded nucleic acid molecules for each type of single-stranded nucleic acid;
The number of single-stranded nucleic acid molecules before being updated by the single-stranded nucleic acid molecule number updating step;
The difference from the number of molecules of the single-stranded nucleic acid after being updated by the single-stranded nucleic acid molecule number updating step is calculated for each type of the single-stranded nucleic acid, and the number of molecules of each single-stranded nucleic acid is estimated based on each difference. A single-stranded nucleic acid convergence determination step for determining whether the result has converged, and
A program for estimating the number of single-stranded nucleic acid molecules. Information indicating a separation rate coefficient, which is a rate at which a single-stranded nucleic acid is separated from a double-stranded nucleic acid, is stored for each type of the double-stranded nucleic acid, the number of molecules of the single-stranded nucleic acid, The nucleic acid that is the sum of the number of molecules of a single-stranded nucleic acid that can be combined with a single-stranded nucleic acid to form a double-stranded nucleic acid and the number of molecules of a double-stranded nucleic acid in which the single-stranded nucleic acid and the single-stranded nucleic acid are combined A computer as a total molecular number estimation device comprising a total molecular number storage unit storing information indicating the total number of molecules for each type of single-stranded nucleic acid,
The information indicating the separation rate coefficient is read from the separation rate coefficient storage unit, the information indicating the total number of molecules is read from the total molecule number storage unit, the read separation rate coefficient and the total number of molecules read out A single-stranded nucleic acid molecule number obtaining step for estimating the number of single-stranded nucleic acid molecules present in the solution for each type of single-stranded nucleic acid by the single-stranded nucleic acid molecule number estimation program according to claim 14,
A single-stranded nucleic acid difference calculating step for calculating, for each type of single-stranded nucleic acid, a difference between the estimated number of molecules of the single-stranded nucleic acid and the known number of molecules of the single-stranded nucleic acid input from the outside of the device; ,
Based on the difference for each type of the single-stranded nucleic acid, a total molecular number convergence determination step for determining whether or not the estimation result of the total number of molecules of the nucleic acid fragment has converged;
When it is determined that the total molecular number convergence determination step has not converged, information indicating each of the separation speed coefficients is read from the separation speed coefficient storage unit, and each of the read separation speed coefficients and the first unit Based on the difference of the second single-stranded nucleic acid that can form a double-stranded nucleic acid by binding to a strand nucleic acid, the total number of nucleic acid fragments having at least one first single-stranded nucleic acid is calculated as the single-stranded nucleic acid. A total molecule number correction step for updating the information indicating the total number of molecules stored in the total molecule number storage unit with information indicating the corrected total number of molecules,
A total molecular number estimation program for executing
The single-stranded nucleic acid molecule number acquisition step reads information indicating the total number of molecules updated by the total number-of-molecules correction procedure from the total number-of-molecules storage unit, and based on the read total number of molecules, A program for estimating the total number of molecules, which causes the single-stranded nucleic acid molecule number estimation apparatus to estimate the number of molecules for each type of single-stranded nucleic acid. In the computer as the separation speed coefficient calculation device,
A request to estimate the number of molecules of a nucleic acid fragment for each type of single-stranded nucleic acid based on each of the separation rate coefficients, which are the rates at which single-stranded nucleic acids are released from double-stranded nucleic acids, and the known number of molecules of each single-stranded nucleic acid Item 15. The total molecular number estimation program according to Item 15,
The number of single-stranded nucleic acid molecules according to claim 14, wherein the number of single-stranded nucleic acids is estimated for each type of single-stranded nucleic acid based on each of the separation rate coefficients and the estimated number of molecules of each nucleic acid fragment. Program and
Based on the estimated number of molecules of each single-stranded nucleic acid, a control step for calculating the separation rate coefficient for each type of the double-stranded nucleic acid,
Detachment speed coefficient calculation program for executing

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