A method for detecting a trinucleotide repeat of a genetic disease with dynamic mutation

Abstract

The application discloses a genetic disease trinucleotide repeat detection method of dynamic mutation and belongs to the technical field of genetic disease molecular detection. Double-stranded DNA of a sample to be detected is obtained and asymmetric amplification is carried out, and a base polarity map is constructed according to the electrophoretic mobility offset of the amplification product; the fluctuation range of the repeat interval is determined according to the polarity offset index corresponding to the base composition type in the repeat unit, the amplification product in the range is screened for forward sequencing and reverse sequencing, and a forward sequencing signal peak map and a reverse sequencing signal peak map are obtained; a complementarity deviation spectrum is constructed according to the forward sequencing signal peak map and the reverse sequencing signal peak map, the trinucleotide repeat number is determined according to the length of the continuous deviation interval in the complementarity deviation spectrum, and the mutation state of the sample to be detected is determined according to the threshold relationship of the trinucleotide repeat number. The method can reduce the interference of non-repeat fragments and the influence of one-way sequencing signal sliding, and more accurate repeat number determination results are obtained.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology for genetic diseases, specifically a method for detecting trinucleotide repeats in dynamically mutated genetic diseases. Background Technology

[0002] Dynamic mutation-related genetic diseases are caused by abnormal amplification of trinucleotide repeat units in specific genes. The accuracy of repeat count determination directly affects the differentiation between normal, pre-mutant, and fully mutant types. Currently, routine techniques for trinucleotide repeat detection include capillary electrophoresis after polymerase chain reaction (PCR), Southern blotting, and Sanger sequencing. PCR-PCR capillary electrophoresis indirectly estimates the repeat count based on product fragment length. However, the electrophoretic mobility of trinucleotide repeat fragments is affected not only by length but also by base composition, secondary structure, and repeat sequence arrangement. Fragments with similar lengths but different sequence compositions may exhibit peak overlap or shift in the electrophoretic spectrum, making it difficult to determine the true boundaries of repeat regions solely based on fragment length. Southern blotting can detect larger repeat amplifications, but the procedure is cumbersome, has limited resolution of fragment length differences, and is prone to genotyping errors in low-abundance samples and samples with repeat counts close to the critical threshold. Sanger sequencing can provide base signals for repetitive regions and flanking sequences, but the low complexity of repetitive sequences easily leads to polymerase slippage, chain termination reaction imbalance, and the formation of local secondary structures, resulting in sliding peaks, overlapping peaks, and signal attenuation in the sequencing signal. Unidirectional sequencing peak patterns often fail to reliably read the number of repeat units in repetitive regions. When detecting high-GC-content repetitive sequences, long repetitive amplifications, or samples with chimeric states, the above methods exhibit more significant biases in repetitive region localization and repetition count readings. When the fluctuation range corresponding to the repetitive region in complex electrophoresis patterns is not effectively separated, fragments entering subsequent sequencing may contain non-repetitive regions, interfering with the counting results. When relying solely on unidirectional sequencing signals, base readout offsets within repetitive regions cannot be corrected by complementary directions, making it difficult to stably determine the start and end positions and lengths of continuous repetitive intervals. In the detection of trinucleotide repeats in dynamic mutant genetic diseases, it is necessary to reduce the interference of non-repetitive regions in complex electrophoresis patterns on subsequent sequencing and improve the counting bias caused by unidirectional sequencing signal slippage within repetitive regions. Summary of the Invention

[0003] To address the challenges of locating repeat regions and the susceptibility of repeat counts to sequencing signal slippage in the detection of trinucleotide repeats in dynamically mutated genetic diseases, this method provides a novel approach for detecting trinucleotide repeats in dynamically mutated genetic diseases. This method constructs a base polarity map from the electrophoretic mobility shift of asymmetric amplification products. The fluctuation range of repeat regions is determined by the polarity shift index corresponding to the base composition type within the repeat unit. Products falling within this range undergo forward and reverse sequencing. A complementarity deviation spectrum is constructed using the forward and reverse sequencing signal peak diagrams. The number of trinucleotide repeats is determined based on the length of consecutive deviation regions, and the mutation status of the sample is determined according to a threshold relationship.

[0004] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a method for detecting trinucleotide repeats in dynamically mutated genetic diseases, comprising: acquiring double-stranded DNA of a sample to be tested; performing asymmetric amplification on the double-stranded DNA to obtain asymmetric amplification products; constructing a base polarity map based on the electrophoretic mobility shift of the asymmetric amplification products; determining the fluctuation range of the repeat interval based on the polarity shift index corresponding to the base composition type within the repeat unit in the base polarity map; screening the asymmetric amplification products within the fluctuation range; performing forward and reverse sequencing on the screened asymmetric amplification products to obtain forward sequencing signal peak diagrams and reverse sequencing signal peak diagrams; constructing a complementarity deviation spectrum based on the forward sequencing signal peak diagrams and the reverse sequencing signal peak diagrams; determining the number of trinucleotide repeats based on the length of continuous deviation intervals in the complementarity deviation spectrum; and determining the mutation status of the sample to be tested based on the relationship between the number of trinucleotide repeats and a threshold. This method utilizes asymmetric amplification and electrophoretic mobility shift characteristics to obtain a base polarity map, and then combines the differences between forward and reverse sequencing signals to construct a complementary deviation spectrum, which can reliably identify repeat regions and accurately calculate the number of trinucleotide repeats.

[0005] As a technical solution of the present invention, when performing asymmetric amplification of the double-stranded DNA, forward and reverse primers are designed, with the annealing temperature of the forward primer being higher than that of the reverse primer. Using the double-stranded DNA as a template, a polymerase chain reaction (PCR) is performed using the forward and reverse primers. During the PCR cycle, the annealing temperature is gradually reduced from a high temperature to a low temperature, allowing the forward primer to preferentially bind to the template strand. After the exponential growth phase of the PCR, the extension time is further increased to allow excessive accumulation of the single-strand extension product from the reverse primer, resulting in asymmetric amplification products. By controlling the difference in annealing temperature and the extension time, asymmetric amplification products enriched with single-stranded products can be obtained, which is beneficial for improving the discriminative power of subsequent migration rate analysis.

[0006] Preferably, when constructing a base polarity map based on the electrophoretic mobility shift of the asymmetric amplification product, the asymmetric amplification product is separated by capillary electrophoresis, the migration time of each fragment in the capillary is detected, and the mobility is calculated based on the migration time; the original electrophoretic spectrum of the asymmetric amplification product is plotted with the mobility benchmark value as the abscissa and the detection signal intensity as the ordinate; the deviation between the measured mobility value and the theoretical mobility value is calculated based on the measured mobility value corresponding to each signal peak in the original electrophoretic spectrum to obtain the mobility shift; and a base polarity map is constructed based on the positive and negative directions and the magnitude of the mobility shift. This base polarity map can reflect the mobility deviation caused by changes in the base composition of repeating units, providing a basis for the identification of repeating intervals.

[0007] Preferably, when determining the fluctuation range of the repeat interval based on the polarity shift index corresponding to the base composition type within the repeating unit in the base polarity map, the base composition type of each repeating unit is extracted from the base polarity map, and the base composition type includes the base sequence arrangement of the trinucleotide repeating unit; the polarity shift index of the repeating unit is calculated based on the distribution ratio of purine bases and pyrimidine bases in the base composition type; a polarity shift sequence is constructed using the polarity shift indices of several consecutive repeating units, and the local extreme points of the polarity shift sequence are obtained; the numerical span between adjacent local maxima and local minima in the polarity shift sequence is determined as the fluctuation range of the repeat interval. Using the extreme value changes of the polarity shift index to determine the fluctuation range allows for a more accurate delineation of the repeat interval boundary.

[0008] Preferably, the asymmetric amplification products within the fluctuation range are screened. When performing forward and reverse sequencing on the screened asymmetric amplification products to obtain forward and reverse sequencing signal peak diagrams, amplification fragments whose electrophoretic mobility shift falls within the fluctuation range are selected from the asymmetric amplification products as the fragments to be sequenced. The fragments to be sequenced are divided into a first group and a second group. The first group is sequenced using forward primers to obtain the original forward sequencing signal; the second group is sequenced using reverse primers to obtain the original reverse sequencing signal. The original forward sequencing signals are arranged according to base position coordinates to generate a forward sequencing signal peak diagram; the original reverse sequencing signals are also arranged according to base position coordinates to generate a reverse sequencing signal peak diagram. By limiting the sequencing fragment range and performing bidirectional sequencing, interference from non-repetitive regions can be reduced, and complementary signals on both sides of the repetitive regions can be obtained.

[0009] Preferably, when constructing a complementarity deviation spectrum based on the forward sequencing signal peak diagram and the reverse sequencing signal peak diagram, the base positions in the forward sequencing signal peak diagram and the base positions in the reverse sequencing signal peak diagram are aligned by base complementarity pairing to obtain the correspondence of aligned base positions; the forward signal intensity value and the reverse signal intensity value corresponding to each aligned base position are extracted; the intensity difference between the forward signal intensity value and the reverse signal intensity value at the same base position is calculated; an intensity difference sequence curve is plotted with the base position coordinates as the horizontal axis and the intensity difference value as the vertical axis, and the intensity difference sequence curve is used as the complementarity deviation spectrum. The complementarity deviation spectrum highlights the consistency deviation of forward and reverse sequencing in repetitive regions, which can improve the anti-interference ability of repetition count calculation.

[0010] Preferably, when determining the number of trinucleotide repeats based on the length of consecutive deviation intervals in the complementarity deviation spectrum, the complementarity deviation spectrum is traversed, and the intensity difference corresponding to each base position is determined sequentially according to the base position to see if it is greater than the deviation threshold. Base positions consecutively greater than the deviation threshold are divided into deviation intervals, and the starting base position, ending base position, and interval length of each deviation interval are recorded. The deviation interval with the largest interval length among all deviation intervals is selected as the target deviation interval. The interval length of the target deviation interval is divided by three to obtain the number of trinucleotide repeats. This allows the deviation signal length to be directly converted into the number of trinucleotide repeats, reducing errors caused by slippage within the repeat sequence.

[0011] Preferably, when determining the mutation status of a sample based on the relationship between the number of trinucleotide repeats and a threshold, the upper limit threshold for the normal repeat count and the lower limit threshold for the pathogenic repeat count corresponding to the genetic disease being tested are obtained. If the number of trinucleotide repeats is less than or equal to the upper limit threshold for the normal repeat count, the sample is determined to be normal. If the number of trinucleotide repeats is greater than the upper limit threshold for the normal repeat count but less than the lower limit threshold for the pathogenic repeat count, the sample is determined to be pre-mutant. If the number of trinucleotide repeats is greater than or equal to the lower limit threshold for the pathogenic repeat count, the sample is determined to be fully mutant. This typing method corresponds to the clinical thresholds of dynamically mutating genetic diseases and can directly provide the mutation status of the sample.

[0012] As a technical solution of the present invention, the migration benchmark value is the fitted curve value of the theoretical migration rate of each known fragment in the standard molecular weight marker. Using the fitted curve of the standard molecular weight marker as a benchmark can reduce the systematic deviation between different electrophoresis batches.

[0013] As a technical solution of the present invention, the polarity shift index is calculated based on the ratio of the total content of adenine and thymine to the total content of guanine and cytosine in the base composition type. This index can quantify the base polarity difference of repeating units and improve the resolution of base polarity maps.

[0014] The technical effects and advantages provided by the present invention in the above technical solution are as follows:

[0015] Asymmetric amplification of double-stranded DNA leads to the over-accumulation of the target single-stranded product. During capillary electrophoresis separation, the migration shift is obtained based on the deviation between the measured and theoretical migration values ​​of each fragment. A base polarity map is constructed from the positive and negative directions and amplitude of the shift. The polarity shift index is calculated based on the distribution ratio of purine and pyrimidine bases within the repeat unit. Sequences are formed using the polarity shift indices of consecutive repeat units, and local maxima and minima are obtained. The numerical span between adjacent extreme values ​​is defined as the fluctuation range of the repeat interval. This method delineates repeat segments through sequence composition-dependent migration shift characteristics, avoiding boundary misjudgments caused by overlapping fragments of similar length when relying solely on fragment length to determine repeat intervals. It can separate segments corresponding to the repeat sequence composition from complex electrophoretic patterns, reducing the number of non-repeatable amplified fragments entering subsequent sequencing steps and concentrating the sequencing fragments more within trinucleotide repeat regions. The selected amplified fragments were subjected to forward and reverse sequencing, respectively. The base positions in the forward and reverse sequencing signal peak diagrams were aligned for complementary pairing. The difference between the forward and reverse signal intensities at the same base position was calculated, and a complementarity deviation spectrum was plotted with the base position coordinates on the x-axis and the intensity difference on the y-axis. The spectrum was then traversed, and base positions with consecutive deviations exceeding a threshold were classified as deviation intervals. The longest deviation interval was selected as the target deviation interval, and its length was divided by three to obtain the trinucleotide repeat count. This method transforms the intensity difference between forward and reverse sequencing signals at the same base position into a deviation interval. The length of consecutive deviation intervals reflects the expansion range of the trinucleotide repeat, reducing the impact of unidirectional sequencing signal slippage, peak shape disorder, and signal attenuation on the readings. The determination of the repeat count no longer relies solely on the peak spacing or peak height in a single direction, and can obtain repeat count calculation results consistent with the complementary relationship between forward and reverse signals. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0017] Figure 1This is a flowchart illustrating a method for detecting trinucleotide repeats in a dynamically mutated genetic disease.

[0018] Figure 2 This is a flowchart of the asymmetric amplification method for trinucleotide repeat regions;

[0019] Figure 3 The curves show the change in fluorescence intensity of the single-strand extension products of the forward and reverse primers as a function of the number of polymerase chain reaction cycles.

[0020] Figure 4 It is the migration shift and base polarity map of asymmetric amplification products by capillary electrophoresis;

[0021] Figure 5 This is a schematic diagram of the polarity shift index of repeating units and the fluctuation range of repeating intervals;

[0022] Figure 6 This is a schematic diagram of the complementarity deviation spectrum and the target deviation interval. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] See Figure 1 This invention provides a method for detecting trinucleotide repeats in dynamically mutated genetic diseases, comprising: acquiring double-stranded DNA of the sample to be tested; performing asymmetric amplification on the double-stranded DNA to obtain asymmetric amplification products; constructing a base polarity map based on the electrophoretic mobility shift of the asymmetric amplification products; determining the fluctuation range of the repeat interval based on the polarity shift index corresponding to the base composition type within the repeat unit in the base polarity map; screening asymmetric amplification products within the fluctuation range; performing forward and reverse sequencing on the screened asymmetric amplification products to obtain forward sequencing signal peak diagrams and reverse sequencing signal peak diagrams; constructing a complementarity deviation spectrum based on the forward and reverse sequencing signal peak diagrams; determining the number of trinucleotide repeats based on the length of the continuous deviation intervals in the complementarity deviation spectrum; and determining the mutation status of the sample to be tested based on the relationship between the number of trinucleotide repeats and a threshold.

[0025] In specific implementation, please refer to Figure 2After obtaining the double-stranded DNA from the sample to be tested, asymmetric amplification was performed. Forward and reverse primers were designed. The binding sites of the forward and reverse primers were selected based on the flanking sequences of the trinucleotide repeat region of the genetic disease to be tested. The annealing temperature of the forward primer was higher than that of the reverse primer. The difference between the annealing temperatures of the forward and reverse primers was set to 5°C to 15°C. The annealing temperatures of the forward and reverse primers were calculated using nearest-neighbor thermodynamic parameters. The length of both the forward and reverse primers was set to 18 to 25 bases. The guanine and cytosine content of both the forward and reverse primers was 40% to 60%.

[0026] In the preparation of the polymerase chain reaction (PCR) system, double-stranded DNA was used as a template, and the forward primer, reverse primer, DNA polymerase, deoxyribonucleoside triphosphate, and reaction buffer were mixed. The final concentration of the forward primer was 0.2 μmol / L, and the final concentration of the reverse primer was 0.02 μmol / L. The molar ratio of the forward primer to the reverse primer was 10:1. The PCR system was placed in a thermal cycler.

[0027] In the cyclic process of polymerase chain reaction (PCR), the initial annealing temperature is set as the annealing temperature of the forward primer. The thermal cycler executes annealing stages according to a preset annealing temperature sequence. The first temperature value in the annealing temperature sequence is the annealing temperature of the forward primer, and the last temperature value is the annealing temperature of the reverse primer. The difference between any two adjacent temperature values ​​is 0.5°C to 2°C. In each annealing stage, the annealing temperature is maintained for 30 to 60 seconds. Under the initial annealing temperature conditions, the forward primer binds to the template strand, while the reverse primer does not. As the annealing temperature gradually decreases, the reverse primer binds to the template strand when the annealing temperature reaches its own annealing temperature. The forward primer preferentially binds to the template strand, and the extension product of the forward primer accumulates in the early stages of the PCR.

[0028] After the exponential growth phase of the polymerase chain reaction (PCR) ends, PCR cycles are continued to extend the extension time. The exponential growth phase is defined as the number of PCR cycles reaching a preset number, set at 25 to 30 cycles. After the exponential growth phase, the extension time is extended from the initial extension time to 2 to 5 times the initial extension time. The initial extension time is set based on the length of the trinucleotide repeat region in the test sample. During the extension phase, both the forward and reverse primer single-strand extension products are synthesized. The reverse primer single-strand extension product accumulates excessively during the extension phase. The asymmetric amplification product contains excessive accumulations of both the reverse and forward primer single-strand extension products.

[0029] See Figure 3 The graph shows the polymerase chain reaction (PCR) cycle number on the horizontal axis and the relative fluorescence unit value on the vertical axis, reflecting the cumulative trends of the forward and reverse primer single-strand extension products, respectively. The solid blue line with squares represents the fluorescence signal of the forward primer single-strand extension product, and the dashed red line with dots represents the fluorescence signal of the reverse primer single-strand extension product. The vertical green dashed line indicates the preset cycle number, and the vertical orange dotted line indicates the start of the extension time extension phase.

[0030] During the initial stage of the polymerase chain reaction up to the preset number of cycles (approximately 25 to 30 cycles), the fluorescence signal of the forward primer single-strand extension product rapidly increases, exhibiting an exponential growth trend, and reaches a plateau at approximately cycle number 26, with the fluorescence intensity stabilizing at around 6000 relative fluorescence units. During this stage, the fluorescence signal of the reverse primer single-strand extension product remains at a low level, without significant accumulation.

[0031] After reaching the preset number of cycles (green dashed line), the extension time extension phase begins (orange dotted line). The fluorescence signal of the reverse primer single-strand extension product then rises rapidly, exhibiting an exponential growth trend, and reaches a peak of approximately 10,000 relative fluorescence units around 40 cycles. At the same time, the fluorescence signal of the forward primer single-strand extension product remains stable without significant growth, indicating that the forward primer extension product has entered a plateau phase.

[0032] In practice, asymmetric amplification products are separated by capillary electrophoresis. The asymmetric amplification products are mixed with standard molecular weight markers containing deoxyribonucleic acid fragments of known length. The mixed asymmetric amplification products and standard molecular weight markers are injected into the capillary of a capillary electrophoresis apparatus. A high-voltage DC electric field is applied across the capillary, causing the fragments in the asymmetric amplification products to migrate within the capillary. The detector records the migration time of each fragment at the detection window of the capillary. The migration rate is calculated based on the migration time.

[0033] The mobility is calculated using the following formula:

[0034]

[0035] in, This indicates the migration rate of a fragment in the asymmetric amplification product, expressed in square centimeters per volt-second. This indicates the effective length of the capillary from the sample inlet to the detection window, in centimeters. This indicates the migration time of the segment within the capillary, in seconds; This represents the electric field strength inside the capillary tube, measured in volts per centimeter. The electric field strength is obtained by dividing the voltage applied across the capillary tube by the total length of the capillary tube.

[0036] Using the migration baseline value as the x-axis and the detection signal intensity as the y-axis, the original electrophoresis spectrum of the asymmetric amplification products was plotted. The migration baseline value is the theoretical migration rate fitted curve value of each known fragment in the standard molecular weight marker. To obtain the migration baseline value, the migration time of each known fragment in the standard molecular weight marker in capillary electrophoresis was substituted into the migration rate calculation formula to obtain the measured migration rate value of each known fragment. Based on the deoxyribonucleic acid length of each known fragment and the measured migration rate value, the least squares method was used to fit the theoretical migration rate fitted curve. The deoxyribonucleic acid length corresponding to each fragment in the asymmetric amplification product was substituted into the theoretical migration rate fitted curve to obtain the theoretical migration rate value corresponding to each fragment. This theoretical migration rate value was used as the migration baseline value. In the original electrophoresis spectrum, the migration baseline values ​​on the x-axis are arranged in ascending order, and the detection signal intensity on the y-axis is the relative fluorescence unit value detected by the fluorescence detector.

[0037] Based on the measured mobility values ​​corresponding to each signal peak in the original electrophoresis spectrum, the deviation between the measured mobility values ​​and the theoretical mobility values ​​is calculated to obtain the mobility shift. The measured mobility value corresponding to the peak apex position of each signal peak is extracted from the original electrophoresis spectrum. The measured mobility value of each signal peak is subtracted from the theoretical mobility value of the corresponding segment; the difference is the mobility shift of that signal peak. The absolute value of the mobility shift is the magnitude of the deviation between the measured and theoretical mobility values, and the sign of the mobility shift indicates the direction of deviation.

[0038] A base polarity map is constructed based on the positive and negative directions and the magnitude of the mobility shifts. The mobility shifts of each signal peak in the original electrophoresis spectrum are arranged in ascending order of their baseline mobility values. A positive mobility shift is recorded as positive polarity in the base polarity map, indicating that the fragment corresponding to that signal peak has a higher proportion of purine bases than pyrimidine bases. A negative mobility shift is recorded as negative polarity, indicating that the fragment corresponding to that signal peak has a higher proportion of pyrimidine bases than purine bases. The larger the absolute value of the mobility shift, the greater the difference in the proportion of purine and pyrimidine bases in the fragment corresponding to that signal peak. The positive and negative directions and the magnitude of the mobility shifts for each signal peak are recorded in the base polarity map, which includes a sequence of positive and negative mobility shifts and a sequence of mobility shift magnitudes.

[0039] See Figure 4In the figure, the horizontal axis represents the baseline mobility value, measured in square centimeters per volt-second, ranging from approximately 0.00005 to 0.00030. The vertical axis represents the detection signal intensity, measured in relative fluorescence units. The solid black line in the figure represents the original electrophoresis spectrum, reflecting the trend of the detection signal intensity of each fragment in the asymmetric amplification product as a function of the baseline mobility value. The dashed blue line represents the theoretical mobility fitting curve of the standard molecular weight marker. The peak of the marker corresponds to a known length of deoxyribonucleic acid fragment, with clear peak shape and uniform distribution, serving as a reference for the mobility baseline. The dashed red line represents the background signal intensity of the asymmetric amplification product.

[0040] In the figure, the green triangles and orange inverted triangles mark the positive and negative polarity signal peaks of the mobility shift, respectively. The signal peak corresponding to the green triangle shows a positive mobility shift, indicating that the proportion of purine bases in this fragment is higher than that of pyrimidine bases, while the signal peak corresponding to the orange inverted triangle shows a negative mobility shift, indicating that the proportion of pyrimidine bases is higher than that of purine bases. The alternating distribution of positive and negative polarity signal peaks reflects the periodic changes in base composition.

[0041] As shown in the figure, the signal peaks exhibit multiple distinct peaks, with peaks corresponding to the mobility baseline values ​​of approximately 0.00007, 0.00010, 0.00015, 0.00018, 0.00020, and 0.00026, respectively. Some peaks are marked with polarity signs. The high kurtosis of the absolute value of the mobility shift indicates a significant difference in the ratio of purines to pyrimidines in the base composition of the corresponding fragment, consistent with the base composition characteristics within a trinucleotide repeat unit.

[0042] In practice, the base composition type of each repeating unit is extracted from the base polarity map. The base polarity map includes the positive and negative direction sequences of the mobility shift and the magnitude sequence of the mobility shift. Starting from the initial signal peak of the base polarity map, every three consecutive signal peaks are divided into a repeating unit. The order of the three consecutive signal peaks is the first, second, and third base of the trinucleotide repeating unit. Based on the positive and negative polarity of the signal peaks and the magnitude of the signal peak shift in the base polarity map, the base type corresponding to each signal peak is determined. The base composition type includes the base sequence arrangement of the trinucleotide repeating unit, specifically the arrangement order of the three consecutive bases.

[0043] In some embodiments, positive polarity signal peaks in the base polarity spectrum correspond to purine bases, and negative polarity signal peaks correspond to pyrimidine bases. Purine bases include adenine and guanine, and pyrimidine bases include thymine and cytosine. The shift amplitude of adenine differs from that of guanine, and the shift amplitude of thymine differs from that of cytosine. By comparing the shift amplitude of signal peaks in the base polarity spectrum with known base shift amplitude reference values ​​obtained from standard base samples under the same capillary electrophoresis conditions, the specific base type corresponding to each signal peak is determined. The base composition type of each trinucleotide repeat unit is obtained by grouping three consecutive signal peaks together.

[0044] The polarity shift index of repeating units is calculated based on the distribution ratio of purine and pyrimidine bases in the base composition type. The polarity shift index is calculated as the ratio of the total content of adenine and thymine to the total content of guanine and cytosine in the base composition type. The formula for calculating the polarity shift index is as follows:

[0045]

[0046] in, This represents the polarity shift index, which is a dimensionless value. This indicates the amount of adenine in a trinucleotide repeat unit. The value range is 0, 1, 2, 3; This indicates the number of thymines in a trinucleotide repeat unit. The value range is 0, 1, 2, 3; This indicates the amount of guanine in a trinucleotide repeat unit. The value range is 0, 1, 2, 3; This indicates the number of cytosine residues in a trinucleotide repeat unit. The value range is 0, 1, 2, 3. The total number of adenine, thymine, guanine, and cytosine in a trinucleotide repeat unit is 3, i.e. .when When it is not equal to 0, calculate the polarity shift index according to the above formula. When the value is 0, the polarity shift index is set to 3.0. The basis for setting the polarity shift index to 3.0 is that the total number of bases in the trinucleotide repeat unit is 3. When the total content of guanine and cytosine is 0, the total content of adenine and thymine is 3. The polarity shift index of 3, representing the total content of adenine and thymine relative to the total number of bases in the trinucleotide repeat unit, indicates the status of the total content of adenine and thymine relative to the total number of bases in the trinucleotide repeat unit.

[0047] A polar shift sequence is constructed using the polar shift indices of a series of consecutive repeating units. In practice, following the order of repeating units in the base polarity map, starting with the first repeating unit, the polar shift indices of five consecutive repeating units are taken sequentially to form the polar shift sequence. The number of consecutive repeating units is set to five because the length of these five repeating units covers at least one local polarity change cycle within the trinucleotide repeat interval. This avoids the situation where too few repeating units would cause fluctuations in the polar shift index of a single repeating unit to affect the detection of local extrema, while also avoiding the situation where too many repeating units would cause the positions of local extrema to deviate from the actual boundaries of the repeat interval. If the total number of repeating units in the base polarity map is less than five, the polar shift indices of all repeating units are used to construct the polar shift sequence. The elemental order in the polar shift sequence is consistent with the order of the repeating units in the base polarity map.

[0048] Obtain the local extrema of the polarity shift sequence. In specific implementation, calculate the difference between two adjacent polarity shift indices in the polarity shift sequence to obtain the first-order difference sequence. The first-order difference sequence contains the... The difference value is the first value in the polarity shift sequence. The polarity offset index minus the first The polarity shift index. When the first-order difference sequence contains the... The sign of the nth difference value is positive and the nth difference value is positive. When the sign of the difference value is negative, the polarity shift sequence will be... The position corresponding to the polarity shift index is taken as a local maximum point. When the first-order difference sequence contains the... The sign of the nth difference value is negative and the nth difference value is negative. When the sign of the first difference value is positive, the polarity shift sequence will be... The positions corresponding to each polarity shift index are designated as local minima. If a term with a difference value of zero exists in the first-order difference sequence, it is skipped when determining the sign change, and the nearest non-zero difference value before and after the term with a difference value of zero is used for sign change determination. For the starting position of the polarity shift sequence, if the first polarity shift index is greater than the second polarity shift index, the position corresponding to the first polarity shift index is designated as a local maximum; if the first polarity shift index is less than the second polarity shift index, the position corresponding to the first polarity shift index is designated as a local minimum. For the ending position of the polarity shift sequence, if the last polarity shift index is greater than the second-to-last polarity shift index, the position corresponding to the last polarity shift index is designated as a local maximum; if the last polarity shift index is less than the second-to-last polarity shift index, the position corresponding to the last polarity shift index is designated as a local minimum. The polarity shift sequence is traversed, and the positions of all local maxima and local minima are recorded.

[0049] The numerical span between adjacent local maxima and local minima in the polarity shift sequence is defined as the fluctuation range of the repeating interval. In practice, adjacent local maxima and local minima are sequentially obtained according to their positional order in the polarity shift sequence. When a local maximum precedes a local minimum, the numerical span is the absolute value of the difference between the polarity shift index corresponding to the local maximum and the polarity shift index corresponding to the local minimum. When a local minimum precedes a local maximum, the numerical span is the absolute value of the difference between the polarity shift index corresponding to the local minimum and the polarity shift index corresponding to the local maximum. This numerical span is defined as the fluctuation range of the repeating interval. Simultaneously, the starting and ending repeating unit positions corresponding to the fluctuation range of the repeating interval are recorded. The starting repeating unit position is the repeating unit number corresponding to the earlier of the adjacent local maxima and local minima, and the ending repeating unit position is the repeating unit number corresponding to the later of the adjacent local maxima and local minimum. If multiple adjacent local maxima and local minima exist, then the fluctuation range of multiple repeating intervals is obtained. The fluctuation range of the repeating intervals includes the upper limit and lower limit of the polarity shift index.

[0050] See Figure 5 In the figure, the horizontal axis represents the repeating unit number, and the vertical axis represents the polarity shift index. The blue continuous curve shows the trend of the polarity shift index with the repeating unit number, reflecting the polarity variation characteristics of the base composition type of each repeating unit within the trinucleotide repeating interval. The curve exhibits periodic fluctuations, showing the periodic variation of the polarity shift index of the repeating unit base composition in spatial distribution.

[0051] In the figure, local maxima are marked with red triangles, and local minima are marked with green inverted triangles. Local maxima and minima alternate, forming distinct peaks and troughs, reflecting the upward and downward fluctuations in the polarity shift sequence. Local maxima correspond to local peaks in the polarity shift index, indicating a relatively high content of purine bases in the repeating unit at that position; local minima correspond to local troughs in the polarity shift index, indicating a relatively high content of pyrimidine bases in the repeating unit at that position.

[0052] The orange dashed line represents the upper limit of the fluctuation range of the repeating interval, with a value of approximately 2.47; the purple dashed line represents the lower limit of the fluctuation range of the repeating interval, with a value of approximately 0.52. The polarity shift index curve shows most fluctuations within this upper and lower limit range, indicating that the polarity changes in the base composition of the repeating interval are confined to this fluctuation range. This range is determined by the numerical span between adjacent local maxima and local minima, reflecting the fluctuation amplitude and boundaries of the repeating interval.

[0053] In practice, amplified fragments whose electrophoretic mobility shifts fall within the fluctuation range of the repeat interval are selected from the asymmetric amplification products and designated as the sequencing fragments. The fluctuation range of the repeat interval is determined by the numerical span between adjacent local maxima and local minima. The electrophoretic mobility shift values ​​of each amplified fragment in the asymmetric amplification product are compared one by one with the lower and upper limits of the fluctuation range of the repeat interval. Amplified fragments whose electrophoretic mobility shift values ​​are greater than or equal to the lower limit of the fluctuation range of the repeat interval and less than or equal to the upper limit of the fluctuation range of the repeat interval are selected as the sequencing fragments.

[0054] In the specific implementation, the fragments to be sequenced are divided into two groups. The fragments are equally divided into two groups according to their molar quantity. The total amount of fragments in the first group is the same as that in the second group. For the first group, a forward primer is used for sequencing to obtain the raw forward sequencing signal. For the second group, a reverse primer is used for sequencing to obtain the raw reverse sequencing signal. The sequencing reaction employs deoxyribonucleic acid polymerase extension.

[0055] In practice, the raw forward sequencing signals are arranged according to their base position coordinates to generate a forward sequencing signal peak diagram. The base position coordinates in the forward sequencing signal peak diagram are numbered consecutively with natural numbers, starting from the first base at the start of the forward sequencing reaction. Similarly, the raw reverse sequencing signals are arranged according to their base position coordinates to generate a reverse sequencing signal peak diagram. The base position coordinates in the reverse sequencing signal peak diagram are numbered consecutively with natural numbers, starting from the first base at the start of the reverse sequencing reaction.

[0056] In practice, a complementarity offset spectrum is constructed based on the forward and reverse sequencing signal peak diagrams. The base positions in the forward and reverse sequencing signal peak diagrams are aligned using complementary base pairing to obtain the corresponding alignment. During this alignment process, the relative base position offset between the forward and reverse sequencing signal peak diagrams is calculated based on the binding positions of the forward and reverse primers on the sequencing fragment. The first base position in the reverse sequencing signal peak diagram is aligned with its corresponding base position in the forward sequencing signal peak diagram, such that adenine in the reverse sequencing signal peak diagram corresponds to thymine in the forward sequencing signal peak diagram, thymine in the reverse sequencing signal peak diagram corresponds to adenine in the forward sequencing signal peak diagram, guanine in the reverse sequencing signal peak diagram corresponds to cytosine in the forward sequencing signal peak diagram, and cytosine in the reverse sequencing signal peak diagram corresponds to guanine in the forward sequencing signal peak diagram. The correspondence between the aligned base positions is recorded as the correspondence between the base position numbers of each pair of forward sequencing signal peaks and the base position numbers of the reverse sequencing signal peaks.

[0057] In practice, the forward and reverse signal intensity values ​​corresponding to each aligned base position are extracted. The forward signal intensity value is the peak height of the corresponding base position in the forward sequencing signal peak diagram. The reverse signal intensity value is the peak height of the corresponding base position in the reverse sequencing signal peak diagram. The peak height is the value obtained by subtracting the baseline of the signal peak from the maximum relative fluorescence unit recorded by the detector.

[0058] In practice, the intensity difference between the forward and reverse signal strengths at the same base position is calculated. The formula for calculating the intensity difference is as follows:

[0059]

[0060] in, It represents the intensity difference between the forward and reverse signal intensity values ​​at the same base position, and the unit is relative fluorescence units; The positive signal intensity value in the positive sequencing signal peak diagram at the same base position is expressed in relative fluorescence units. This represents the reverse signal intensity value in the reverse sequencing signal peak diagram at the same base position, expressed in relative fluorescence units.

[0061] In practice, an intensity difference sequence curve is plotted with base position coordinates on the horizontal axis and intensity difference values ​​on the vertical axis. Base position coordinates are continuously taken starting from the initial base position of the aligned forward sequencing signal peak. The intensity difference at each base position is plotted as a point on the intensity difference sequence curve. The intensity differences at all base positions are sorted by their base position coordinates and then connected to form the intensity difference sequence curve. This intensity difference sequence curve is used as the complementarity deviation spectrum.

[0062] In practice, the number of trinucleotide repeats is determined based on the length of consecutive deviation intervals in the complementarity deviation spectrum. The complementarity deviation spectrum is traversed, and the intensity difference corresponding to each base position is determined sequentially to see if it exceeds the deviation threshold. The deviation threshold is obtained by selecting the intensity difference sequence corresponding to the non-repetitive interval in the complementarity deviation spectrum, calculating the standard deviation of the intensity difference sequence for the non-repetitive interval, and setting the deviation threshold to three times the standard deviation of the intensity difference sequence for the non-repetitive interval. The basis for setting the deviation threshold to three times the standard deviation of the intensity difference sequence for the non-repetitive interval is that the intensity difference between the positive and negative signal intensity values ​​within the non-repetitive interval is caused by random sequencing error. Random sequencing error follows a normal distribution, and three times the standard deviation corresponds to a 99.7% confidence range; intensity differences exceeding three times the standard deviation are considered deviations.

[0063] In practice, consecutive base positions with values ​​greater than the deviation threshold are divided into deviation intervals. The starting base position, ending base position, and interval length of each deviation interval are recorded. The interval length equals the ending base position minus the starting base position plus one. All base positions in the complementarity deviation spectrum are traversed. When a base position with an intensity difference greater than the deviation threshold is encountered, this base position is designated as the starting base position of the deviation interval. When a subsequent base position with an intensity difference less than or equal to the deviation threshold appears, the base position preceding this base position with the intensity difference greater than the deviation threshold is designated as the ending base position of the deviation interval. This process is repeated until all base positions in the complementarity deviation spectrum have been determined.

[0064] In practice, the deviation interval with the largest interval length among all deviation intervals is selected as the target deviation interval. When multiple deviation intervals have the same interval length and are all the maximum values, the deviation interval with the earliest base position is selected as the target deviation interval.

[0065] In practice, the length of the target deviation interval is divided by three to obtain the number of trinucleotide repeats. When the length of the target deviation interval is not divisible by three, the integer part of the quotient obtained by dividing the length of the target deviation interval by three is taken as the number of trinucleotide repeats.

[0066] See Figure 6 In the figure, the horizontal axis represents the base position coordinates, and the vertical axis represents the intensity difference (in relative fluorescence units). The curve is the intensity difference sequence curve in the complementarity deviation spectrum. The red dashed line in the figure represents the set deviation threshold, which is approximately 15, equivalent to three times the standard deviation of the intensity difference sequence in the non-repetition interval. The green dashed line marks the starting base position of the target deviation interval, approximately at position 600; the orange dashed line marks the ending base position of the target deviation interval, approximately at position 730.

[0067] As shown in the figure, within the base position range of approximately 600 to 730, the intensity difference sequence curve is significantly higher than the deviation threshold, forming a continuous intensity difference deviation interval. This indicates a significant deviation between the forward and reverse sequencing signal peaks, consistent with the technical characteristic of determining the deviation interval based on the complementarity deviation spectrum in this embodiment. The length of this deviation interval is approximately 130 bases, which is relatively long and continuous, indicating a high number of repetitions of the trinucleotide repeat amplification fragment within this region.

[0068] Outside this range, specifically in the regions with base positions less than 600 and greater than 730, the fluctuations in the intensity difference curves did not exceed the deviation threshold. This indicates that the difference in the intensity of forward and reverse sequencing signals within the non-repetitive region is mainly caused by random sequencing errors and does not constitute a significant deviation, thus verifying the rationality of the deviation threshold setting.

[0069] In practice, the upper limit threshold for the normal repeat count and the lower limit threshold for the pathogenic repeat count corresponding to the genetic disease to be tested are obtained. These thresholds are pre-set according to the clinical diagnostic guidelines for the genetic disease to be tested and stored in the threshold configuration unit of the testing system. Based on the name of the genetic disease to be tested corresponding to the sample, the upper limit threshold for the normal repeat count and the lower limit threshold for the pathogenic repeat count are read from the threshold configuration unit.

[0070] In some embodiments, both the upper limit threshold for normal repetitions and the lower limit threshold for pathogenic repetitions are positive integers. The upper limit threshold for normal repetitions is less than the lower limit threshold for pathogenic repetitions.

[0071] In practice, a mutation status determination index is constructed based on the number of trinucleotide repeats, the upper limit threshold for the number of normal repeats, and the lower limit threshold for the number of pathogenic repeats. The formula for calculating the mutation status determination index is as follows:

[0072]

[0073] in, This represents the mutation state determination index, which is a dimensionless value. This represents the number of trinucleotide repeats, which is a positive integer. The number of trinucleotide repeats is obtained by dividing the length of the target deviation interval by three. This represents the upper limit of the normal number of repetitions. The upper limit of the normal number of repetitions is a positive integer and is determined by the clinical diagnostic guidelines for the genetic disease being tested. This represents the lower limit threshold for the number of pathogenic repeats. The lower limit threshold for the number of pathogenic repeats is a positive integer and is determined by the clinical diagnostic guidelines for the genetic disease being tested. Greater than .

[0074] As can be understood, the numerator of the mutation status index represents the difference between the number of trinucleotide repeats and the upper threshold of the normal repeat count, while the denominator represents the difference between the lower threshold of the pathogenic repeat count and the upper threshold of the normal repeat count. When the number of trinucleotide repeats equals the upper threshold of the normal repeat count, the mutation status index equals 0. When the number of trinucleotide repeats equals the lower threshold of the pathogenic repeat count, the mutation status index equals 1.

[0075] Optionally, when the upper threshold for the normal repeat count is equal to the lower threshold for the pathogenic repeat count, the mutation status determination index is not calculated, and the trinucleotide repeat count is directly compared with the threshold. If the trinucleotide repeat count is less than or equal to the upper threshold for the normal repeat count, the sample is determined to be normal; if the trinucleotide repeat count is greater than the upper threshold for the normal repeat count, the sample is determined to be fully mutant. The condition where the upper threshold for the normal repeat count is equal to the lower threshold for the pathogenic repeat count is determined by the diagnostic guidelines for the genetic disease being tested.

[0076] In practice, when the upper limit of the normal repeat count threshold is less than the lower limit of the pathogenic repeat count threshold, the mutation status determination index is used for assessment. If the mutation status determination index is less than or equal to 0, the trinucleotide repeat count is less than or equal to the upper limit of the normal repeat count threshold, and the sample is determined to be normal. If the mutation status determination index is greater than 0 and less than 1, the trinucleotide repeat count is greater than the upper limit of the normal repeat count threshold and less than the lower limit of the pathogenic repeat count threshold, and the sample is determined to be pre-mutant. If the mutation status determination index is greater than or equal to 1, the trinucleotide repeat count is greater than or equal to the lower limit of the pathogenic repeat count threshold, and the sample is determined to be fully mutant.

[0077] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A method for detecting trinucleotide repeats in dynamically mutated genetic diseases, characterized in that, include: Obtain double-stranded DNA from the sample to be tested, and perform asymmetric amplification on the double-stranded DNA to obtain asymmetric amplification products; Based on the electrophoretic mobility shift of the asymmetric amplification products, a base polarity map is constructed. The fluctuation range of the repeating interval is determined based on the polarity shift index corresponding to the base composition type within the repeating unit in the base polarity diagram. The asymmetric amplification products within the fluctuation range are screened, and the screened asymmetric amplification products are subjected to forward sequencing and reverse sequencing to obtain forward sequencing signal peak diagrams and reverse sequencing signal peak diagrams. Based on the forward sequencing signal peak diagram and the reverse sequencing signal peak diagram, a complementary deviation spectrum is constructed; The number of trinucleotide repeats is determined based on the length of the continuous deviation interval in the complementary deviation spectrum; The mutation status of the sample to be tested is determined based on the relationship between the number of trinucleotide repeats and the threshold.

2. The method for detecting trinucleotide repeats in dynamically mutated genetic diseases according to claim 1, characterized in that, Asymmetric amplification of the double-stranded DNA includes: Design forward and reverse primers, wherein the annealing temperature of the forward primer is higher than that of the reverse primer; Using the double-stranded DNA as a template, a polymerase chain reaction is performed using the forward primer and the reverse primer; During the polymerase chain reaction cycle, the annealing temperature is controlled to gradually decrease from a high temperature to a low temperature, so that the forward primer preferentially binds to the template strand; After the exponential growth phase of the polymerase chain reaction ends, the extension time is further increased to allow excessive accumulation of the single-strand extension product of the reverse primer, thereby obtaining the asymmetric amplification product.

3. The method for detecting trinucleotide repeats in dynamically mutated genetic diseases according to claim 2, characterized in that, When constructing a base polarity map based on the electrophoretic mobility shift of the asymmetric amplification product, the following steps are included: The asymmetric amplification products were separated by capillary electrophoresis, and the migration time of each fragment in the capillary was detected. The migration rate was calculated based on the migration time. Using the mobility baseline value as the x-axis and the detection signal intensity as the y-axis, the original electrophoretic spectrum of the asymmetric amplification product is plotted. Based on the measured mobility values ​​corresponding to each signal peak in the original electrophoresis spectrum, the deviation between the measured mobility values ​​and the theoretical mobility values ​​is calculated to obtain the mobility offset. Based on the positive and negative directions and the magnitude of the mobility offset, a base polarity map is constructed.

4. The method for detecting trinucleotide repeats in dynamically mutated genetic diseases according to claim 3, characterized in that, When determining the fluctuation range of the repeating interval based on the polarity shift index corresponding to the base composition type within the repeating unit in the base polarity diagram, the following are included: The base composition type of each repeating unit is extracted from the base polarity map, and the base composition type includes the base sequence arrangement of the trinucleotide repeating unit; The polarity shift index of the repeating unit is calculated based on the distribution ratio of purine and pyrimidine bases in the base composition type. A polarity offset sequence is constructed by using the polarity offset indices of a series of repeating units, and the local extreme points of the polarity offset sequence are obtained. The numerical span between adjacent local maxima and local minima in the polarity offset sequence is determined as the fluctuation range of the repeating interval.

5. The method for detecting trinucleotide repeats in dynamically mutated genetic diseases according to claim 4, characterized in that, The process of screening asymmetric amplification products within the specified fluctuation range, and performing forward and reverse sequencing on the screened asymmetric amplification products to obtain forward and reverse sequencing signal peak diagrams includes: Amplified fragments whose electrophoretic mobility shift falls within the fluctuation range are selected from the asymmetric amplification products and used as fragments to be sequenced. The fragments to be sequenced are divided into a first group and a second group. The first group is sequenced using forward primers to obtain the original forward sequencing signal. The second group was used to perform a sequencing reaction using reverse primers to obtain the original reverse sequencing signal; The original forward sequencing signals are arranged according to base position coordinates to generate a forward sequencing signal peak diagram; The original reverse sequencing signals are arranged according to base position coordinates to generate a reverse sequencing signal peak diagram.

6. The method for detecting trinucleotide repeats in dynamically mutated genetic diseases according to claim 5, characterized in that, When constructing a complementary offset spectrum based on the forward sequencing signal peak diagram and the reverse sequencing signal peak diagram, the following steps are included: The base positions in the forward sequencing signal peak diagram and the base positions in the reverse sequencing signal peak diagram are aligned by base complementary pairing to obtain the correspondence of the aligned base positions. Extract the forward and reverse signal intensity values ​​corresponding to each aligned base position; Calculate the intensity difference between the positive signal intensity value and the negative signal intensity value at the same base position; Plot an intensity difference sequence curve with the base position coordinates as the horizontal axis and the intensity difference as the vertical axis, and use the intensity difference sequence curve as the complementarity deviation spectrum.

7. The method for detecting trinucleotide repeats in dynamically mutated genetic diseases according to claim 6, characterized in that, Determining the number of trinucleotide repeats based on the length of consecutive deviation intervals in the complementary deviation spectrum includes: Traverse the complementary deviation spectrum and determine whether the intensity difference corresponding to each base position is greater than the deviation threshold in order of base position. Divide consecutive base positions that are greater than the deviation threshold into a deviation interval, and record the starting base position, ending base position, and interval length of each deviation interval; Select the deviation interval with the largest interval length from all deviation intervals as the target deviation interval; Divide the length of the target deviation interval by three to obtain the number of trinucleotide repeats.

8. The method for detecting trinucleotide repeats in dynamically mutated genetic diseases according to claim 7, characterized in that, When determining the mutation status of a sample to be tested based on the relationship between the number of trinucleotide repeats and the threshold, the following methods are included: Obtain the upper limit threshold for the normal repeat count and the lower limit threshold for the pathogenic repeat count corresponding to the genetic disease to be tested; If the number of trinucleotide repeats is less than or equal to the upper limit threshold of the normal number of repeats, the sample to be tested is determined to be normal. If the number of trinucleotide repeats is greater than the upper limit threshold of the normal repeat count and less than the lower limit threshold of the pathogenic repeat count, then the sample to be tested is determined to be a pre-mutant. If the number of trinucleotide repeats is greater than or equal to the lower limit threshold of the number of pathogenic repeats, the sample to be tested is determined to be a fully mutant.

9. The method for detecting trinucleotide repeats in dynamically mutated genetic diseases according to claim 3, characterized in that, The migration benchmark value is the theoretical migration rate fitting curve value of each known fragment in the standard molecular weight marker.

10. The method for detecting trinucleotide repeats in dynamically mutated genetic diseases according to claim 4, characterized in that, The polarity shift index is calculated based on the ratio of the total content of adenine and thymine to the total content of guanine and cytosine in the base composition type.

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