WO2019127928A1

WO2019127928A1 - Kit for detecting genetic variation in human chromosome 15q11-13 and application thereof

Info

Publication number: WO2019127928A1
Application number: PCT/CN2018/079453
Authority: WO
Inventors: 周巍; 金云舟
Original assignee: 上海五色石医学研究股份有限公司
Priority date: 2017-12-27
Filing date: 2018-03-19
Publication date: 2019-07-04
Also published as: CN107937495A

Abstract

The present invention provides a kit for detecting genetic variation in human chromosome 15q11-13 and an application thereof. A multiplex PCR amplification system for simultaneously amplifying 16 STR loci distributed on human 15 chromosome was designed. The primer set for PCR amplification comprises SEQ ID Nos. 1-32. The kit for genotyping detection comprises a container for a primer composition and a container for a PCR stock solution. The container for a primer composition comprises a stock solution of a primer composition of SEQ ID Nos. 1-32.

Description

Human chromosome 15q11-13 genetic variation detection kit and application thereof

Technical field

The invention belongs to the technical field of human nucleic acid in vitro detection, and particularly relates to the detection of genotypes of highly polymorphic STR loci in human genomic DNA, in particular to a multi-polymerase chain reaction on human chromosome 15 STR locus for detection kits for genotyping and genetic linkage analysis.

Background technique

Prader-Willi syndrome (PWS, OMIM #176270) is also known as low muscle tone-low intelligence-gonad hypoplasia-obesity syndrome. Children in the neonatal period show difficulty feeding and slow growth. Generally, an uncontrolled diet begins at around 2 years of age, resulting in continued weight gain and severe obesity, slow development of exercise and language function, and mental retardation. Angelman syndrome (AS, OMIM #105830) is also known as "Happy Puppet Syndrome". These children have severe developmental delays, often with smiles on the face, but with mechanical movements, mental retardation, and significant Impaired language skills, often accompanied by epilepsy. Both of the above diseases have serious prognosis and are highly disabling, and there is currently no targeted and effective treatment plan.

At present, the molecular genetic mechanism of PWS and AS has been studied more clearly. Among the children with PWS, the two main types of mutation are the parental chromosome 15q11-13 deletion and the maternal chromosome 15 monopodal diploid; in AS children, the 15q11-13 microdeletion occurs in the mother. The source is on chromosome 15, and UPD is the single parent diploid formed by the parent source chromosome 15.

Molecular diagnosis is the most important means of confirming PWS and AS and related differential diagnosis.

After many years of PWS and AS clinical molecular diagnostic practice, the European Union has been evaluated by the European Molecular Genetic Quality Control Network and the UK External Quality Assessment System. After many revisions, the Executive Committee of the British Clinical Molecular Genetics Association released the latest version of PWS and AS in 2010. Guide to molecular diagnostic practice, this guide has been approved by EMQN and has been widely recognized by experts and scholars in related fields. In China, in June 2015, the Endocrine Genetics Group of the Pediatrics Branch of the Chinese Medical Association released the consensus on the diagnosis and treatment of Prader-Willi syndrome in China (2015).

Whether it is the PWS/AS molecular diagnostic strategy recommended by CMGS or the PWS molecular diagnostic strategy given by the consensus of Chinese PWS diagnosis and treatment, comprehensive application of multiple detection methods is required. Among these detection methods, pyrosequencing, MS-MLPA, and FSIH methods have corresponding commercial, research-only reagents. Since both single-parent two-fold chromosomal variants are common in both PWS and AS, some scholars have studied PWS and AS diagnostic methods based on STR linkage analysis, but no mature commercial reagents have been available yet.

The current practical difficulties faced by molecular diagnostic strategies are the lack of detection tools that can simultaneously perform 15q11-13 microdeletions and single parental diploid linkage analysis. Therefore, in clinical practice, there is an urgent need for a detection tool capable of simultaneously providing detection of the above various types of mutations, thereby providing efficiency in clinical diagnosis work.

Summary of the invention

The object of the present invention is to provide a detection kit capable of simultaneously performing single-parent diploids of chromosome 15 and micro-deletions of 15q11-13 and its application in view of the current clinical needs.

The first aspect of the present invention provides a composite PCR amplification system for simultaneously genotyping 16 STR loci distributed on human chromosome 15 in a PCR reaction, according to each STR locus on chromosome 15. The distribution of the 16 short tandem repeat polymorphisms, ie, the STR loci, is divided into three groups, and each combination is combined with the corresponding PCR amplification primers as follows:

The first group: 9 STR loci distributed between the first break point (BP1) and the third break point (BP3) in the 15q11-13 region: the corresponding PCR amplification primer containers are D15SM01, D15SM02, D15SM03, D15SM04, D15SM05, D15SM06, D15SM07, D15SM08 and D15SM09; the corresponding primer nucleotide sequence and fluorescein are: SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18;

That is, the primer nucleotide sequences corresponding to D15SM01 are: SEQ ID No. 1 and SEQ ID No. 2; the primer nucleotide sequences corresponding to D15SM02 are: SEQ ID No. 3 and SEQ ID No. 4; primer core corresponding to D15SM03 The nucleotide sequences of the primers are: SEQ ID No. 5 and SEQ ID No. 6; the primer nucleotide sequences corresponding to D15SM04 are: SEQ ID No. 7 and SEQ ID No. 8; the primer nucleotide sequence corresponding to D15SM05 is: SEQ The primer nucleotide sequences corresponding to ID No. 9 and SEQ ID No. 10; D15SM06 are: SEQ ID No. 11 and SEQ ID No. 12; the primer nucleotide sequences corresponding to D15SM07 are: SEQ ID No. 13 and SEQ The primer nucleotide sequences corresponding to ID No. 14; D15SM08 are: SEQ ID No. 15 and SEQ ID No. 16; the primer nucleotide sequences corresponding to D15SM09 are: SEQ ID No. 17 and SEQ ID No. 18;

The second group is the three STR loci distributed between the third break point (BP3) and the fourth break point (BP4) in the 15q11-13 region: the corresponding PCR amplification primer containers are D15SM010, D15SM11 and D15SM012; corresponding primer nucleotide sequence and fluorescein are: SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23 and SEQ ID No. 24 Shown

That is, the primer nucleotide sequences corresponding to D15SM10 are: SEQ ID No. 19 and SEQ ID No. 20; the primer nucleotide sequences corresponding to D15SM11 are: SEQ ID No. 21 and SEQ ID No. 22; the primer core corresponding to D15SM12 The nucleotide sequence is: SEQ ID No. 23 and SEQ ID No. 24;

The third group: 4 STR loci distributed at the distal end of the long arm of chromosome 15: the corresponding PCR amplification primer containers are D15SM013, D15SM014, D15SM15 and D15SM016 in sequence; the corresponding primer nucleotide sequence and fluorescein are in turn Shown as SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31 and SEQ ID No. ,

That is, the primer nucleotide sequences corresponding to D15SM13 are: SEQ ID No. 25 and SEQ ID No. 26; the primer nucleotide sequences corresponding to D15SM14 are: SEQ ID No. 27 and SEQ ID No. 28; the primer core corresponding to D15SM15 The nucleotide sequences are: SEQ ID No. 29 and SEQ ID No. 30; the primer nucleotide sequences corresponding to D15SM16 are: SEQ ID No. 31 and SEQ ID No. 32;

Wherein SEQ ID No. 1, SEQ ID No. 13, SEQ ID No. 15 and SEQ ID No. 19 are modified by fluorescein HEX; SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 9 and SEQ ID No. 12 was modified with fluorescein FAM; SEQ ID No. 8, SEQ ID No. 23, SEQ ID No. 29 and SEQ ID No. 32 were modified with fluorescein ROX; SEQ ID No. 17, SEQ ID No. 21. SEQ ID No. 26 and SEQ ID No. 27 were modified with fluorescein TAMRA.

In another preferred embodiment, the PCR amplification primers and the fluorescein-modified sequences in the composite PCR amplification system corresponding to the 16 STR loci are as shown in Table 1:

Table 1

In the present invention, the lengths of PCR amplification products of each group of STR loci do not overlap each other. Adjust the working concentration of PCR primers in each STR locus so that the peak height difference between homozygotes or heterozygote in the same group is within 40%. The working concentration of the PCR amplification primer is

In the present invention, the primer composition consists of a dry powder or a solution of a PCR primer of the nucleotide sequence shown in SEQ ID No. 1 to SEQ ID No. 32.

The second aspect of the present invention provides the use of the above composite PCR amplification system for non-diagnostic detection of chromosome 15q11-13 genetic variation in human DNA samples, that is, 16 short tandem repeat polymorphism genetic marker genotyping for DNA samples And linkage non-diagnostic analysis: detection of human genomic DNA samples using human complex genomic DNA samples selected from human blood, blood spots, semen, spots, saliva, saliva, amniotic fluid, hair, One or more of muscle tissue and bone.

A third aspect of the present invention provides a human chromosome 15q11-13 genetic variation test kit comprising a container (i.e., a primer composition container) and a PCR reaction mother liquid container comprising the PCR amplification system of the first aspect of the present invention, among them:

The container containing the PCR amplification system contains the primer composition storage solution shown in SEQ ID No. 1 to SEQ ID No. 32, and the concentration of each primer in the storage solution is 10 times of the concentration of the corresponding primer working solution;

The PCR reaction mother liquid container contains a PCR reaction mother solution at a reaction concentration twice the PCR reaction, and the PCR reaction mother liquid contains DNA polymerase, magnesium ion, dNTP, and the like.

A fourth aspect of the present invention provides a method for using the detection kit of the third aspect of the present invention (preferably, the method is for non-diagnostic purposes in vitro), and the details are as follows:

(1) Extract human genomic DNA, and the concentration of genomic DNA by ultraviolet spectrometry is

(2) Multiplex PCR amplification: The total reaction system was 20 μL, and the reaction system consisted of the following:

(3) The PCR reaction is carried out on a common PCR instrument (such as ABI9700, ABI9600, Bio-Rad C1000, etc.), and the PCR reaction conditions are as follows: after starting at 95 ° C for 10 minutes, 95 ° C for 30 seconds, 60 ° C for 60 seconds, 72 ° C for 60 seconds , a total of 28 cycles, then 72 ° C for 60 minutes, and then 4 ° C;

(4) 1 μL of the PCR product was subjected to capillary electrophoresis on a genetic analyzer according to a conventional operation.

(5) The data of the electrophoresis can be obtained by using special data analysis software (such as GeneMapper) to obtain the classification map and data of each STR locus.

(6) Analysis of human chromosome 15q11-13 genetic variation by linkage analysis of biological mother-child or father-son sample test results.

In the present invention, the DNA template refers to human genomic DNA. Human genomic DNA can be extracted from the following tissues or samples by various conventional methods (such as Chelex-100 method, magnetic bead method, phenol chloroform method, and various commercial human genomic DNA extraction kits): human blood, Blood spots, semen, spots, saliva, saliva spots, amniotic fluid, hair, muscle tissue, bones, etc. Preferably, in the 20 μL PCR amplification system, the amount of the DNA template is in the range of 0.5 ng to 5 ng to obtain better amplification and typing results.

In the present invention, the PCR amplification products of the 16 STR locus complex PCR amplification systems provided can be analyzed by capillary electrophoresis using a genetic analyzer (such as AB3130XL, AB3500 Genetic Analyzer), and the data after electrophoresis can be analyzed by GeneMapper and other data analysis software. On the analysis, the typing map and data of each STR locus were obtained.

In the present invention, the human chromosome 15q11-13 genetic variation is analyzed by linkage analysis, and the flow is:

(1) Determine whether the test sample has a maternal source or a parental single parent diploid of chromosome 15, as follows: Analyze the test sample by comparing each genetic marker type map and data of the sample with its biological mother or father sample. The biological source of each genetic marker, and whether there is a maternal/parental single parent diploid (ie, the typing results of all genetic markers in the test sample are identical to their biological mother or father).

(2) Determining whether there is a maternal/parental deletion of the 15q11-13 region in the test sample, as follows: by the typing results of the first set of 9 genetic markers, combined with the results of the analysis in step (1), It was judged whether there was a maternal or paternal deletion of the 15q11-13 region (ie, all nine genetic markers were shown to be homozygous and were derived only from their biological mother or father).

According to a fifth aspect of the invention, the use of the detection kit of the third aspect of the invention for detecting genetic variation of human chromosome 15q11-13 is provided. Preferably, the use is non-diagnostic in vitro.

A sixth aspect of the present invention provides the use of the composite PCR amplification system of the first aspect of the present invention for preparing a human chromosome 15q11-13 genetic variation kit.

The beneficial effects of the invention:

In the process of establishing a multiplex PCR amplification system, as the number of detected STR loci increases, the mutual interference between the pairs of primers also increases. The optimized design of the invention realizes single tube amplification of 16 STR loci, and the STR loci in each fluorescent channel and in different fluorescent channels are balanced and the typing pattern is good.

By detecting the genetic linkage analysis between the sample and its biological mother/father, it is possible to simultaneously detect the single-parent diploid of chromosome 15 and the micro-deletion of 15q11-13 region, providing a rapid and convenient technique for the clinical diagnosis and treatment of PWS and AS. tool.

The invention also has the beneficial effects that the genetic characteristics of the STR locus can clearly distinguish the sample contamination, and effectively avoid the pollution between the parent source and the unrelated samples.

DRAWINGS

Figure 1 is a fragmentation map of 16 STR loci obtained by using the kit provided by the present invention as a parent source 15q11-13 microdeletion sample.

2 is a 16 STR locus typing map obtained from the biological mother sample of the parent source 15q11-13 microdeletion sample of FIG. 1 using the kit provided by the present invention.

Detailed ways

For a better understanding of the present invention, the following is a description of specific examples of 16 STR locus typing in human blood samples (not limited to human blood samples). It is to be understood that the following specific examples are merely illustrative of the invention and are not intended to limit the invention.

In this example, the amplification reaction was carried out on an ABI 9700 thermal cycler, electrophoresis and detection were performed on an ABI 3500 Genetic Analyzer, and data analysis was performed using GeneMapper ID v3.2 software. Other reagents and materials used, such as internal standards, POP7, capillary electrophoresis buffers, Hi-Di, allelic ladders, are conventional materials commonly used by those skilled in the art.

The primer composition in the PCR reaction system reagent of the present invention comprises nucleotide primer sequences of SEQ ID No. 1 to SEQ ID No. 32, which are designed by the inventors by primers, and are routinely used by DNA synthesis companies in accordance with the art. Synthetic method for synthesis, and then formulated into working concentration

As used herein, the terms "PCR amplification primer set", "primer set" are used interchangeably and refer to a collection of primers having nucleotide sequences from SEQ ID No. 1 to SEQ ID No. 32, wherein SEQ ID No. 1. SEQ ID No. 13, SEQ ID No. 15 and SEQ ID No. 19 sequences were modified with fluorescein HEX; sequences of SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 9 and SEQ ID No. 12. Modified by fluorescein FAM; SEQ ID No. 8, SEQ ID No. 23, SEQ ID No. 29 and SEQ ID No. 32 sequences were modified with fluorescein ROX; SEQ ID No. 17, SEQ ID No. 21, SEQ ID The sequence of No. 26 and SEQ ID No. 27 was modified with fluorescein TAMRA.

Example

1: Human blood sample genomic DNA preparation

Test samples were donated by volunteers with informed consent. Peripheral venous blood was taken 1 mL according to medical routine, and EDTA was anticoagulated. Genomic DNA was extracted using QIAGEN's Human Peripheral Blood Genomic Extraction Kit, eluting a volume of 100 μl, quantified by UV spectrometer, and the genomic DNA concentration was diluted to 1 ng/μL.

2: Preparation of PCR system

The PCR reaction system was prepared according to the following system (total reaction system was 20 μL):

, PCR reaction mother liquor container. Multiply the total reaction number by the single reaction requirement in the above reaction system to calculate the required amount of PCR reaction mother liquor, primer composition amount, PCR reaction auxiliary solution amount and ddH ₂ O amount, and mix the above reagents in a 1.5 mL EP tube. Evenly, dispense and number 19 μL per PCR reaction tube, then add 1 μL of the sample DNA template according to the sample number, and mix again.

3: PCR reaction

The PCR reaction was carried out using an ABI 9700 PCR machine.

The PCR conditions were as follows: after starting at 95 ° C for 10 minutes, 95 ° C for 30 seconds, 60 ° C for 60 seconds, 72 ° C for 60 seconds for a total of 30 cycles, then 72 ° C for 60 minutes, and then 4 ° C incubation.

4: Capillary electrophoresis typing detection

(1) Take (1 μL internal standard + 10 μL Hi-Di) × (number of samples + 1) to prepare a mixed solution, and mix and mix in a well of a 96-well plate in 10 μL per tube. One μL of the allele ladder was added to one of the wells.

(2) Take 1 μL of the PCR product into the corresponding 96-well plate wells by sample number.

(3) The sample was denatured at 95 ° C for 4 minutes and then rapidly cooled on ice for 4 minutes.

(4) Place the sample in the sample tray of the genetic analyzer and perform capillary electrophoresis according to the conventional parameters (see the Genetic Analyzer Manufacturer's Operating Instructions).

(5) At the end of capillary electrophoresis, the experimental data was analyzed by GeneMapper software to obtain a typing map, as shown in Fig. 1 and Fig. 2.

1 is a 16 STR locus typing map obtained by using the kit provided by the present invention as a parent source 15q11-13 microdeletion sample; FIG. 2 is a biological mother sample of the paternal 15q11-13 microdeletion sample using the present invention. The 16 STR locus typing profiles obtained from the kits provided. According to Figure 1, D15SM01～D15SM09 are homozygous, and there are multiple heterozygotes in D15SM10～D15SM16, suggesting that the sample has microdeletions in the 15q11-13 region; compared with each STR locus of its biological mother sample. Analysis, D15SM01 ~ D15SM09 these 9 STR loci sites are consistent with their biological mothers, suggesting that the STR locus in the 15q11-13 region is derived from its biological mother. Combining two points, the sample shown in Figure 1 is the paternal source deletion of the 15q11-13 region, which is Prader-Willi syndrome (PWS).

The human chromosome 15q11-13 genetic variation detection kit proposed by the invention can be stably implemented in a laboratory with a PCR instrument and genetic analysis, and the detection time is 3-4 hours. At the same time, the reagents provided by the invention can be conveniently used in the production of biotechnology companies and in biomedical testing institutions and forensic DNA testing institutions for testing, and have the conditions for industrialization and popularization and application.

The invention has been described with reference to specific embodiments thereof. It will be apparent to those skilled in the art that various modifications and changes may be made to the present invention, including but not limited to: changing different sets of fluorescein labels, changing fluorescein-labeled primers (eg, by labeling upstream) The primers are changed to labeled downstream primers, and the genomic group arrangement is changed according to the range of alleles of each STR locus. The PCR amplification conditions and primer concentration are optimized according to other PCR reaction mother liquids, and the recommended reaction system is changed. . It is apparent that the above-described modifications and variations are possible to those skilled in the art, and such modifications and changes may be made without departing from the spirit and scope of the invention.

Claims

A composite PCR amplification system for simultaneously genotyping 16 STR loci distributed on human chromosome 15, characterized in that the 16 is distributed according to the distribution of each STR locus on chromosome 15. A short tandem repeat polymorphism genetic marker is divided into three groups, the combination of which is combined with the corresponding PCR amplification primers as follows:

The first group: 9 STR loci distributed between the first break point (BP1) and the third break point (BP3) in the 15q11-13 region: the corresponding PCR amplification primer containers are D15SM01, D15SM02, D15SM03, D15SM04, D15SM05, D15SM06, D15SM07, D15SM08 and D15SM09; the corresponding primer nucleotide sequence and fluorescein are: SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18;

The second group is the three STR loci distributed between the third break point (BP3) and the fourth break point (BP4) in the 15q11-13 region: the corresponding PCR amplification primer containers are D15SM010, D15SM11 and D15SM012; corresponding primer nucleotide sequence and fluorescein are: SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23 and SEQ ID No. 24 Shown

The third group: 4 STR loci distributed at the distal end of the long arm of chromosome 15: the corresponding PCR amplification primer containers are D15SM013, D15SM014, D15SM15 and D15SM016 in sequence; the corresponding primer nucleotide sequence and fluorescein are in turn Shown as SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31 and SEQ ID No. ,

The sequences in which the above primers were modified with fluorescein are shown in Table 1.
The composite PCR amplification system according to claim 1, wherein the working concentration of the PCR amplification primer is
The composite PCR amplification system according to claim 1, wherein the primer composition consists of a dry powder or a solution of a PCR primer of the nucleotide sequence shown in SEQ ID No. 1 to SEQ ID No. 32.
A human chromosome 15q11-13 genetic variation detection kit, comprising: a primer composition container, a PCR reaction mother liquid container, wherein:

The primer composition container comprises primers SEQ ID No. 1 to SEQ ID No. 32 composition stock solution, the stock solution concentration is 10 times of the corresponding working concentration;

The PCR reaction mother liquid container has a PCR reaction mother solution at a reaction concentration twice the PCR reaction, and the PCR reaction mother liquid contains DNA polymerase, magnesium ion, and dNTP.
A non-diagnostic method for detecting a test kit according to claim 4, comprising the steps of:

(1) Extract human genomic DNA, and the concentration of genomic DNA by ultraviolet spectrometry is

(2) Multiplex PCR amplification: The total reaction system was 20 μL, and the reaction system consisted of the following:

(3) The PCR reaction was carried out in a common PCR instrument. The PCR reaction conditions were as follows: after starting at 95 ° C for 10 minutes, 95 ° C for 30 seconds, 60 ° C for 60 seconds, 72 ° C for 60 seconds, a total of 28 cycles, and then 72 ° C for 60 minutes. Then kept at 4 ° C;

(4) 1 μL of the PCR product was subjected to capillary electrophoresis on a genetic analyzer;

(5) analyzing the data after electrophoresis using special data analysis software to obtain the typing map and data of each short tandem repeat polymorphism genetic marker;

(6) Analysis of human chromosome 15q11-13 genetic variation by linkage analysis of biological mother-child or father-son sample test results.
The method of use according to claim 5, wherein the DNA template is human genomic DNA.
The method of using according to claim 5, wherein the linkage analysis comprises the following steps:

(1) Judging whether the test sample has the parent source of the chromosome 15 or the parental single parent diploid, specifically including:

Analyze the biological source of each genetic marker in the test sample by comparing each genetic marker typing map and data of the sample with its biological mother or father sample, and whether there is a maternal/parental single parent diploid, ie, the test sample The genotyping results of all genetic markers are completely consistent with their biological mothers or fathers;

(2) Determining whether there is a maternal/parental source of the 15q11-13 region in the test sample, including:

By the result of the typing of the nine genetic markers in (1) of claim 1, combined with the results of the analysis in step (1), it is judged whether there is a maternal or paternal deletion of the 15q11-13 region, that is, 9 appeals. Whether the genetic markers are shown to be homozygous and are derived only from their biological mother or father.
The use of the composite PCR amplification system of claim 1 for the preparation of a human chromosome 15q11-13 genetic variation kit.
Use of the composite PCR amplification system of claim 1 for non-diagnostic detection of chromosome 15q11-13 genetic variation in human DNA samples.
Use of the test kit of claim 4 for detecting genetic variation in human chromosome 15q11-13.