KR20010105503A - Rapid Multiplex Genotyping Kit System for the detection of Y-chromosome STRs - Google Patents

Abstract

The subject of the present invention are variants of ciliary neurotrophic factor with enhanced receptor selectivity (CNTFR), useful for the treatment of diseases and disorders including motor neuron diseases and muscle degenerative diseases. Another subject of the invention is to provide a method for identifying the above mentioned CNTF variants. The hCNTF variants with the amino acid substitutions in accordance with the present invention, have a reduced ability, as compared to the human CNTF, to elicit biological effects through soluble CNTFR, without affecting its ability to activate membrane-bound neuronal CNTF receptors, thereby improving its therapeutic properties. Fig. 1 shows the reduced CNTFR binding affinity of a CNTF variant according to the invention (IA-CNTF; SEQ ID NO:2). It is evident that the binding affinity of this variant to the CNTFR is reduced as compared to the wild-type human CNTF molecule.

Description

Rapid Multiplex Genotyping Kit System for the detection of Y-chromosome STRs}

The present invention relates to methods and kits (including allelic ladders) that can economically, quickly and accurately analyze mutations in STRs (Short Tandem Repeats) genes located on human Y-chromosomes. Analytical methods used in the field of personal identification and forensic science, consisting of amplification using a multiplex polymerase chain reaction (PCR) to compare at the same time, followed by comparison with the obtained allelic ladder And kits.

With the recent development of molecular biological techniques, DNA profiles analysis results have been applied to the field of personal identification and forensics in many countries including Korea, Asia, USA and Europe. Currently in most cases internationally, methods for analyzing DNA profiles via autosomal gene markers (STRs) are mainly utilized, and such autosomal STR assay kit systems have been developed commercially. On the other hand, since the non-recombining portion of the Y-chromosome (NRPY) region of the Y-chromosome does not intersect with the X-chromosome, mutations of various genes located in this region are compared with the male by a haplotype. It can be used as a very useful tool in the field of personal identification and forensic science involved. Since the Y-STR markers located on NRPY show a wide variety of genetic variations according to the individual, it is necessary in the personal identification and forensic science fields to analyze and determine the genetic variation of Y-STRs by standardized techniques. have. Unlike the autosomal, no Y-chromosome STR assay and assay kit system (allelic ladders) have been developed.

Accordingly, it is an object of the present invention to provide a method for analyzing Y-STRs necessary in the field of personal identification and forensic science related to men.

Another object of the present invention is to provide a Y-STRs analysis kit for personal identification associated with men.

1 is an electrophoretic image shown by Triplex system I ( DYS19 / DYS389I / DYS389II ) among the multiplex systems according to the present invention.

Figure 2 is an electrophoretic image represented by Triplex system II ( DYS390 / DYS391 / DYS393 ) of the Multiplex system according to the present invention

3 is an electrophoretic image shown by Triplex system III ( DYS19 / DYS388 / DYS392 ) of the Multiplex system according to the present invention.

For this purpose, alleles ( DYS19 : 7 alleles, DYS388 : 7 alleles, DYS389Ⅰ : 5 alleles, DYS389Ⅱ) identified by PCR and sequencing at 8 STR loci in different human populations (approximately 2,000). : 7 alleles, DYS390 : 8 alleles, DYS391 : 5 alleles, DYS392 : 10 alleles, DYS393 : 7 alleles) were selected and cloned into plasmid vectors, and then amplified these alleles from each clone by PCR method. I was. The PCR products were then separated and purified to develop allelic ladders that can be standardized for allelic determination of Y-STRs.

Y-STRs analysis method using the multiplex PCR system of the present invention is at least three or more Y-STR loci that can be amplified simultaneously from the group consisting of DYS19 , DYS388 , DYS389I , DYS389II , DYS390 , DYS391 , DYS392 , and DYS393 locus Obtaining one or more analytical DNA samples having these; PCR amplifying a Y-STR sequence using specific primers in the DNA sample; And evaluating the amplified alleles by detecting the amplified products by electrophoresis and comparing them with allelic ladders, thereby determining the respective genotypes of the analyzed locus combinations in the DNA sample.

The multiplex STR system consisting of the at least three or more loci is preferably a triplex STR system, and the triplex STR system of the present invention is preferably one selected from the group consisting of the following three locus combinations: It is characterized by including a combination of the above loci:

DYS19 / DYS389Ⅰ / DYS389Ⅱ

DYS390 / DYS391 / DYS393

DYS19 / DYS388 / DYS392

The present invention also provides a container comprising a container having a combination of at least three or more loci selected from the group consisting of DYS19 , DYS388 , DYS389I , DYS389II , DYS390 , DYS391 , DYS392 , and DYS393 loci and primers corresponding to each of the selected loci. STRs analysis kit.

Primers corresponding to the locus of the assay kit have the following sequence pairs:

If the locus is DYS19 upstream 5'-CTACTgAgTTTCTgTTATAgT-3 ',

Downstream 5'-ATggCATgTAgTgAggACA-3 ';

Upstream 5′-gTgAgTTAgCCgTTTAgC gA-3 ′, downstream 5′-CAgATCgCAACCACTgCg-3 ′ when the locus is DYS388 ;

Upstream 5′-CCAACTCTC ATCTgTATTATCTAT-3 ′, downstream 5′-TCTTATCTCCACCCACCAgA-3 ′ when the loci are DYS389I and DYS389II ;

Upstream 5′-TATATTTTACACATTTTTggg CC-3 ′, downstream 5′-TgACAgTAAAATgAACACATTgC-3 ′ when the locus is DYS390 ;

Upstream 5′-CTATTCATTCAATCATACAC CCA-3 ′, downstream 5′-gATTCTTTgTggTgggTCTg-3 ′ when the locus is DYS391 ;

Upstream 5′-TCATTAATCTAgCTTTTAAAAA CAA-3 ′, downstream 5′-A gACCCAgTTgATgCAATgT-3 ′ when the locus is DYS392 ;

Upstream 5′-gTggTCTTCTACTTgTgTCAA TAC-3 ′ and downstream 5′-AACTCAAgTCCAAAAAATgAgg-3 ′ when the locus is DYS393 .

The assay kit of the present invention is preferably characterized by visualizing genotypes via silver staining.

The configuration of the present invention, that is, the "Y-STRs analysis kit" is as follows.

[Triplex Silver Detection System for 8 Types of Y-STR Markers]

Except for Taq DNA polymerase and DNA to be analyzed, the present invention consists of a kit of 10 × primer pairs, Y-STR 10 × buffer, 2 × loading solution, positive control DNA, and allelic ladder that can be PCR 50 times for each system. do.

1) Y-Triplex system kitⅠ: DYS19 / DYS389Ⅰ / DYS389Ⅱ

2) Y-Triplex system kitⅡ: DYS390 / DYS391 / DYS393

3) Y-Triplex system kit III: DYS19 / DYS388 / DYS392

Y-STR Triplex System Y-STR locus Allele size range (bp) Repeat count Control DNA Allele Size Kit I DYS389Ⅱ 355-379 23-29 27 DYS389Ⅰ 243-259 8-12 11 DYS19 182-206 13-18 16 Kit II DYS390 199-227 20-27 23 DYS391 275-291 8-12 10 DYS393 116-140 11-17 ^* 13 Kit III DYS392 236-263 7-16 11 DYS19 182-206 13-18 16 DYS388 125-143 11-17 13

^* DYS393--16 , 17 allele: Korean specific allele of the present invention that has not been reported worldwide.

Example 1-3

Human genomic DNA used in the embodiment of the present invention is a blood donor from Dankook University donors, Dankook University students, cooperative researchers in Japan, China, Mongolia, Vietnam, Philippines, Indonesia, Thailand, etc. The one that was supplied and extracted was used. The DNA sample is amplified in one reaction vessel. PCR amplification was performed using a total volume of 25 ?? of 10 × PCR buffer, 0.2 mM dNTPs, 0.25 units of Taq polymerase, 1 to 10 ng DNA template, and 0.015-0.5 ?? M of each corresponding primer. The reaction solution was carried out in l. The PCR cycle adopts the following amplification protocol.

Preheat at 94 ° C. for 3 minutes;

30 repetitions of 30 seconds at 94 ° C, 30 seconds at 58 ° C, 45 seconds at 72 ° C; And

Final extension for 5 minutes at 72 ° C.

The amplified products were subjected to electrophoresis using an electrophoresis apparatus at 50W until the xylene cyanol dye (Xylene Cyanol Dye) was about 40-60 mm at the bottom of the gel (20 cm x 43 cm). The gel was a 6T4, 7M urea acrylamide gel and the running buffer and the gel buffer used TBE buffer.

After electrophoresis, the gel was subjected to silver staining to identify a band. The gel was first washed with 10 ethanol for 10 minutes. Then, washed three times with distilled water, and reacted for 0.2 minutes in 0.2 silver nitrate and washed once again with distilled water. The washed gel was stopped with 10 acetic acid solution according to the appearance of the band while reducing in 0.28 M anhydrous sodium carbonate and 0.019 formalin solution. This dyed gel was adsorbed onto 3M paper, dried in a gel drier and stored. Test results are shown in FIGS.

Since Y-STR markers represent a wide variety of genetic variations, the genetic variation of Y-STRs needs to be invented and developed by standardized techniques, particularly in the field of personal identification and forensic science involving men. In general, strong events and sex crimes are often caused by men, so the DNA information of Y-chromosome can be a very suitable marker for solving this problem. In particular, there is an advantage that can specifically analyze the DNA of a man. Currently, kit systems for analyzing autosomal gene markers (STRs) have been developed and commercialized at home and abroad. Unlike autosomals, STR assay kit systems (allelic ladders) of Y-chromosomes have not been developed yet. Therefore, the development of the present invention will contribute to the advancement and competitiveness of the domestic biotechnology industry, and it is expected to increase the efficiency and reliability of the analytical method as well as increase employment and economic added value. In addition, by inducing continuous technology development in the future, faster and more economical genetic typing techniques such as bio-chip development, DNA profiling business of nationwide who can search for separated families and replace fingerprints in preparation for unification of South and North Korea. You can do that.

Claims (6)

Obtaining one or more analytical DNA samples having at least three or more Y-STR loci that can be amplified simultaneously selected from the group consisting of DYS19 , DYS388 , DYS389I , DYS389II , DYS390 , DYS391 , DYS392 , and DYS393 loci; PCR amplifying a Y-STR sequence using specific primers in the DNA sample; And Evaluating the amplified alleles by comparing the amplified products with allelic ladders developed by electrophoresis, thereby determining the respective genotypes of the analyzed locus combinations in the DNA sample. Y-STRs Analysis The method of claim 1, wherein the multiplexed STR system consisting of at least three loci consists of a combination of at least three loci selected from the group consisting of the following loci: DYS19 / DYS389Ⅰ / DYS389Ⅱ DYS390 / DYS391 / DYS393 DYS19 / DYS388 / DYS392 A Y-STRs analysis kit comprising a container having a combination of at least three or more loci selected from the group consisting of DYS19 , DYS388 , DYS389I , DYS389II , DYS390 , DYS391 , DYS392 , and DYS393 loci and primers corresponding to each of the selected loci. 4. The Y-STRs analysis kit according to claim 3, wherein the multiplexed STR system consisting of at least three loci consists of a combination of at least three loci selected from the group consisting of the following loci: DYS19 / DYS389Ⅰ / DYS389Ⅱ DYS390 / DYS391 / DYS393 DYS19 / DYS388 / DYS392 The Y-STRs assay kit according to claim 3 or 4, wherein the primers corresponding to the locus of the assay kit are the following sequence pairs: If the locus is DYS19 upstream 5'-CTACTgAgTTTCTgTTATAgT-3 ', Downstream 5'-ATggCATgTAgTgAggACA-3 '; Upstream 5′-gTgAgTTAgCCgTTTAgC gA-3 ′, downstream 5′-CAgATCgCAACCACTgCg-3 ′ when the locus is DYS388 ; Upstream 5′-CCAACTCTC ATCTgTATTATCTAT-3 ′, downstream 5′-TCTTATCTCCACCCACCAgA-3 ′ when the loci are DYS389I and DYS389II ; Upstream 5′-TATATTTTACACATTTTTggg CC-3 ′, downstream 5′-TgACAgTAAAATgAACACATTgC-3 ′ when the locus is DYS390 ; Upstream 5′-CTATTCATTCAATCATACAC CCA-3 ′, downstream 5′-gATTCTTTgTggTgggTCTg-3 ′ when the locus is DYS391 ; Upstream 5′-TCATTAATCTAgCTTTTAAAAA CAA-3 ′, downstream 5′-A gACCCAgTTgATgCAATgT-3 ′ when the locus is DYS392 ; Upstream 5′-gTggTCTTCTACTTgTgTCAA TAC-3 ′ and downstream 5′-AACTCAAgTCCAAAAAATgAgg-3 ′ when the locus is DYS393 . 4. The Y-STRs assay kit according to claim 3, wherein the assay kit preferably visualizes genotypes through silver staining.