CN109839417B

CN109839417B - Human autosomal STR (short tandem repeat) gene locus typing method based on micro-fluidic chip

Info

Publication number: CN109839417B
Application number: CN201711211209.2A
Authority: CN
Inventors: 秦建华; 苏文涛; 姜雷
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2017-11-28
Filing date: 2017-11-28
Publication date: 2021-08-27
Anticipated expiration: 2037-11-28
Also published as: CN109839417A

Abstract

The invention discloses a human autosomal STR (short tandem repeat) gene locus typing method based on a microfluidic chip, belonging to the field of DNA (deoxyribonucleic acid) analysis. The method comprises the steps of selecting a buffer system, selecting an electrophoresis chip, selecting an autosomal STR locus, performing electrophoresis and the like. The invention separates STR loci on 5 human autosomes in 12 minutes based on the combination of chip electrophoresis technology and a laser-induced fluorescence detection system, and completes the autosomal STR typing test of real blood samples. The chip electrophoresis has high resolution and rapid separation and analysis capability for autosomal STR typing, so that the chip electrophoresis is hopeful to become one of analysis means for rapidly, simply and efficiently separating and detecting human chromosome STR loci.

Description

Human autosomal STR (short tandem repeat) gene locus typing method based on micro-fluidic chip

Technical Field

The invention relates to the field of DNA analysis, in particular to a human autosomal STR (short tandem repeat) gene locus typing method based on a microfluidic chip.

Background

Short Tandem Repeat (STR), also known as microsatellite DNA (micro satellite), or simple repeat (SSR), is a type of DNA tandem repeat with length polymorphism that is widely present in eukaryotic genomes. The core sequence is 2-6 bp (Am J Forensic Med and Path,1994,15(4):269-282), and is arranged in a repeated tandem manner, the repeated times are usually 15-30 times, the length of the polymorphic fragment is usually 100-300bp, and accounts for about 10% of the whole genome DNA. Most STR loci are polymorphic (Am J Hum Genet,1991,49: 746-. STR loci are wide in distribution, large in number, simple in structure, easy to amplify, high in polymorphism and specificity, and widely applied to the field of forensic physical evidence research as DNA genetic markers at present.

Autosomal STR loci are widely present in the human genome and are numerous, and thus, in practice, when multiplex detection is performed in combination with multiple STRs loci, hundreds of millions of genotype combinations are generated, and each genotype combination has a low distribution frequency in the population, so that individual identification can be achieved at a high probability level. At present, capillary electrophoresis technology is mostly adopted as a separation system for commercialized instruments for autosomal STR typing, PMT or CCD is adopted as a photoelectric detection system, and the time for STR locus detection exceeds 40 minutes. However, for some emergency cases, it is crucial to obtain accurate STR typing results quickly. In addition, with the increase of the amount of DNA samples collected by cases and the rapid advance of national DNA database construction, the number of DNA samples to be detected is increased dramatically, which is not only an examination on manpower and financial resources of a DNA laboratory of a forensic doctor, but also an examination on a technical method for the examination. Therefore, the development of a rapid STR typing technology is urgently needed to meet the requirements of DNA workers in forensic science.

The micro-fluidic chip technology, or micro total analysis system (mu-TAS), is a very important scientific technology in the 21 st century and has great application prospect. The characteristics of high integration and flexible combination can integrate a plurality of analysis processes, so that the rapid STR analysis becomes possible. An article of chip capillary electrophoresis was published by J.Ramsey et al in 1994, and a series of papers on chip for high-speed DNA sequencing and PCR amplification were published by Mathies et al in 1995-1997. Schmalzing et al used microchip electrophoresis to achieve high precision separation of 4 STR sites (Proc Natl Acad Sci 1997,94(19): 10273-10278). Yeung et al show a chip electrophoresis apparatus, and a four-color fluorescence collector for STR typing is configured, which can complete the separation of 16 STR sites within less than 30min, and the resolution reaches 1bp, which shows the feasibility and superiority of the microfluidic chip electrophoresis technology for forensic STR typing (J.Forensic Sci.,2006,51(4): 740-. However, these studies still require long time, high temperature (. gtoreq.60 ℃) and different isolation patterns for the genotyping of autosomal STR.

Disclosure of Invention

The invention aims to provide an autosomal STR (short tandem repeat) electrophoretic typing method for quickly separating and analyzing human autosomal gene loci based on a chip electrophoresis technology combined with a laser-induced fluorescence detection system.

The technical solution of the invention is as follows: a human autosomal STR gene locus typing method based on a microfluidic chip comprises the following steps:

(1) selecting an electrophoresis system: the sample injection buffer solution system comprises 1 XTTE buffer solution composed of 50mmol/L of Tris (hydroxymethyl) aminomethane (Tris), 50mmol/L N-Tris (hydroxymethyl) methyl-3-aminopropane sulfonic acid (TAPS) and 1mmmol/L of Ethylene Diamine Tetraacetic Acid (EDTA), and the pH value is 8.3; the sample separation system consists of POP 4 or POP 7 glue;

(2) selecting an electrophoresis chip: the chip comprises a sample pool, a sample waste liquid pool, a buffer liquid waste liquid pool and a cross pipeline, wherein the left end and the right end of a cross arm of the cross pipeline extend towards the vertical direction respectively and are communicated with the buffer liquid waste liquid pool and the buffer liquid pool respectively; arranging a detection point on a cross arm between the cross part of the cross pipeline and the buffer solution waste liquid pool;

(3) chip surface modification: firstly, placing the chip in a vacuum drier for vacuumizing for about 10 minutes; taking out the chip, immediately adding 1M NaOH into the chip channel, keeping the temperature of 65 ℃ for 15min, draining the NaOH, adding 1M HCl, keeping the temperature at room temperature for 15min, adding 0.1-0.5% pHEA after draining, keeping the temperature at room temperature for 1 h, and finally draining the microchannel by acetonitrile and placing the microchannel at room temperature for later use;

(4) selecting an autosomal STR locus, namely selecting five autosomal loci with large polymorphism and small allele span of Chinese population as research objects based on the consideration of gene polymorphism (GD >0.6) and allele span in the Chinese population; the five autosomal loci are TPOX, vWA, D5S818, FGA and D2S1338, respectively; dividing 5 loci into two groups, respectively grouping and marking by HEX and TAMRA, marking by a third color fluorescent dye for a molecular weight internal standard, and forming a 5 autosomal STR locus multiplex amplification system as an electrophoresis sample to be detected;

(5) electrophoresis: soaking and washing the substrate for 2 minutes by using 1 XTTE buffer solution; after the buffer solution is added into the sample waste liquid pool, the buffer solution pool and the buffer solution waste liquid pool, the sample to be detected is injected into the sample pool; a sample introduction stage, wherein the sample cell is 0V, the sample waste liquid cell is + 500-plus 800V, the buffer liquid cell is +0-200V, the buffer liquid waste liquid cell is + 300-plus 800V, and the voltage is switched to enter a separation stage after sample introduction is carried out for 5-60 seconds; during separation, the buffer solution waste liquid pool is +3000-4000V, the buffer solution pool is 0V, the sample pool and the sample waste liquid pool are both applied with +500-1100V, and the operating temperature is 25 ℃; and obtaining the peak time and relative content data of 5 autosomal STR gene loci in the serum sample.

The size of the chip is 63mm multiplied by 30mm, the width of the chip pipeline is 90 μm, the depth is 10-30 μm, the length from the chip sample pool to the cross point is 5mm, the effective separation length is 160mm, and the diameters of the sample pool, the sample waste liquid pool, the buffer liquid pool and the buffer liquid waste liquid pool are all 2 mm.

The invention separates STR loci (TPOX, vWA, D5S818, FGA and D2S1338) on 5 human autosomes in 12 minutes based on the chip electrophoresis technology combined with a laser-induced fluorescence detection system, and completes the autosomal STR typing test of a real blood sample. The chip electrophoresis has high resolution and rapid separation and analysis capability for autosomal STR typing, so that the chip electrophoresis is hopeful to become one of analysis means for rapidly, simply and efficiently separating and detecting the human chromosome STR gene loci.

Drawings

Figure 1 is a schematic diagram of the structure of an electrophoresis chip,

wherein: 1, a sample cell; 2, a sample waste liquid pool; 3, a buffer liquid pool; 4, a buffer solution waste liquid pool; 5, detecting points; 6 a cross pipe.

FIG. 2 is an electropherogram of an autosomal STR allelic typing standard.

FIG. 3 shows the result of electrophoresis of autosomal STR allelic sites of human blood sample No. 1 in example 1.

FIG. 4 shows the result of electrophoresis of autosomal STR allelic sites of human blood sample No. 2 in example 1.

Detailed Description

The invention is further illustrated by the following figures and examples.

Example 1

Electrophoretic analysis of autosomal STR allelic typing standard

1) Selection of electrophoresis buffer system:

the sample injection buffer solution system comprises 1 XTTE buffer solution composed of 50mmol/L of Tris (hydroxymethyl) aminomethane (Tris), 50mmol/L N-Tris (hydroxymethyl) methyl-3-aminopropane sulfonic acid (TAPS) and 1mmmol/L of Ethylene Diamine Tetraacetic Acid (EDTA), and the pH value is 8.3; the sample separation system consists of POP 4 or POP 7 glue;

2) preparation of autosomal STR allelic typing standards

Based on the consideration of gene polymorphism (GD >0.6) and allele span in Chinese population, five autosomal loci with large polymorphism and small allele span in Chinese population are selected as research objects. An autosomal kit was co-produced under the co-operation with Wuxi Zhongde Mei Co., Ltd at loci including TPOX, vWA, D5S818, FGA and D2S 1338. The 5 loci are divided into two groups, and are respectively marked by HEX and TAMRA groups, the molecular weight internal standard is marked by a third fluorescent dye, and a 5 autosomal STR locus multiplex amplification system is formed and used as an electrophoresis sample to be detected.

3) Chip electrophoresis

The chip structure is shown in fig. 1. The chip comprises a sample pool 1, a sample waste liquid pool 2, a buffer liquid pool 3, a buffer liquid waste liquid pool 4 and a cross pipeline 6;

the left end and the right end of a cross arm of the cross pipeline 6 extend towards the vertical direction respectively and are communicated with a buffer solution waste liquid pool 4 and a buffer solution pool 3 respectively, the upper end of a longitudinal arm of the cross pipeline 6 is communicated with the sample pool 1, and the lower end of the longitudinal arm of the cross pipeline 6 is communicated with the sample waste liquid pool 2 after passing through a U-shaped extension section; a detection point 5 is arranged on a cross arm between the cross part of the cross pipeline and the buffer solution waste liquid pool;

the chip is made of quartz glass through photoetching, wet etching and low-temperature bonding, the size of the chip is 63mm multiplied by 30mm, the width of a chip pipeline is 90 mu m, the depth of the chip pipeline is 30 mu m, the length from a chip sample pool to a cross point is 5mm, the effective separation length is 160mm, and the diameters of the sample pool, the sample waste liquid pool, the buffer liquid pool and the buffer liquid waste liquid pool are all 2 mm. The sample pool and the waste sample liquid pool are sample feeding channels, and the buffer liquid pool and the waste buffer liquid pool are separation channels.

Before analysis, chip surface modification is carried out, namely 1M NaOH is introduced into a chip channel, the temperature is kept at 65 ℃ for 15min, the NaOH is drained, 1M HCl is added, the room temperature is kept for 15min, 0.25 percent pHEA is added after the drainage, the room temperature is kept for 1 h, and finally acetonitrile is used for draining the microchannel and placing the microchannel at the room temperature for standby. After the separation buffer solution is added into the sample waste liquid pool 2, the buffer liquid pool 3 and the buffer liquid waste liquid pool 4, the sample to be detected is injected into the sample pool 1. A sample introduction stage, wherein the sample cell is 0V, the sample waste liquid cell is + 500-plus 800V, the buffer liquid cell is +0-200V, the buffer liquid waste liquid cell is + 300-plus 800V, and the voltage is switched to enter a separation stage after sample introduction is carried out for 5-60 seconds; during separation, the buffer solution waste liquid pool is +3000-4000V, the buffer solution pool is 0V, the sample pool and the sample waste liquid pool are both applied with +500-1100V, and the operating temperature is 25 ℃.

4) Results interpretation profiles and data were processed using Microsoft excel, origin8.0 and SPASS11.5 software packages. Making a standard electrophoresis pattern by an autosomal STR allelic gene typing standard substance, and then selecting a tested blood sample for comparison analysis to obtain parameters such as the peak emergence time, the relative content and the like of an autosomal STR gene locus of the blood sample. The allelic site represented by each peak has been reported.

Example 2

Electrophoretic analysis of autosomal STR alleles from blood samples

1) Electrophoresis buffer system

The sample injection buffer solution system comprises 1 XTTE buffer solution composed of 50mmol/L of Tris (hydroxymethyl) aminomethane (Tris), 50mmol/L N-Tris (hydroxymethyl) methyl-3-aminopropane sulfonic acid (TAPS) and 1mmmol/L of Ethylene Diamine Tetraacetic Acid (EDTA), and the pH value is 8.3; the sample separation system consists of POP 4 or POP 7 glue.

2) Preparation of autosomal STR allelic typing standards

3) Chip electrophoresis: same as example 1, step (3).

4) And (4) interpretation of results:

FIG. 2 is an electropherogram of an autosomal STR allelic typing standard. The chip electrophoresis mainly uses electroosmotic flow as the driving force of an autosomal STR zone, and gene loci with different charge-mass ratios are separated under the action of the electroosmotic flow during electrophoresis. The highest resolution of the micro-fluidic chip electrophoresis for separating five allelic gene typing standard substances is less than or equal to 4 bp. Five gene site single-stranded DNA fragments with the total length of less than 250bp are subjected to baseline separation on chip electrophoresis, and the separation time is less than 12 min.

Wherein the No. 1 blood sample autosomal STR allele parting electrophoretic separation analysis

Electrophoresis buffer system, preparation of blood autosomal STR allele typing, and chip electrophoresis same autosomal STR allele typing standard substance analysis method;

and (4) interpretation of results: FIG. 3 is an electropherogram of an autosomal STR allelic typing standard. The chip electrophoresis mainly uses electroosmotic flow as the driving force of autosomal STR, and gene loci with different charge-mass ratios are separated under the action of the electroosmotic flow during electrophoresis. The highest resolution of the micro-fluidic chip electrophoresis for separating five allelic gene typing standard substances is less than or equal to 4 bp. Five gene site single-stranded DNA fragments with the total length of less than 250bp are subjected to baseline separation on chip electrophoresis, and the separation time is less than 12 min.

Wherein the No. 2 blood sample autosomal STR allele parting electrophoretic separation analysis

and (4) interpretation of results: FIG. 4 is an electropherogram of an autosomal STR allelic typing standard. The chip electrophoresis mainly uses electroosmotic flow as the driving force of autosomal STR, and gene loci with different charge-mass ratios are separated under the action of the electroosmotic flow during electrophoresis. The highest resolution of the micro-fluidic chip electrophoresis for separating five allelic gene typing standard substances is less than or equal to 4 bp. Five gene site single-stranded DNA fragments with the total length of less than 250bp are subjected to baseline separation on chip electrophoresis, and the separation time is less than 12 min.

Claims

1. A human autosomal STR gene locus typing method based on a microfluidic chip is characterized by comprising the following steps: comprises the following steps:

(1) selecting an electrophoresis system: the sample injection buffer solution system comprises 1 XTTE buffer solution composed of 50mmol/L of tris (hydroxymethyl) aminomethane, 50mmol/L of L N-tris (hydroxymethyl) methyl-3-aminopropane sulfonic acid and 1mmmol/L of ethylene diamine tetraacetic acid, and the pH value is 8.3; the sample separation system consists of POP 4 or POP 7 glue;

(2) selecting an electrophoresis chip: the chip comprises a sample pool, a sample waste liquid pool, a buffer liquid waste liquid pool and a cross pipeline; the left end and the right end of a cross arm of the cross pipeline extend in the direction vertical to the longitudinal arm respectively and are communicated with the buffer solution waste liquid pool and the buffer solution pool respectively, the upper end of the longitudinal arm of the cross pipeline is communicated with the sample pool, and the lower end of the longitudinal arm of the cross pipeline is communicated with the sample waste liquid pool after passing through the U-shaped extension section; arranging a detection point on a cross arm between the cross part of the cross pipeline and the buffer solution waste liquid pool;

(3) chip surface modification: firstly, placing the chip in a vacuum drier for vacuumizing for 10 minutes; taking out the chip, immediately adding 1M NaOH into the chip channel, keeping the temperature at 65 ℃ for 15min, draining the NaOH, adding 1M HCl, keeping the temperature at room temperature for 15min, adding 0.1-0.5% of poly N-hydroxyethyl acrylamide after draining, keeping the temperature at room temperature for 1 h, and finally draining the microchannel with acetonitrile and placing the microchannel at room temperature for later use;

(4) selecting an autosomal STR locus, namely selecting five autosomal locus points for marking to form a 5 autosomal STR locus composite amplification system; as electrophoresis to-be-detected sample; the five autosomal locus points are TPOX, vWA, D5S818, FGA and D2S1338, respectively; the mark is specifically as follows: dividing 5 loci into two groups, marking TPOX and D2S1338 by HEX, marking vWA, D5S818 and FGA by TAMRA, marking the molecular weight internal standard by a third-color fluorescent dye ROX, and forming a 5 autosomal STR locus composite amplification system as an electrophoresis sample to be detected;

(5) electrophoresis: soaking and washing the substrate for 2 minutes by using 1 XTTE buffer solution; after the buffer solution is added into the sample waste liquid pool, the buffer solution pool and the buffer solution waste liquid pool, the sample to be detected is injected into the sample pool; a sample introduction stage, wherein a sample pool is 0V, a sample waste liquid pool is +800V, a buffer liquid pool is +200V, and a buffer liquid waste liquid pool is +700V, and voltage is switched to enter a separation stage after sample introduction is carried out for 10 seconds; during separation, the buffer solution waste liquid pool is +3600V, the buffer solution pool is 0V, the sample pool and the sample waste liquid pool are both applied with +900V, and the operation temperature is 25 ℃; the time to peak for the five autosomal locus sites was obtained.

2. The method for genotyping human autosomal STR genetic loci based on a microfluidic chip according to claim 1, wherein the method comprises the following steps: the size of the chip is 63mm multiplied by 30mm, the width of a chip pipeline is 90 μm, the depth of the chip pipeline is 30 μm, the length from a chip sample pool to a cross point is 5mm, the effective separation length is 160mm, and the diameters of the sample pool, the sample waste liquid pool, the buffer liquid pool and the buffer liquid waste liquid pool are all 2 mm.