RU2799797C2 - Method of obtaining molecular str markers of the human y chromosome for identifying an unknown individual and determining biological relationship by multiplex amplification, and a kit of oligonucleotides for implementing the method - Google Patents

Abstract

FIELD: molecular biology; biotechnology; forensic medicine; forensics.

SUBSTANCE: invention relates to the genetic research in the field of human identification. A method of obtaining molecular STR markers of the human Y chromosome for identifying an unknown individual and determining biological relationship by multiplex amplification and a set of synthetic oligonucleotide primers for multiplex polymerase chain reaction (PCR) are proposed. The genotyping kit includes a composition formed by 15 pairs of synthetic original oligonucleotide primers for loci in the human genome, including 17 STR markers of the Y chromosome, which allows multiplex PCR using primers with the following sequences of SEQ ID NO: 1 — SEQ ID NO 30. Synthetic oligonucleotide primers are designed to successfully amplify selected loci in a single multiplex reaction and subsequent “massive parallel sequencing” (NGS) sequencing.

EFFECT: invention can also be used for DNA testing to identify an unknown individual and determine biological relationship in the Russian population with high sensitivity, specificity and accuracy compared to existing analogues.

6 cl, 1 tbl, 1 ex

Description

The invention relates to the field of molecular biology, biotechnology, forensic medicine and forensics, genetic research in the field of human identification.

Currently, DNA analysis is one of the most evidence-based methods for studying biological material in the production of a forensic identification examination and has been used in forensic science since 1985. DNA analysis makes it possible to study sections of the genome specific to each person, and thus obtain a unique genetic "passport" or genetic profile of an individual. Individualizing traits, determined at the DNA level, are characterized by almost absolute stability, that is, they remain unchanged in the human body throughout life. In recent decades, various marker systems localized both on autosomes and on non-recombining regions of the Y chromosome and X chromosomes and in mitochondrial DNA have been widely used for the purposes of genetic identification of a person.

In the early 1990s, short tandem repeats (STRs) were introduced as a new type of polymorphic DNA marker and have since become the gold standard for human identification in DNA databases, criminal cases, paternity and kinship analysis, and missing person identification. Autosomal STRs and Y-chromosome STRs (Y-STRs) have been used successfully in forensic DNA analysis for many years.

The study of the non-recombining part of the Y-chromosome, specific only for men, is widely used in forensic and archaeological research, especially in cases where the analysis of standard autosomal markers turns out to be of little information. Today, analysis of markers consisting of short tandem repeats in the non-recombining region of the Y chromosome (Y-STR) is widespread in forensics, which allows you to specifically characterize the paternal line of the man whose sample is being studied. Moreover, the genotypes of Y-STR markers can be used in the study of a sample of mixed male and female DNA, which is problematic when analyzing autosomal markers. A similar problem often arises when studying mixed samples [1–4].

Thus, the analysis of Y-STR markers allows:

- Determine the paternal line of the test sample;

- to provide material for the investigation in the search and identification of the identity of an unknown man [1, 2, 5].

In addition, the analysis of Y-STR markers is used to establish the fact of male kinship, which is relevant in the study of controversial historical and archaeological cases, as well as in situations of missing people and identifying male victims of disasters. Moreover, using the databases being developed today, the Y-STR markers can also be used to suggest the geographic affiliation of the paternal line of an unknown man [1, 2].

The main method for genotyping STR markers is capillary electrophoresis. Based on this method, a large number of commercial kits containing different amounts of standard STR markers of the Y chromosome are used. Currently, there are a number of commercial kits that allow you to determine the Y-STR haplotype using multiplex PCR. These sets differ in the composition and number of STR markers and include highly polymorphic, standardized markers with a low mutation rate. With their use, modern databases were created containing the frequencies of Y-STR haplotypes. The largest of these databases is the YHRD [Y-STR Haplotype Reference Database (YHRD) v4.0 [6]. Below are the most known sets containing different numbers of STR markers of the Y chromosome:

- Yfiler (Thermo Fisher) - 16 Y chromosome STR markers

- Yfiler Plus (Thermo Fisher) - 24 STR Y chromosome markers

- PowerPlexY23 (Promega) - 23 Y chromosome STR markers

- Powerplex 35GY 8C (Promega) - 11 Y chromosome STR markers

- SurelD (compass / Health Gene Technologies, China) - 17 STR markers of the Y chromosome

Expressmarker 16+10Y (AGCU Scien Tech, China) - 10 Y chromosome STR markers

Expressmarker 16+18Y (AGCU Scien Tech., China) - 18 Y chromosome STR markers.

Recently, there have been developments of identification kits that use the most modern and technologically advanced approach based on the use of "massive parallel sequencing" (NGS). This approach is used, for example, in the set DNA Signature Prep Kit (Verogen, San Diego, CA, USA) [7], which contains 24 STR markers of the Y chromosome, amplified in a set with a large number of other molecular markers. The disadvantages of existing imported kits for DNA identification include their significant high cost, due to the need to use expensive consumables (specific DNA polymerases, PCR solutions, buffer solutions). To eliminate these shortcomings, a necessary condition is to increase the information content of the proposed set and its cost-effectiveness, as well as adaptation to the specifics of biological material samples used in forensic examinations.

For the successful use of Y chromosome STR markers in forensic science and forensic medicine, it is necessary to develop oligonucleotide primer compositions that provide simultaneous amplification (by multiplex PCR) of loci of the smallest possible size containing only STR markers for the Y chromosome for sequencing by "massive parallel sequencing" (NGS).

To solve this problem, the authors proposed a set of 15 pairs of synthetic oligonucleotide primers containing 17 STR markers of the human Y chromosome for analysis by multiplex amplification, followed by sequencing by "massive parallel sequencing" (NGS). The invention discloses the developed original synthetic oligonucleotide primers that allow amplification of STR loci, human Y chromosomes.

The genotyping kit includes a composition formed by 15 pairs of synthetic original oligonucleotide primers for loci in the human genome, including 17 loci with STR markers on the Y chromosome, allowing for multiplex PCR (primer nucleotide sequence SEQ ID NO: 1 - SEQ ID NO 30 (shown in Table 1).

The advantages of this kit is that the authors of the invention have developed a system of oligonucleotide primers with a minimum size of PCR products, which allows simultaneous analysis of many samples by the "mass parallel sequencing" method and includes only STR markers of the human Y chromosome.

Implementation of the invention

An example of genotyping a biological sample

The example shows a method for obtaining selected STR markers of the human Y chromosome from biological samples containing human genomic DNA by performing multiplex PCR analysis followed by "massive parallel sequencing" of the resulting multiplex PCR products and genotyping of selected markers using an adapted STRinNGS program [8].

Obtaining DNA

DNA can be obtained from any human biological material, any human tissue containing cell nuclei, for example, leukocytes, hair follicles, buccal epithelial cells, etc. DNA suitable for PCR can be isolated from the image using any previously widely used DNA extraction method. The authors recommend using ready-made kits for DNA extraction from various human tissues (for example, those provided by Qiagen).

Obtaining molecular STR markers of the Y chromosome includes four steps:

- Multiplex PCR of genomic DNA using a mixture of specific oligonucleotide primers;

- Preparation of fragment libraries from the obtained PCR products for sequencing on the Illumina platform;

- Carrying out large-scale parallel sequencing of the obtained libraries;

The oligonucleotide primer set is adapted to use DNA in an amount of 10 ng to 1 ng of DNA. If necessary, the DNA sample should be diluted in EB buffer. It is also necessary to carry out in parallel a control reaction (negative control), where deionized water is added instead of DNA.

Amplification of genome regions containing the studied markers

Amplification of genome regions containing the studied markers can be carried out using any kit designed for multiplex PCR; the authors used the Multiplex PCR kit from Qiagen. Multiplex PCR was performed on a GeneAmp PCR System 9700 Thermal Cycler from Applied Biosystems.

The composition of the reaction mixture for multiplex PCR:

- Oligonucleotide solution for multiplex PCR (final concentration from 1 nM to 0.5 µM

- Mixture of polymerase and buffer

- Deionized water

- DNA under investigation.

Amplification should be carried out in test tubes, strips, or plates designed for PCR.

In a separate sterile test tube, mix all the necessary components for one reaction, according to the calculation table (test tube with MasterMix solution).

* The suggested calculation for preparing the reaction mixture is suitable for working with DNA samples with concentrations above 10 ng/µl. When working with DNA samples with a lower concentration, the calculation of the amount of deionized water introduced into the reaction mixture should be carried out according to the formula (23-X), where X is the volume of the analyzed DNA preparation, which provides the required amount of DNA in the reaction mixture. For example, a DNA sample with a concentration of 250 pg/µl. It is necessary to add at least 4 µl of the DNA preparation (1 ng in total) and 19 µl of deionized water to the reaction mixture.

When using such an amplification system (2-mix buffer and polymerase), the minimum DNA concentration that can be used is 40 pg/µl.

After preparation, the mixture for amplification is placed in the cycler and the program is set:

95°C 15 minutes

94°C - 30 seconds

57°C - 90 seconds 35 cycles

72°C - 90 seconds

72°С - 10 minutes

4°С∞.

The presence of amplification products of the studied DNA and the absence of amplification products in the negative control are checked using agarose gel electrophoresis. Amplification products are purified using appropriate mini-columns (for example, Qiagen MinElute system) in accordance with the instructions for the reagent kit used. Purified PCR products obtained as a result of multiplex amplification and containing STR markers of the Y chromosome are used as templates for preparing fragment genomic libraries for sequencing.

Preparation of fragment libraries

First, it is necessary to measure the concentration of purified PCR fragments obtained at the previous stage of multiplex amplification on NanoDrop or Qubit (ThermoFisher) devices in accordance with the instructions for the device. For the preparation of fragment libraries, it is optimal to use about 300 ng of DNA of purified PCR products of multiplex amplification. The preparation of fragment genomic libraries is carried out using Illumina® reagent kits. TruSeq® DNA PCR-Free library prep kit according to the kit instructions. To sequence multiple samples of fragment libraries at the same time, you must use indexed adapters (for example, Dual index adapters Illumina). For each library sample, a separate indexable adapter is used, which must be recorded in a table for further consideration when analyzing the results of sequencing of several fragment library samples. For the preparation of fragment libraries, it is recommended/possible to use 1/2 the volume of all reagents included in the Illumina® kit. TruSeq® DNA PCR-Free library prep kit specified in the kit instructions.

Steps 1-3 are recommended but not required.

1. Purification of fragment libraries: Apply the entire volume of the eluate to one well of a 2% agarose gel with SYBR Gold stain (for example, using ready-made 2% E-gel (Invitrogen)). Perform electrophoresis using a 50 bp Ladder molecular weight marker for 10 minutes (if using the e-gel system (Invitrigen)).

2. Using a sterile disposable scalpel, cut out gel pieces containing library fragments from 220 bp. and more. Luminous fragments less than 150 bp in length. correspond to adapter dimers and should not be used for further analysis.

3. Purify the excised DNA fragments using the MinElute gele extraction kit (Qiagen). Elute in 20-22 µl of EB buffer (Qiagen).

Fragment libraries prepared as described above can be directly used for sequencing on the Illumina technology platform, having previously assessed the quality of the obtained fragment libraries.

Fragment libraries can be qualitatively and quantitatively evaluated using standard methods recommended by sequencing platform manufacturer Illumina. To streamline your workflow, we recommend the following steps: Measure fragment library concentrations on the Qubit using the HS Reagent Kit. For further sequencing, it is recommended to use fragment libraries with a concentration of at least 2 nM/µl.

Prior to sequencing, libraries should be multiplexed in equimolar amounts (library pooling for multiple samples). It is not allowed to pool libraries with the same indexes.

The prepared fragment libraries are sequenced by the method of parallel large-scale sequencing on the Illumina technology platform in the single-ended sequencing mode with a long read of at least 350 b.i. The recommended instrument is the MiSeq benchtop sequencer (Illumina). Genotyping was performed using the adapted program STRinNGS[8].

The authors analyzed 10 DNA samples from individuals and control DNA M2800 (Promega) in various DNA dilutions. As a result of the work, all the studied samples had amplified all the selected loci. The genotypes of the STR marker loci of the Y chromosome for the M2800 control DNA (Promega) determined using the adapted STRinNGS program coincided.

Bibliography

1. Roewer L.Y chromosome STR typing in crime casework // Forensic Sci. Med. Pathol. 2009 Vol. 5, #2. P. 77-84.

2. Kayser M. Forensic use of Y-chromosome DNA: a general overview // Hum. Genet. Springer Berlin Heidelberg, 2017. Vol. 136, no. 5. P. 621-635.

3. Hall A., Ballantyne J. The development of an 18-locus Y-STR system for forensic casework // Anal. bioanal. Chem. 2003 Vol. 376, no. 8. P. 1234-1246.

4. Purps J. et al. A global analysis of Y-chromosomal haplotype diversity for 23 STR loci // Forensic Sci. Int. Genet. 2014. Vol. 12. P. 12-23.

5. Willuweit S., Roewer L. The new chromosome haplotype reference database // Forensic Sci. Int. Genet. Elsevier Ireland Ltd, 2015. Vol. 15. P. 43-48.

6. http://www.yhrd.org.]

7. et al. Developmental validation of the MiSeq FGx Forensic Genomics System for Targeted Next Generation Sequencing in Forensic DNA Casework and Database Laboratories DNA typing Forensic DNA SWGDAM validation Next-Generation Sequencing (NGS) Massively parallel seq. 2017.

8. Carina Jonck, Xiaoqin Qian, Halimureti Simayijiang, Claus STRinNGS v2.0: Improved tool for analysis and reporting of STR sequencing data, Forensic Science International: Genetics, Volume 48, 2020, 102331, ISSN 1872-4973, https://doi.org/10.1016/j.fsigen.2020.102331.

Oligonucleotide sequence listing

SEQID NO:1

TAATACTTCGGGCCATGG

SEQ ID NO:2

GACTACTGAGTTTCTGTTATA

SEQ ID NO:3

GCATGGGTGACAGAGCTAG

SEQ ID NO:4

CCAATTACATAGTCCTCCTTTC

SEQ ID NO:5

CCAACTCTCATCTGTATTATCT

SEQ ID NO:6

TTATCCCTGAGTAGTAGAAGA

SEQ ID NO:7

AGTGGGAGAAATGGATGACAG

SEQ ID NO:8

TATTTTTACACATTTTTGGGCC

SEQ ID NO:9

TTCATTCAATCATACACCCATA

SEQ ID NO:10

GAGGGATAGGTAGGCAGGC

SEQ ID NO:11

ACCTACCAATCCCATTCCTTA

SEQ ID NO:12

AGAGGCAGTCATCGCAGTG

SEQ ID NO:13

TGGTCTTCTACTTGTGTCAATACAG

SEQ ID NO:14

AACTCAAGTCCAAAAAATGAGGT

SEQ ID NO:15

TGGGACTATGGGCGTGAG

SEQ ID NO:16

AGACCCTGTCATTCACAGATG

SEQ ID NO:17

TGGGGAATAGTTGAACGGTAAA

SEQ ID NO: 18

GAGGTTGTGGTGAGTCGAG

SEQ ID NO:19

TCGAGTTGTTATGGTTTTAGGTC

SEQ ID NO:20

GCTGGAATTCTTTACCCATCA

SEQ ID NO:21

AGAAAGGGAGATAGAGACATGG

SEQ ID NO:22

CTTCCTTAACGTGAATTTCCTCA

SEQ ID NO:23

GGGACCTTGTGATAATGTAAGATA

SEQ ID NO:24

CCATCAACTCAGCCCAAAACT

SEQ ID NO:25

GAGCAACAGGAATGAAACTCC

SEQ ID NO:26

GCCACCACGCCCACCCT

SEQ ID NO:27

ACCAGCCCAAATATCCATCA

SEQ ID NO:28

TGGAATGCTCTCTTGGCTTC

SEQ ID NO:29

TATGCTGAGGAGAATTTCCAA

SEQ ID NO:30

TATTCATCCATCTAATCTATC.

Claims (6)

1. A method for obtaining molecular STR markers of the human Y chromosome for identifying an unknown individual and determining biological relationship by the method of multiplex amplification, characterized in that for genotyping a composition formed by 15 pairs of synthetic original oligonucleotide primers is used for carrying out a multiplex polymerase chain reaction (PCR) with nucleotide sequences SEQ ID NO: 1 - SEQ ID NO: 30, flanking DNA regions, containing 17 STR markers of the human Y chromosome; PCR products corresponding to the selected primers are markers. 2. The method according to claim 1, characterized in that the PCR products obtained as a result of multiplex amplification, containing markers for identifying an unknown individual and determining biological relationship, are used as a template for preparing genomic libraries for determining the nucleotide sequence. 3. The method according to p. 1, where the determination of the nucleotide sequence of the amplification products (PCR products) is carried out by sequencing according to the method of "massive parallel sequencing" (NGS). 4. The method according to claim 1 for identifying an unknown individual and determining the biological relationship of an individual originating from Russian populations. 5. A method for identifying an unknown individual and determining biological relationship, including genetic studies of a biological sample containing DNA isolated from any biological material in accordance with standard methods that provide high-quality purification of DNA from proteins and impurities, using multiplex polymerase chain reaction (multiplex PCR) and subsequent determination of the nucleotide sequence of amplification products containing STR-markers of the Y-chromosome specific for loci in the human genome, obtained by the method according to item 1. 6. A set of oligonucleotides for implementing the method according to claim 1, used to detect the presence of molecular STR markers of the Y chromosome, intended to identify an unknown individual and determine biological relationship, including synthetic original primers with nucleotide sequences SEQ ID NO: 1 - SEQ ID NO: 30.