Sequence structure of 12 novel Y chromosome microsatellites and PCR amplification strategies

doi:10.1016/S0379-0738(01)00405-4

Forensic Science International

Volume 122, Issue 1, 15 October 2001, Pages 19-26

https://doi.org/10.1016/S0379-0738(01)00405-4 Get rights and content

Abstract

In this work, we present sequencing data for 12 recently reported Y STR loci (DYS434, DYS435, DYS436, DYS437, DYS438, DYS439, GATA A10, GATA 7.1, GATA 7.2, GATA C4, GATA H4, GATA A4), as well as the PCR multiplex strategies we implemented for their detection.

Sequenced allelic ladders were constructed and a nomenclature for these new systems is proposed based on the sequence structure and following ISFG recommendations.

GATA A4 and DYS439 are likely the same STR. They have the same STR structure and the alleles are always the same in the same individuals.

Sequence polymorphisms were observed in the GATA C4 and DYS437 STRs. The variation in DYS437 was associated with a specific population group and is very interesting not only for forensic genetics but also for anthropological studies.

Introduction

The use of Y-chromosome polymorphisms, especially STRs, has become common place in forensic laboratories [1], [2], [3], [4], [5], [6]. Despite the general scarcity of Y-specific polymorphisms [7], the non-pseudoautosomal region of the Y chromosome contains several examples of polymorphisms including biallelic markers, STRs, the minisatellite MSY1 and the alphoid system [8]. Among all these markers, the Y STRs are the most frequently used for forensic purposes. However, compared to the large number of autosomal STR loci that have been described, the number of Y chromosome specific STRs is much lower.

Even fewer Y-STR loci — namely DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392 and DYS393 — currently meet the criteria necessary for their application in forensic genetics [9], [10].

Recently, several new Y microsatellites have been reported: GATA A10, GATA A7.1, GATA A7.2, GATA C4, GATA H4, GATA A4 described by White et al. [11] and DYS434, DYS435, DYS436, DYS437, DYS438, DYS439 described by Ayub et al. [12]. All these systems have tetranucleotide repeat motifs with the exception of the trimeric DYS436 and the pentameric DYS438.

In this work, we report data of significance for population and forensic studies. This includes sequence analysis of the different alleles of each STR, and the construction of sequenced allelic ladders. Based on the sequencing results obtained, a nomenclature is proposed for these systems following the recommendations of the DNA Commission of the International Society for Forensic Genetics (ISFG) [13].

Section snippets

DNA samples

Population samples were obtained from unrelated individuals from major populations groups (Europeans, Asians, Afro-American and Afrocaribbean). A few samples from primates were also sequenced (Pan troglodytes, Macacus rhesus).

Genomic DNA was extracted using a phenol–chloroform procedure and quantified using either the Quantiblot kit (Perkin-Elmer, Foster City, CA) or fluorescence detection with DyNAQuant 200 (APB, Uppsala, Sweden).

Multiplex amplification

Two multiplexes were designed (see Table 1). Multiplex I

Sequencing data and nomenclature

Sequencing results and consensus structure of the Y chromosome STRs described by White et al. [11] are shown in Table 2. In general they are simple STRs usually with a block of different types of constant repetitive stretches and one variable stretch.

GATA A7.2 (GenBank accession number G42671) is a simple STR consisting of blocks of (TAGA)_n repeats with the general structure (GATA)₂…(TAGA)₂…(TAGA)_n.

GATA A10 (GenBank accession number G42674) is another simple STR with several constant repetitive

Acknowledgements

This work was supported by grants from the Xunta de Galicia XUGA 20806B97 and PIPGIDT99PXI20807B.

The technical assistance of Meli Rodriguez is highly appreciated.

References (17)

L Gusmão et al.
Robustness of the Y STRs DYS19, DYS389I and II, DYS390 and DYS393: optimization of a PCR pentaplex
Forensic Sci. Int.
(1999)
M Prinz et al.
Multiplexing of Y chromosome specific STRs and performance for mixed samples
Forensic Sci. Int.
(1997)
L Gusmao et al.
Y chromosome specific polymorphisms in forensic analysis
Legal Medicine
(1999)
L Roewer et al.
A new method for the evaluation of matches in non-recombining genomes:application to Y-chromosomal short tandem repeat (STR) haplotypes in european males
Forensic Sci. Int.
(2000)
P White et al.
New, male-specific microsatellite markers from the human Y chromosome
Genomics
(1999)
L Gusmao et al.
Alternative primers for DYS391 typing: advantages of their application to forensic genetics
Forensic Sci. Int.
(2000)
B.M Dupuy et al.
DXYS267: DYS393 and its X chromosome counterpart
Forensic Sci. Int.
(2000)
M.A Jobling et al.
The Y chromosome in forensic analysis and paternity testing
Int. J. Legal Med.
(1997)

There are more references available in the full text version of this article.

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The DNA Commission of the International Society of Forensic Genetics (ISFG) regularly publishes guidelines and recommendations concerning the application of DNA polymorphisms to the problems of human identification. A previous recommendation published in 2001 has already addressed Y-chromosome polymorphisms, with particular emphasis on short tandem repeats (STRs). Since then, the use of Y-STRs has become very popular, and a numerous new loci have been introduced. The current recommendations address important aspects to clarify problems regarding the nomenclature, the definition of loci and alleles, population genetics and reporting methods.
Y chromosomal STR haplotypes in the northeastern China Han population
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Persisting fetal microchimerism does not interfere with forensic Y-chromosome typing
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Forensic Y-chromosome typing applies Y-chromosomal polymorphisms to the analysis of male/female mixed stains such as vaginal swabs in rape cases. The sensitivity of this approach exceeds that of cytological techniques combined with autosomal DNA typing. Y-chromosome typing is based on the assumption that Y-chromosomal DNA found in tissue or secretions of women must originate from a male individual, usually the perpetrator. Nevertheless, it was shown recently that fetal cells can migrate into the female body during pregnancy and can persist for decades (“persisting fetal microchimerism”). The body of a woman after a pregnancy with a male embryo can thus display a small fraction of fetal cells with Y-chromosomes. Using high sensitivity PCR protocols (reamplification with nested primers and up to 60 PCR cycles) fetal cells were previously identified in a number of maternal tissues including skin, blood, muscle and solid organs. It is, however, not clear at present, whether these cells can occur in vaginal secretions, and whether they are capable of producing false positive results in forensic Y-chromosome typing. To evaluate these questions, 66 blood samples of women with at least one son and nine vaginal swabs of women without sexual intercourse in the last 2 weeks were amplified for a stretch of the SRY gene. Eight thyroid gland tissues with already established male fetal microchimerism were used as positive control samples. Blood samples of 10 young girls without history of pregnancy were used as negative controls. Using a PCR with 10 ng of extracted DNA and 30 PCR cycles (“routine sensitivity assay”) none of the samples yielded positive results. However, in a PCR with 200 ng of extracted DNA and 45 PCR cycles (“high sensibility assay”), 14% of the blood samples of mothers and 33% of the vaginal swabs amplified for SRY. Our results thus show that increasing the sensitivity of the PCR method and the amount of template DNA produce positive results while protocols used for routine Y-chromosomal typing with small amounts of DNA (approximately 10 ng of DNA) and with a limited number of PCR cycles (approximately 30) can clearly eliminate this peril.
High-throughput Y-STR typing of U.S. populations with 27 regions of the Y chromosome using two multiplex PCR assays
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Two Y-chromosome short tandem repeat (STR) multiplex polymerase chain reaction (PCR) assays were used to generate haplotypes for 19 single copy and 3 multi-copy Y-STRs. A total of 27 PCR products were examined in each sample using the following loci: DYS19, DYS385 a/b, DYS388, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS426, DYS437, DYS438, DYS439, DYS447, DYS448, DYS450, DYS456, DYS458, DYS460, DYS464 a/b/c/d, H4, and YCAII a/b. The first multiplex is the Y-STR 20plex previously described by Butler et al. [Forensic Sci. Int. 129 (2002) 10]. The second multiplex is a novel Y-STR 11plex and includes DYS385 a/b, DYS447, DYS448 and the new markers DYS450, DYS456, DYS458, and DYS464 a/b/c/d. These two multiplexes were tested on 647 males from three United States population sample sets: 260 African Americans, 244 Caucasians, and 143 Hispanics. Haplotype comparisons between common loci included in the 20plex and 11plex assays as well as commercially available kits found excellent agreement across a sampling of the population samples. The multi-copy loci DYS464, DYS385, and YCAII were the most polymorphic followed by the following single copy Y-STRs: DYS458, DYS390, DYS447, DYS389II, DYS448, and DYS456. Samples containing the most common type in the European database could be well resolved with additional markers beyond the minimal haplotype loci.
Molecular characterization and Austrian Caucasian population data of the multi-copy Y-chromosomal STR DYS464
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DYS464 is a multi-copy STR system with four positions on the Y-chromosome (DYS464a, b, c, and d) which was recently identified and characterized [Forensic Sci. Int. 130 (2002) 97]. The aims of our study were to perform a population study, to estimate the mutation rate and an extensive sequence analysis in order to confirm the nomenclature. Fourteen different alleles were found in an Austrian population sample with an allele length varying from 9 to 19 repeats. All alleles were cloned and sequenced. Alleles 9–19 showed the general repeat structure (CCTT)_n … (CCTT)₂ … (CCTT)₃ … (CCTT)₄ … (CCTT)₂ … (CCTT)₂. The nomenclature is based on the number of repeated units of the variable (CCTT)_n-stretch only. In 13% of the samples intermediate alleles, namely 14.3A, 14.3B and 15.3 were detected. In these alleles the variable repeat block is interrupted by a CTT motif (14.3A: (CCTT)₃CTT(CCTT)₁₁; 14.3B and 15.3: (CCTT)₇CTT(CCTT)_7/8). A comparison with GenBank entries revealed the existence of a length variant due to a deletion of one cytosine in the 5′ flanking region of the first repeat block. We designed an alternative forward primer to circumvent possible ambiguities in the allele designation. A total of 54 different genotypes were identified in 135 men corresponding to a discrimination capacity (DC) of 40% and a gene diversity (GD) of 0.97. These values are much higher than those of other Y-chromosomal short tandem repeats (Y-STRs). DYS464 has the same haplotype diversity (HD) as the combination of the five Y-STR loci with the lowest gene diversities of the Y-STR core set. On the other hand, a combination of the three most diverse loci (DYS464, DYS385 and DYS390) has the same capacity to distinguish between paternal lineages than the complete minimal haplotype (minHT) consisting of eight Y-STR loci. In our population sample the addition of DYS464 to the minHT increases the number of different haplotypes from 110 to 122. The mutation-rate estimate based on the 70 meioses analyzed amounts to 2.86×10⁻² (95% confidence interval 3.5×10⁻³ to 9.95×10⁻²). This value is approximately 10 times higher than the average mutation-rate estimate for Y-STRs.
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Sequence structure of 12 novel Y chromosome microsatellites and PCR amplification strategies

Abstract

Introduction

Section snippets

DNA samples

Multiplex amplification

Sequencing data and nomenclature

Acknowledgements

Forensic Sci. Int.

Forensic Sci. Int.

Legal Medicine

Forensic Sci. Int.

Genomics

Forensic Sci. Int.

Forensic Sci. Int.

The Y chromosome in forensic analysis and paternity testing

Int. J. Legal Med.