Haplotype-specific Extraction Research Articles

Validation of haplotype-specific extraction for separating a mitochondrial DNA model mixture and application to simulated casework

Haplotype-specific extraction (HSE) is a new field of application for the separation of mitochondrial DNA (mtDNA) mixtures and is developed to identify the mtDNA haplotypes of the contributors subsequently by sequencing. Here we show the validation of HSE with an exemplary mitochondrial DNA mixture into its individual haplotypes according to our laboratory standards. These specify several critical areas of assay performance to be tested, such as sensitivity, robustness and mixture studies comprising varying proportions of their components,degraded samples and samples of different qualities and material. Wereport the successful and unambiguous analysis of the exemplary separated mitochondrial DNA mixture under various conditions as well as simulated casework samples, which manifest as mixed nucleotide calls at single base positions previously. Here we demonstrate that the HSE assay is high sensitive, stable against degradation and applicable in a wide range of sample qualities. Based on our findings from the validation study, we believe that this assay has great potential power and may be useful for distinguishing among the mtDNA of individuals and their geographical origin in mixed DNA samples.

New application for haplotype-specific extraction: Separation of mitochondrial DNA mixtures

A particularly challenging aspect of forensic casework is the accurate interpretation of samples that contain DNA from more than one contributor, since DNA analysis of specific identification markers often results in ambiguous mixed profiles. Physically separating the various haploid components of a diploid or polyploid DNA sample or of a specimen containing DNA from multiple individuals can resolve this problem. We adapted and combined previously developed methods of haplotype-specific extraction (HSE) to separate and analyze mitochondrial DNA (mtDNA) in samples containing DNA from two individuals. After optimizing conditions and probes, we performed HSE to separate mitochondrial DNA mixtures, which manifest as mixed nucleotide calls at single base positions. We were able to subsequently identify the DNA of the two individuals by sequencing. Based on our findings from this proof-of-concept experiment, this novel assay will be useful for distinguishing among the mtDNA of individuals in mixed DNA samples.

Separation of Y-chromosomal haplotypes from male DNA mixtures via multiplex haplotype-specific extraction

In forensic analysis, the interpretation of DNA mixtures is the subject of ongoing debate and requires expertise knowledge. Haplotype-specific extraction (HSE) is an alternative method that enables the separation of large chromosome fragments or haplotypes by using magnetic beads in conjunction with allele-specific probes. HSE thus allows physical separation of the components of a DNA mixture. Here, we present the first multiplex HSE separation of a Y-chromosomal haplotype consisting of six Yfiler short tandem repeat markers from a mixture of male DNA.

New Prediction Model for Probe Specificity in an Allele-Specific Extension Reaction for Haplotype-Specific Extraction (HSE) of Y Chromosome Mixtures

Allele-specific extension reactions (ASERs) use 3′ terminus-specific primers for the selective extension of completely annealed matches by polymerase. The ability of the polymerase to extend non-specific 3′ terminal mismatches leads to a failure of the reaction, a process that is only partly understood and predictable, and often requires time-consuming assay design. In our studies we investigated haplotype-specific extraction (HSE) for the separation of male DNA mixtures. HSE is an ASER and provides the ability to distinguish between diploid chromosomes from one or more individuals. Here, we show that the success of HSE and allele-specific extension depend strongly on the concentration difference between complete match and 3′ terminal mismatch. Using the oligonucleotide-modeling platform Visual Omp, we demonstrated the dependency of the discrimination power of the polymerase on match- and mismatch-target hybridization between different probe lengths. Therefore, the probe specificity in HSE could be predicted by performing a relative comparison of different probe designs with their simulated differences between the duplex concentration of target-probe match and mismatches. We tested this new model for probe design in more than 300 HSE reactions with 137 different probes and obtained an accordance of 88%.

On the trail of anti‐CDE to unexpected highlights of the RHD*weak 4.3 allele in the Upper Austrian population

The application of a commercial available microcolumn system for ABO/RH determination lead to irregular results in CDE typing of seemingly D- blood samples. In this study, we introduce a comprehensive serological and molecular work-up of a novel haplotype carrying the RHD*weak 4.3 in combination with an aberrant RHCE*ce. The molecular background was characterized by RHD and RHCE-specific DNA sequencing, RHD cDNA sequencing and RHD zygosity testing. Haplotype-specific extraction and inheritance analysis were initiated to determine the linkage of the polymorphisms. The genetic admixture was studied by whole genome SNP array analysis. Serology was done using commercial available standard techniques and by in-house sera likewise. All samples (n = 29) were shown to harbour an altered RHD(T201R, F223V, P291R) allele known as RHD*weak 4.3 associated with a RHCE*ce(W16C, A36T, L245V) gene formation, expressing C(X) and VS. Both anti-C(X) and anti-V/VS were detected as contaminating antibodies in a commercial available microcolumn system for ABO/RH determination accounting for the positive results in CDE typing. Compared with other population data, the samples were clearly identified as Caucasian. The RHD*weak 4.3 allele with markedly reduced antigen D expression was shown to be associated with an altered RHCE gene formation leading to the expression of C(X) and VS. Its frequency was estimated 1 in 854 among apparently D- Upper Austrian blood donors.

Individual specific extraction of DNA from male mixtures—First evaluation studies

In forensic work, the interpretation of DNA profiles becomes complicated when samples contain more than one contributor because the simultaneous amplification of individual identification markers results in mixed profiles. To overcome this problem, we present haplotype-specific extraction (HSE) as a more straightforward method to analyze a DNA mixture. HSE has been developed to clarify ambiguous HLA alleles by separating diploid samples into their haploid components to facilitate HLA typing. We have started to establish new protocols and strategies to adapt HSE for the separation of male DNA mixtures in forensic analysis. First results have shown an improved enrichment of male DNA from a single contributor. We have also evaluated a new, optimized buffer composition by testing different concentrations of its components. Improved separation of a male DNA mixture is detected using AmpFℓSTR® Yfiler short tandem repeat analysis.

Molecular genetic analysis of a new B112 allele of ABO blood group

To analyze the molecular genetic basis for a new B112 allele of ABO blood group and the pedigree of the proband. The ABO group antigens on red cells of the proband were identified by monoclonal antibodies. The ABO antibody in serum was detected by using standard A, B and O cells. The exons 5-7 of ABO gene for the proband was amplified by polymerase chain reaction and the amplified products were digested with double enzymes and sequenced for exons 6 and 7. A magnetic bead-based, haplotype specific extraction was used to separate the diploid sample of the proband into its haploid components. The exons 6 and 7 of the two single ABO haplotypes were then amplified and sequenced separately. The samples of the parents of the proband were collected, and the blood group serological test and sequence analysis for exons 6 and 7 of ABO gene were performed. The serum characteristic of the proband was consisted with the normal B phenotype. The DNA sequencing of exons 6 and 7 showed 261G/del, 297A/G, 526C/G, 559C/T, 657C/T, 703G/A, 796C/A, 803G/C and 930G/A heterozygotes and was assigned for B/O genotype. After separation of the two single strands of the proband with haplotype specific extraction, a B112 and an O01 allele were identified after sequencing. The B112 allele had one nucleotide change (C to T) at position 559 compared with B101, which resulted in an amino acid change at position 187 (Arg to Cys). The B112 in the proband was identified to inherit from his mother after pedigree analysis, the ABO blood group serological characteristics and sequences of exons 6 and 7 of the mother were identical to that of the proband. A novel B112 allele of ABO group system with 559C>T was identified. It had normal B antigen expression, suggesting that Arg118Cys of alpha-1, 3 galactosyltransferase did not affect its enzyme activity.

Comprehensive polymorphism analysis of ABO using allele‐specific separation by bead technology and subsequent sequencing

The ABO system is the most important blood group system in transfusion and transplantation medicine. Over the last decade ABO genotyping was introduced to complement serological analysis but because of the enormous diversity at the ABO locus, DNA-based typing is highly sophisticated. In some genotype combinations and especially when dealing with hybrid genes, cis/trans linkage analysis is necessary. Therefore, we developed a simple and reliable approach to isolate ABO haplotypes. The described method physically separates diploid genomic DNA using probe-dependent allele hybridization followed by magnetic bead separation. After this haplotype-specific extraction (HSE), sequencing based typing (SBT) was performed to analyze coding and non-coding respectively regulatory regions of ABO.

Identification of a New HLA-B Allele (B*1576) by Haplotype Specific Extraction

Routine typing of a potential bone marrow donor by sequence-specific oligonucleotide probes (SSOP) and sequence based typing (SBT) produced inconclusive subtyping results, suggesting a new allele. A magnetic bead-based method, haplotype specific extraction (HSE), was used to separate the diploid sample into its haploid components. The sample was then re-typed using standard SBT, revealing a new human leukocyte antigen (HLA) allele, since named B*1576. HSE used in conjunction with standard SBT is a convenient and simple tool for resolving ambiguous and novel allele combinations without the need for amplification or subcloning.

Haplotype‐specific extraction: a universal method to resolve ambiguous genotypes and detect new alleles – demonstrated on HLA‐B

Haplotype-specific extraction (HSE) allows the collection of individual alleles by separating diploid samples into their haploid components. The separation step is performed using magnetic beads in conjunction with allele-group-specific probes. The haplo-separated DNA samples can be directly typed with downstream applications such as sequence-specific oligonucleotide (SSO) typing, sequence-specific primer (SSP) typing, and sequence-based (SB) typing. Here we show that HSE permits the direct sequencing of an allele in its individual, separated state, including previously unknown alleles. Allele pair combinations that cannot be resolved by SSP, SSO, or generic SB typings can be unambiguously typed after the alleles are separated by HSE, which allows for new alleles to be easily detected without cloning. We show how HSE was performed to separate samples with locus-specific ambiguities in human leukocyte antigen (HLA)-B, which could not be resolved by means of generic SB typing for either sample. After haplotype-specific separation of the respective allele pairs, novel polymorphisms in the HLA-B*56 and HLA-B*44 alleles were clearly detected by SB typing.

68-OR: Haplotype linkage of HLA-B and HLA-C by tiled HSE (Haplotype-Specific Extraction)

68-OR: Haplotype linkage of HLA-B and HLA-C by tiled HSE (Haplotype-Specific Extraction)

Multiplexed haploseparation of genomic DNA for HLA-A, HLA-B and HLA-DRB1 by haplotype-specific extraction (HSE)

Multiplexed haploseparation of genomic DNA for HLA-A, HLA-B and HLA-DRB1 by haplotype-specific extraction (HSE)

A novel HLA-B*420502 allele identified by PCR-SSO/SSP routine typing and confirmed by Sequencing-based typing*

A novel human leukocyte antigen-B (HLA-B) allele, B*420502, was identified in a patient with leukemia (Caucasoid, Czech ancestry) and his mother during intrafamily search for the hematopoietic stem cell donor. The novel allele was initially detected by HLA typing at low resolution using both sequence specific primers and sequence specific oligonucleotides techniques that resulted in unique reaction patterns. The alleles of the HLA-B locus were separated by the haplotype-specific extraction technique. Sequencing of those alleles revealed a novel allele, B*420502, that is identical with B*420501 except a T-->G exchange (synonymous mutation) at position 618.

Separation and SNP-typing of genomic DNA by Haplotype-specific extraction and TaqMan

Separation and SNP-typing of genomic DNA by Haplotype-specific extraction and TaqMan

Amplification of haplo-separated genomic DNA by haplotype-specific extraction (HSE) and whole-genome amplification (WGA)

Amplification of haplo-separated genomic DNA by haplotype-specific extraction (HSE) and whole-genome amplification (WGA)