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Multiplexed, ligation-dependent probe amplification for rapid and inexpensive HLA-DQB1 allelotyping.

ABSTRACT: Many effective options exist to accurately type DNA for human leukocyte antigen (HLA) alleles. However, most of the existing methods are excessively costly in terms of overall monetary costs, DNA requirements, and proprietary software. We present a novel assay capable of resolving heterozygous HLA-DQB1 allelotypes at two digits, with even greater specificity for the HLA-DQB1*06 allele family, by using the multiplexed ligation-dependent probe amplification technology. This assay provides more specific allele data than genome-wide analysis and is more affordable than sequencing, making it a useful intermediate for researchers seeking to accurately allelotype human DNA samples.

SUBMITTER: Akers NK

PROVIDER: S-EPMC3431962 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Multiplexed, ligation-dependent probe amplification for rapid and inexpensive HLA-DQB1 allelotyping.

Akers N K NK Curry J D JD Smith M T MT Bracci P M PM Skibola C F CF

Tissue antigens 20110718 4

Many effective options exist to accurately type DNA for human leukocyte antigen (HLA) alleles. However, most of the existing methods are excessively costly in terms of overall monetary costs, DNA requirements, and proprietary software. We present a novel assay capable of resolving heterozygous HLA-DQB1 allelotypes at two digits, with even greater specificity for the HLA-DQB1*06 allele family, by using the multiplexed ligation-dependent probe amplification technology. This assay provides more spe ...[more]

PMID: 21762399

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Project description:BackgroundWith the zebrafish increasingly being used in immunology and infectious disease research, there is a need for efficient molecular tools to evaluate immune gene expression in this model species. RT-MLPA (reverse transcription - multiplex ligation-dependent probe amplification) provides a sensitive and reproducible method, in which fluorescently labelled amplification products of unique lengths are produced for a defined set of target transcripts. The method employs oligonucleotide probes that anneal to adjacent sites on a target sequence and are then joined by a heat-stable ligase. Subsequently, multiplex PCR with universal primers gives rise to amplicons that can be analyzed with standard sequencing equipment and relative quantification software. Allowing the simultaneous quantification of around 40 selected markers in a one-tube assay, RT-MLPA is highly useful for high-throughput screening applications.FindingsWe employed a dual-colour RT-MLPA probe design for chemical synthesis of probe pairs for 34 genes involved in Toll-like receptor signalling, transcriptional activation of the immune response, cytokine and chemokine production, and antimicrobial defence. In addition, six probe pairs were included for reference genes unaffected by infections in zebrafish. First, we established assay conditions for adult zebrafish infected with different strains of Mycobacterium marinum causing acute and chronic disease. Addition of competitor oligonucleotides was required to achieve peak heights in a similar range for genes with different expression levels. For subsequent analysis of embryonic samples it was necessary to adjust the amounts of competitor oligonucleotides, as the expression levels of several genes differed to a large extent between adult and embryonic tissues. Assay conditions established for one-day-old Salmonella typhimurium-infected embryos could be transferred without further adjustment to five-day-old M. marinum-infected larvae. RT-MLPA results were compared with results of previous transcriptome analyses and with real-time PCR data, demonstrating a good correlation between all expression analysis methods.ConclusionsThe RT-MLPA assay developed in this study provides a rapid, cheap, and robust analysis tool for simultaneous quantification of a set of 34 innate immune response genes. With adjustment of conditions, the assay is suitable for infection studies in both adult and embryonic zebrafish. Application of RT-MLPA will facilitate high-throughput screening of immune responses in the zebrafish model.

Dataset Information

Multiplexed, ligation-dependent probe amplification for rapid and inexpensive HLA-DQB1 allelotyping.

Publications

Multiplexed, ligation-dependent probe amplification for rapid and inexpensive HLA-DQB1 allelotyping.

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