UMMS Affiliation

Department of Pathology

Date

9-19-2007

Document Type

Article

Subjects

Amino Acid Motifs; Antigens, Viral; Cells, Cultured; Complementarity Determining Regions; *Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; *Genes, T-Cell Receptor beta; HLA-A2 Antigen; Humans; *Immunologic Memory; Influenza A virus; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes; Peptide Fragments; Sequence Homology, Nucleic Acid; T-Cell Antigen Receptor Specificity; T-Lymphocyte Subsets; T-Lymphocytes, Cytotoxic; Viral Matrix Proteins

Disciplines

Genetics and Genomics | Life Sciences | Medicine and Health Sciences

Abstract

BACKGROUND: Providing quantitative microarray data that is sensitive to very small differences in target sequence would be a useful tool in any number of venues where a sample can consist of a multiple related sequences present in various abundances. Examples of such applications would include measurement of pseudo species in viral infections and the measurement of species of antibodies or T cell receptors that constitute immune repertoires. Difficulties that must be overcome in such a method would be to account for cross-hybridization and for differences in hybridization efficiencies between the arrayed probes and their corresponding targets. We have used the memory T cell repertoire to an influenza-derived peptide as a test case for developing such a method.

RESULTS: The arrayed probes were corresponded to a 17 nucleotide TCR-specific region that distinguished sequences differing by as little as a single nucleotide. Hybridization efficiency between highly related Cy5-labeled subject sequences was normalized by including an equimolar mixture of Cy3-labeled synthetic targets representing all 108 arrayed probes. The same synthetic targets were used to measure the degree of cross hybridization between probes. Reconstitution studies found the system sensitive to input ratios as low as 0.5% and accurate in measuring known input percentages (R2 = 0.81, R = 0.90, p < 0.0001). A data handling protocol was developed to incorporate the differences in hybridization efficiency. To validate the array in T cell repertoire analysis, it was used to analyze human recall responses to influenza in three human subjects and compared to traditional cloning and sequencing. When evaluating the rank order of clonotype abundance determined by each method, the approaches were not found significantly different (Wilcoxon rank-sum test, p > 0.05).

CONCLUSION: This novel strategy appears to be robust and can be adapted to any situation where complex mixtures of highly similar sequences need to be quantitatively resolved.

Rights and Permissions

Citation: BMC Genomics. 2007 Sep 19;8:329. Link to article on publisher's site

DOI of Published Version

10.1186/1471-2164-8-329

Comments

© 2007 Wang et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Related Resources

Link to Article in PubMed

Journal Title

BMC genomics

PubMed ID

17880719

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