Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, Australia.
Methods Mol Biol. 2024;2826:201-218. doi: 10.1007/978-1-0716-3950-4_15.
The immunoglobulin heavy constant gamma (IGHG) gene cluster encoding immunoglobulin G (IgG) subclasses is highly polymorphic, resulting in amino acid variation along the antibody constant heavy chain referred to as allotypes. IGHG1 and IGHG3 are the two most polymorphic IgG subclasses in humans, with 4 classical IgG1 allotypes and 13 allotypes described for IgG3, though recent studies suggest greater allelic diversity, especially in underrepresented ethnic populations. Polymerase chain reaction (PCR) and Sanger sequencing of IGHG amplicons allow for the identification of the single nucleotide polymorphisms (SNPs) responsible for the observed amino acid substitutions. Here, we provide a detailed protocol for the amplification of IGHG1 and IGHG3 segments by PCR, sample preparation for Sanger sequencing, and analysis of sequencing data to identify SNPs associated with different IgG1 and IgG3 allotypes.
免疫球蛋白重链恒定区 γ（IGHG）基因簇编码免疫球蛋白 G（IgG）亚类，高度多态性，导致沿抗体重链恒定区的氨基酸变异，称为同种异型。IGHG1 和 IGHG3 是人类中最具多态性的 IgG 亚类，IgG1 有 4 种经典同种异型，IgG3 有 13 种同种异型，但最近的研究表明，等位基因多样性更大，尤其是在代表性不足的种族群体中。聚合酶链反应（PCR）和 IGHG 扩增子的 Sanger 测序可鉴定导致观察到的氨基酸取代的单核苷酸多态性（SNP）。在这里，我们提供了一个详细的方案，用于通过 PCR 扩增 IGHG1 和 IGHG3 片段、Sanger 测序的样品制备以及分析测序数据以鉴定与不同 IgG1 和 IgG3 同种异型相关的 SNP。
