Sequencing of Antibody Chains by 5' SMART RACE

Sequencing of mRNAs encoding heavy and light chains of antibodies was performed using 5′ SMART RACE method as described previously [49 (link)] with slight modifications. The first strand of cDNA was synthesized using Mint kit (Evrogen, Moscow, Russia) with primers for k-chain (TTG TCG TTC ACT GCC ATC AAT C), λ-chain (GGG GTA CCA TCT ACC TTC CAG), and heavy-chain (CTG GAC AGG GAT CCA GAG TTC CA) according to the manufacturer’s protocol. Next, cDNA encoding immunoglobulins was amplified by conventional PCR using M1 forward primer (AAG CAG TGG TAT CAA CGC AGA GT) and revers primers specific for k-chain (ACA TTG ATG TCT TTG GGG TAG AAG), λ-chain (ATC GTA CAC ACC AGT GTG GC), and heavy-chain (GGG ATC CAG AGT TCC AGG TC). Obtained DNA was purified and cloned into pKAN-T (Evrogen, Moscow, Russia) vector that was subsequently sequenced. At least 3 different clones were sequenced for each chain of the antibodies. Immunoglobulin sequences were analyzed using IgBLAST (https://www.ncbi.nlm.nih.gov/igblast/), BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE), SignalP-5.0 (http://www.cbs.dtu.dk/services/SignalP/) and Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) software.

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Antipova N.V., Larionova T.D., Siniavin A.E., Nikiforova M.A., Gushchin V.A., Babichenko I.I., Volkov A.V., Shakhparonov M.I, & Pavlyukov M.S. (2020). Establishment of Murine Hybridoma Cells Producing Antibodies against Spike Protein of SARS-CoV-2. International Journal of Molecular Sciences, 21(23), 9167.

Publication 2020

Mint kit

Antibodies chain Antibodies light chains Cdna Clones Immunoglobulin Mint Mrnas Pkan Primers Vector

Corresponding Organization : Institute of Bioorganic Chemistry

Other organizations : National Research University Higher School of Economics, Peoples' Friendship University of Russia, Ministry of Health of the Russian Federation, City Clinical Hospital

Top 5 similar protocols

Variable analysis

independent variables

5' SMART RACE method
Primers for k-chain, λ-chain, and heavy-chain
M1 forward primer
Reverse primers specific for k-chain, λ-chain, and heavy-chain

dependent variables

Sequencing of mRNAs encoding heavy and light chains of antibodies

control variables

Manufacturer's protocol for Mint kit
Cloning into pKAN-T vector
Sequencing of at least 3 different clones for each chain of the antibodies

controls

Positive control: None specified
Negative control: None specified

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