Macdev tech center kit clone 511/14/2022 In addition, we suspect one particular clone contained a transcript from myeloma cells used for cell fusion because the sequence was obtained from multiple hybridomas. In a few cases, clones had sequence homology to the target antibodies, yet coded for ORFs unlikely to encode functional antibodies due to the presence of stop codons in their sequences. Amplification of various subclasses of mouse antibody genes. Sequence analysis showed that 90% of the H-chain clones and 50% of the L-chain clones accurately coded for the N-terminal amino acid sequence for their respective target antibody proteins.įigure 4. PCR products were obtained from all antibody subclasses. We then performed the same type of gene sequence analysis of the variable antibody region for six additional hybridoma strains. Sequences were confirmed to contain ORFs corresponding to the amino acid sequences of the H and L chains obtained from analysis of the N-terminal amino acid sequence of the monoclonal antibody. Confirmation of successful amplification of antibody genes from mouse hybridomas.Īmplified products were cloned into pUC118 and then sequenced. First-strand cDNA synthesis and 5′-RACE PCR were performed according to the recommended protocols for both the heavy (H) and light (L) chains, and amplified products were confirmed by agarose gel electrophoresis of a portion of the reaction mixture.įigure 3. Total RNA was prepared and used as input for the SMARTer RACE cDNA Amplification Kit. Experimental workflow for 5′-RACE PCR of the antibody variable domain using the SMARTer RACE cDNA Amplification Kit.Ī mouse hybridoma strain was cultured and confirmed to produce a monoclonal antibody with H and L chains consisting of γ2a and κ chains, respectively. By using the SMARTer RACE method to perform first-strand cDNA synthesis from total RNA, directly followed by 5′-RACE PCR, it is possible to obtain not only 5′-terminal sequences but also cDNA fragments that contain full-length sequences of the variable regions.įigure 2. In preparation for cloning mouse hybridoma antibody genes, a primer for reverse transcription (RT primer) and a reverse primer for RACE PCR (5′-RACE primer) were designed for H, L (κ), or L (λ) chains based on the sequence of all mouse IgG classes registered in the NCBI nucleotide database. Rapid cloning of antibody genes derived from a mouse hybridoma Amplification of cDNA fragments with a high-fidelity polymerase ensures the accurate replication of nucleotide sequences needed for effective analysis. The robust system yields reliable results even with samples that are contaminated with genomic DNA and is appropriate for use in a wide range of applications. Optimization of the SMARTer RACE components and protocol dramatically reduce non-specific background and increase amplification efficiency. Furthermore, the addition of the SMART sequence at the 5′ end enables the use of this site for downstream amplification and cloning. Most other RACE-PCR methods do not capture the 5′ end and therefore miss out on important information. This process incorporates our unique SMART ( Switching Mechanism at the 5′ end of RNA Template) technology, which allows the reverse transcriptase to reach the absolute 5′ end of the transcript. With the SMARTer RACE cDNA Amplification Kit, the SMARTer II A Oligonucleotide is added to the end of the cDNA during reverse transcription. Advantages of SMARTer technology in RACE applications SMARTer RACE kits bring complete 5′-end information and sensitivity to this process. A target region can then be cloned using the 5′ RACE ( Rapid Amplification of cDNA Ends) method, and subsequently sequenced. An effective way to analyze these variable domains is to design a degenerate primer in a constant region downstream of the variable region, where the sequence is relatively conserved. The inherent variability in the upstream 5′ terminus of the H and L chains results in low sequence homology. When analyzing the variable region, designing universal primers that will amplify all possible antibody sequences is extremely difficult. Since it is the variable region on the N-terminal side of the H and L chains that determines antibody-antigen affinity, analysis of this region is of great interest. This process generates unique antigen receptors. Antibody molecules have heavy (H) and light (L) chain components with both constant and variable (V) regions V(D)J recombination is a nearly random rearrangement of the variable sequence with other gene segments. This diversity is achieved through V(D)J recombination that occurs in developing lymphocytes. The immune system produces 10 8 varieties of antibodies to defend against invading substances from the outside world. Solutions for sequence analysis of variable domains
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