Antibodies, A Laboratory Manual, Second Edition, Edited by Edward A. Greenfield

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Creation of Recombinant Antibodies Using 5-RACE

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Because of the complexity of the V gene family and the number of possible sequences for amplification, there may be a distinct advantage to using a PCR method that does not require a specific 5 primer to amplify the gene segment. 5-RACE, originally described by Frohman et al. (1998), is just such a method. Doenecke et al. (1997) successfully used 5-RACE to amplify mouse Ig genes that could not be amplified using degenerate oligonucleotides. In the original 5-RACE method, mRNA is a template for cDNA synthesis using either oligo(dT) priming or a gene-specific primer. Terminal deoxynucleotide transferase (TdT) is used to tail the first strand with a region of known sequence [such as poly(A)]. Once tailed, the second strand can be amplified using poly(T). Many modern variants of 5-RACE are currently available as kits, such as RLM-RACE (Invitrogen), SMARTer RACE (Clontech Laboratories), and ExactSTART (Epicentre). The following 5-RACE protocol is based on Schramm et al. (2000). It uses the terminal deoxynucleotide transferase (TdT) activity of reverse transcriptase, which allows non-templated addition of nucleotides to the end of the nascently made cDNA first strand. This method is similar in many respects to that of Matz et al. (1999). Additional information regarding 5-RACE is available in Protocol 9 in Chapter 7 of Green and Sambrook (2012). Note that the use of a template-switching method for creation of full-length cDNAs may be subject to patent considerations based on Clontech patent USP 5,962,272.

Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition

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