Method for obtaining immunoglobulin encoding nucleic acid

摘要

The current invention is directed to a method for obtaining a nucleic acid encoding an immunoglobulin variable domain from a single cell comprising the following steps: —performing a first polymerase chain reaction with three to six 5′-primer and one 3′-primer, performing with the product of the first polymerase chain reaction a second polymerase chain reaction with thirteen to sixteen 5′-primer and one 3′-primer, whereby the distance of the binding locations of the primer employed in the second polymerase chain reaction is reduced compared to the first polymerase chain reaction.

主权项

1. A method for obtaining a nucleic acid from a single cell, wherein said nucleic acid encodes an immunoglobulin variable domain selected from a heavy chain variable domain, a kappa light chain variable domain, and a lambda light chain variable domain, comprising: (a) performing a first polymerase chain reaction (PCR) to obtain a first PCR product; and (b) performing a second PCR with the first PCR product, whereby the distance of the binding locations of the primers employed in the second PCR is reduced compared to the distance in the first PCR, wherein:
i) for obtaining the nucleic acid encoding the immunoglobulin heavy chain variable domain, the first PCR is performed with 5′ primers comprising the nucleic acids of SEQ ID NO: 05 and/or 06, 07 and/or 08, 09, 10 and/or 11, 12, and 13, and with a 3′ primer comprising the nucleic acid of SEQ ID NO:104 or 105 or 106, and the second PCR is performed with 5′ primers comprising the nucleic acids of SEQ ID NO: 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, and 141 and/or 142 and with a 3′ primer comprising the nucleic acid of SEQ ID NO: 104 or 105 or 106 or 143, and ii) for obtaining the nucleic acid encoding the immunoglobulin kappa light chain variable domain, the first PCR is performed with 5′ primers comprising the nucleic acids of SEQ ID NO: 16, 17, 18, and 19, and with a 3′ primer comprising the nucleic acid of SEQ ID NO: 115, and the second PCR is performed with 5′ primers comprising the nucleic acids of SEQ ID NO: 53 and/or 54, 55 and/or 56, 57 and/or 58, 59, 60, 61 and/or 62, 63 and/or 64, 65, 66, 67, 68, 69, 70, and 144, and with a 3′ primer(s) comprising the nucleic acid(s) of SEQ ID NO: 115 and/or 145, and iii) for obtaining the nucleic acid encoding the immunoglobulin lambda light chain variable domain, the first PCR is performed with 5′ primers comprising the nucleic acids of SEQ ID NO: 21, 22, 23 and/or 24 and/or 25 and/or 26, and with a 3′ primer(s) comprising the nucleic acid(s) of SEQ ID NO:120 and/or 121 and/or 122 and/or 123 and/or 124 and/or 125, and the second PCR is performed with 5′ primers comprising the nucleic acids of SEQ ID NO: 72, 73 and/or 74, 75, 76, 77 and/or 78, 79, 80, 81, 82 and/or 83, 84 and/or 85, 86, 87 and/or 88, 89, 90 and/or 91, and 92, and a 3′ primer(s) comprising the nucleic acid(s) of SEQ ID NO: 120 and/or 121 and/or 122 and/or 123 and/or 124 and/or 125.