A PCR-based high-throughput screen with multiround sample pooling: Application to somatic cell gene targeting

Hiroyuki Konishi, Josh Lauring, Joseph P. Garay, Bedri Karakas, Abde M. Abukhdeir, John P. Gustin, Yuko Konishi, Ben H. Park

Research output: Contribution to journalArticlepeer-review

Abstract

Here, we describe a method of systematic PCR screening with multiround sample pooling for the isolation of rare PCR-positive samples. As an example, we have applied this protocol to the recovery of gene-targeted clones in human somatic cells comprising only 0.02-0.17% of cells transduced with targeting vectors. Initially, cells infected with targeting vectors are seeded and grown in fourteen 96-well tissue culture plates. Samples are then collected from these plates and subjected to two rounds of pooling to yield twelve 'superpools' used for an initial PCR. After identifying PCR-positive samples, de-pooling is carried out with successive rounds of PCR screening, using samples of decreasing complexity. Single-cell cloning is subsequently performed to isolate gene-targeted clones. The entire protocol can be completed in 4-8 weeks depending on the proliferative capacity of the cell line.

Original languageEnglish (US)
Pages (from-to)2865-2874
Number of pages10
JournalNature protocols
Volume2
Issue number11
DOIs
StatePublished - Nov 15 2007

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Fingerprint Dive into the research topics of 'A PCR-based high-throughput screen with multiround sample pooling: Application to somatic cell gene targeting'. Together they form a unique fingerprint.

Cite this