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Preparation and screening of an arrayed human genomic library generated with the P1 cloning system
N S Shepherd1, B D Pfrogner, J N Coulby
1Cancer Research Program, DuPont Merck Pharmaceutical Company, Wilmington, DE 19880-0328.
Summary
Researchers created a human genomic library using bacteriophage P1 cloning, yielding over 130,000 clones for genetic research. This resource aids in identifying specific human genes and understanding genome structure.
Area of Science:
- Genomics
- Molecular Biology
- Biotechnology
Background:
- High-throughput screening requires comprehensive genomic libraries.
- Bacteriophage P1 cloning offers a robust method for large DNA insert cloning.
Purpose of the Study:
- To construct and characterize a 3-fold coverage genomic library of the human haploid genome.
- To establish a resource for identifying specific human genes and loci.
Main Methods:
- Genomic DNA from human foreskin fibroblasts was partially digested and size-fractionated.
- DNA inserts were cloned into the pAd10sacBII vector and packaged into P1 phage.
- Recombinant clones were robotically arrayed, cultured, and stored for library creation.
Main Results:
- A library of approximately 130,000-140,000 recombinant clones (DMPC-HFF#1 series A) was generated.
- Cloned inserts averaged 80 kb, ranging from 49 kb to 95 kb.
- PCR screening identified 17 clones containing specific genes, including HGPRT, p53, and DNA ligase I.
Conclusions:
- The constructed P1-based human genomic library is a valuable resource for genetic studies.
- The library's high coverage and large insert size facilitate gene identification and mapping.
- Robotic handling and pooling strategies enable efficient screening and characterization.