Screening Large‐Insert Libraries by Hybridization

Three types of hybridization screening are discussed in this unit colony hybridization, DNA hybridization, and YAC Alu‐PCR‐product hybridization. The first protocol describes methods for arraying large‐insert clones. Subsequent protocols discuss hybridizing YAC and BAC/PAC colony blots. An alternate protocol provides a guide for preparing and hybridizing with, high‐specific‐activity oligonucleotide probes called “overgo” probes. Support protocols indicate the procedures for identifying the plate and well address of positive clones by manual and software‐assisted methods. Hybridization of Alu‐PCR‐product probes to Alu‐PCR products of YAC clones are provided. An alternate protocol describes a method for preparing DNA from individual YACs and for producing and using Alu‐PCR product blots from individual YACs. Additional support protocols provide guidelines for preparing Alu‐PCR probes from cosmids, YACs, BACs/PACs, and somatic cell hybrids, and describe the use of 96‐well plates to radiolabel and spin‐column‐purify large numbers of probes. A method for YAC walking is also provided.Three types of hybridization screening are discussed in this unit colony hybridization, DNA hybridization, and YAC Alu‐PCR.