Tremendous strides in decoding the human genome have been made over the last ten years. Large, chromosome-scale, genome-scale EST-sequencing and mapping techniques have dramatically expanded the base of identified genes and sequenced DNA. Gene isolation and cloning techniques that emerged earlier on, however, still remain effective mainstays for constructing dense transcript maps to isolate coding sequences and disease-associated genes contained within a specific chromosomal region. Positional Cloning by Exon Trapping and cDNA Selection examines two powerful methods for locating the coding region of a given gene by isolating gene fragments from individual clones or pools of genomic clones. This comprehensive guide details the exon-trapping and cDNA selection processes step by step-from isolating genomic templates and nuclear splicing, to verifying generated clones and sequenced data, to analyzing exon libraries and cDNA sublibraries. Procedures covered include: * Exon-trapping systems and descriptions of pSPL1 and pSPL3 vectors. * Exon amplification protocols for preparing vectors for cloning; subcloning genomic DNA into vectors; transformation, analysis, and transfection of sublibraries; RNA transcription; and PCR amplification and PCR product cloning. * Evaluation of exon libraries by PCR colony testing, identifying artifactual clones using Southern blot, and sequencing and mapping back candidate exons. * Isolation of genomic templates, such as COSMID-, P1-, PAC, and YAC DNA. * cDNA selection experiment protocols including cDNA screening, hybridization, and biotinylation; preparation of genomic and cDNA sources; and PCR cloning. * Clone analysis and analysis by hybridization. Positional Cloning by Exon Trapping and cDNA Selection offers researchers, scientists, and graduate students an invaluable tool for probing gene distribution and molecular organization. Most importantly, it provides a critical approach to isolating specific disease genes within a targeted genomic area.
It is now 10 years since the first edition of YAC Protocols was published in 1996. YAC Protocols was first produced to address the huge demand within the research community for a lab-based text that described in detail the wide range of uses for large insert yeast artificial chromosome (YAC) DNA clones. In doing this, the original editor, David Markie, and the many different contri- tors who provided descriptions of the protocols they used and developed, did a magnificent job. Indeed many of the techniques described within the first e- tion require little change and have stood up admirably to the test of time. Since the first edition, the use of YACs has proved invaluable for addressing a wide range of new biological problems ranging from those of basic biochemistry to assisting in the mapping and sequencing of the human genome. The requirement for a second edition of YAC Protocols was prompted by a number of major advances in biology since the publication of the first edition. These advances have included the sequencing of the human genome, and the genomes of a wide variety of other organisms, and the increased use of transgenic animals for understanding the molecular basis of human and animal disease.