NDLI: Methods for high throughput validation of amplified fragment pools of BAC DNA for constructing high resolution CGH arrays

Content Provider	Springer Nature : BioMed Central
Author	Watson, Spencer K deLeeuw, Ronald J Ishkanian, Adrian S Malloff, Chad A Lam, Wan L
Abstract	Background The recent development of array based comparative genomic hybridization (CGH) technology provides improved resolution for detection of genomic DNA copy number alterations. In array CGH, generating spotting solution is a multi-step process where bacterial artificial chromosome (BAC) clones are converted to replenishable PCR amplified fragments pools (AFP) for use as spotting solution in a microarray format on glass substrate. With completion of the human and mouse genome sequencing, large BAC clone sets providing complete genome coverage are available for construction of whole genome BAC arrays. Currently, Southern hybridization, fluorescent in-situ hybridization (FISH), and BAC end sequencing methods are commonly used to identify the initial BAC clone but not the end product used for spotting arrays. The AFP sequencing technique described in this study is a novel method designed to verify the identity of array spotting solution in a high throughput manner. Results We show here that Southern hybridization, FISH, and AFP sequencing can be used to verify the identity of final spotting solutions using less than 10% of the AFP product. Single pass AFP sequencing identified over half of the 960 AFPs analyzed. Moreover, using two vector primers approximately 90% of the AFP spotting solutions can be identified. Conclusions In this feasibility study we demonstrate that current methods for identifying initial BAC clones can be adapted to verify the identity of AFP spotting solutions used in printing arrays. Of these methods, AFP sequencing proves to be the most efficient for large scale identification of spotting solution in a high throughput manner.
Related Links	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/1471-2164-5-6.pdf
Ending Page	8
Page Count	8
Starting Page	1
File Format	HTM / HTML
ISSN	14712164
DOI	10.1186/1471-2164-5-6
Journal	BMC Genomics
Issue Number	1
Volume Number	5
Language	English
Publisher	BioMed Central
Publisher Date	2004-01-14
Access Restriction	Open
Subject Keyword	Life Sciences Microarrays Proteomics Animal Genetics and Genomics Microbial Genetics and Genomics Plant Genetics and Genomics Bacterial Artificial Chromosome Comparative Genomic Hybridization Bacterial Artificial Chromosome Clone Bacterial Artificial Chromosome Array Bacterial Artificial Chromosome Clone Sequence
Content Type	Text
Resource Type	Article
Subject	Biotechnology Genetics
Journal Impact Factor	3.5/2023
5-Year Journal Impact Factor	4.1/2023

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