Region-specific microdissection library and single-copy microclones for human chromosome 2p11–p13

The short arm of human chromosome 2, comprising approximately 93 million bp, has been divided into four regions to construct region-specific microdissection libraries to facilitate physical mapping and gene cloning. These four regions include 2p23-p25 (designated 2P1), 2p21-p23 (2P2), 2p14-p16 (2P3), and 2p11-p13 (2P4). Together with three previously constructed microdissection libraries of 2P1, 2P2 and 2P4, a fourth library for the region 2p14-p16 (2P3) has been constructed and characterized to complete all four region-specific libraries for the entire 2p. The 2P3 library is very large, potentially comprising 1,000,000 recombinant microclones with insert sizes ranging between 50 and 800 bp and a mean of 250 bp. Approximately 40% of the microclones contain unique sequences. Of the 77 single-copy microclones analyzed, 66 clones (86%) hybridized to both human and chromosome 2 DNAs, indicating that they were derived from human and are chromosome 2 specific. The hybridizing HindIII genomic fragments for the 66 microclones have also been determined.

Construction and characterization of region-specific microdissection libraries and single-copy microclones for short arm of human chromosome 2

Tong

et al. 1994

Analysis of region-specific library constructed by sequence-independent amplification of microdissected fragments surrounding weaver (wv) gene on mouse chromosome 16

Wei

Dlouhy

Zhu

et al. 1994

The C3-C4 region of mouse chromosome 16 was microdissected and amplified directly by sequence-independent amplification (SIA). The SIA product was proved to originate from the microdissected region by fluorescence in situ hybridization (FISH) and was cloned into the PCR II vector (mean insert size 506 bp). Colony hybridization showed that about 59% of the clones contained either unique or low copy number sequences. Southern blot analysis of 100 unique clones demonstrated that 50 clones hybridized with single (33 clones) or multiple (17 clones) bands on blots of DNA from a hamster-mouse hybrid cell line that contains mouse chromosome 16, 13 clones hybridized with mouse but not with the hamster-mouse hybrid DNA, 19 clones contained repetitive sequences, and the remaining 18 clones failed to yield bands. One third of the 100 unique clones hybridized to human genomic DNA. Thirty-three clones were sequenced. None of them was found in GenBank. Our results demonstrate that this relatively simple method of microdissection and cloning can produce a library of good quality.

Three region-specific microdissection libraries for the long arm of human chromosome 2, regions q33-q35, q31-q32, and q23-q24

Tong

Whittier

et al. 1995

Three region-specific libraries have been constructed from the long arm of human chromosome 2, including regions 2q33-35 (2Q2 library), 2q31-32 (2Q3) and 2q23-24 (2Q4). Chromosome microdissection and the MboI linker-adaptor microcloning techniques were used in constructing these libraries. The libraries comprised hundreds of thousands of microclones in each library. Approximately half of the microclones in the library contained unique or low-copy number sequence inserts. The insert sizes ranged between 50 and 800 bp, with a mean of 130-190 bp. Southern blot analysis of individual unique sequence microclones showed that 70-94% of the microclones were derived from the dissected region. 31 unique sequence microclones from the 2Q2 library, 31 from 2Q3, and 30 from 2Q4, were analyzed for insert sizes, the hybridizing genomic HindIII fragment sizes, and cross-hybridization to rodent species. These libraries and the short insert microclones derived from the libraries should be useful for high resolution physical mapping, sequence-ready reagents for large scale genomic sequencing, and positional cloning of disease-related genes assigned to these regions, e.g. the recessive familial amyotrophic lateral sclerosis assigned to 2q33-q35, and a type I diabetes susceptibility gene to 2q31-q33.