Generation of full coverage libraries from microdissected DNA: Optimization for FISH of DNA microclones from an amplified domain

Journal Article · Thu Sep 01 04:00:00 EDT 1994 · American Journal of Human Genetics

OSTI ID:134789

Cummings, L; Bittner, M L; McGill, J R ^[1]

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1. NIH, Bethesda, MD (United States); and others

Chromosome microdissection/microcloning is increasingly important in the molecular analysis of chromosome rearrangements. Despite a number of publications using this technology, no detailed report examining representation of the starting template DNA have appeared. Based upon fluorescence in situ hybridization (FISH) performance of microdissected DNA probes derived from genetically amplified regions (dmins, hsrs) form compact intense signals readily interpretable even in interphase nuclei. Microdissected probes from non-amplified DNA typically produce much more diffuse signals in interphase nuclei. The difference in fluorescence intensity may arise from the more abundant template of the amplified domain at the time of dissection. We will report on the construction and characterization of microclone libraries using FISH to interphase nuclei as an indicator of template representation. Factors influencing cloning efficiency (e.g., vector cloning schemes, transformation vs. electroporation, generation of large PCR fragments, etc.) are being optimized to generate microclone libraries more fully representative of the dissected region. Retention of representation during microclone library generation is being examined for both a highly amplified starting material (dmin amplified for cmyc), and for a microdissected normal chromosome 8. It is expected that comparisons of the signal intensity between the uncloned and cloned dmin-derived PCR products will assist in the establishment of full coverage DNA microclone libraries and optimization of these products for FISH.

• OSTI ID: 134789
• Report Number(s): CONF-941009--
• Journal Information: American Journal of Human Genetics, Journal Name: American Journal of Human Genetics Journal Issue: Suppl.3 Vol. 55; ISSN AJHGAG; ISSN 0002-9297
• Country of Publication: United States
• Language: English

Related Subjects

44 INSTRUMENTATION
INCLUDING NUCLEAR AND PARTICLE DETECTORS
55 BIOLOGY AND MEDICINE
BASIC STUDIES
ANIMAL CELLS
CELL NUCLEI
CHROMOSOMAL ABERRATIONS
DETECTION
DNA
DNA HYBRIDIZATION
DNA-CLONING
EFFICIENCY
FLUORESCENCE
HUMAN CHROMOSOMES
MITOSIS
OPTIMIZATION
POLYMERASE CHAIN REACTION
PROBES