Genomics 1992 Aug;13(4):1322-4

A method for the rapid sequence-independent amplification of microdissected chromosomal material.

Bohlander SK, Espinosa R 3d, Le Beau MM, Rowley JD, Diaz MO

Section of Hematology/Oncology, University of Chicago, Illinois 60637.

We have developed a simple, efficient method by which microdissected material can be amplified directly in the collection container in a few hours. The procedure involves two initial rounds of DNA synthesis with T7 DNA polymerase, using a primer that contains a random pentanucleotide sequence at its 3' end and a defined sequence at its 5' end, followed by PCR amplification with the defined sequence as the primer. The resulting products can be biotinylated and used for fluorescence in situ hybridization (FISH) to confirm their chromosomal location. As few as 17 dissected chromosomal regions provide sufficient material for a specific FISH signal on the appropriate band of metaphase chromosomes. We have obtained a chromosome 6q25-qter-specific painting probe in this way.

MeSH Terms:

• Base Sequence
• Chromosome Mapping
• Chromosomes, Human*
• DNA
• Fluorescence
• Gene Amplification*
• Human
• Karyotyping
• Metaphase
• Molecular Sequence Data
• Nucleic Acid Hybridization
• Support, Non-U.S. Gov't
• Support, U.S. Gov't, Non-P.H.S.
• Support, U.S. Gov't, P.H.S.

Substances:

• DNA

Grant support:

• CA42557/CA/NCI
• CA49133/CA/NCI

PMID: 1505965, UI: 92372035