Detection of individual human chromosomes by chromosome in situ suppression hybridization using PCR-amplified bacteriophage library probes.
We used the polymerase chain reaction (PCR) to prepare chromosome-specific probes from the bacteriophage lambda library LAO1NS01, prepared at the Los Alamos National Laboratory from flow sorted human chromosome 1. By using oligonucleotide primers flanking the EcoRI insertion site of the Charon 21A vector, we were able to amplify the human sequences preferentially in the library up to 9.1 kb (maximum insert size). The product of the PCR reaction was nick translated with incorporation of biotinylated residues and used with fluorescence in situ hybridization to observe metaphase chromosomes by fluorescence microscopy. This technique allows for a relatively easy method for preparation of chromosome-specific library probes for "chromosome painting." The quality of the results obtained by this method compares favorably to those obtained by using bulk-purified library inserts. This method offers potential advantages in terms of cost and ease of use.
Burde
S
S
Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, NY 14642.
Leary
J F
JF
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002878:Chromosomes, Human, Pair 1; D004279:DNA, Viral / Q000235:genetics; D015698:Genomic Library; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000378:metabolism*; D016133:Polymerase Chain Reaction
D004279:DNA, Viral; D015345:Oligonucleotide Probes