Chromosome painting using chromosome-specific probes from flow-sorted pig chromosomes

Abstract

Biotinylated chromosome-specific probes were prepared from flow-sorted pig chromosomes 1 13,18, X, and Y using the degenerate oligonucleotide-primed polymerase chain reaction. Probes prepared in this way can be used to confirm the identity of chromosomes in the bivariate pig flow karyotype and in pig × mouse somatic cell hybrids.

Isolation of rat chromosome-specific paint probes by bivariate flow sorting followed by degenerate oligonucleotide primed-PCR

“…Telenius et al (1992aTelenius et al ( , 1992b described a method in which a different PCR protocol (5 cycles with a low annealing temper ature, 35 cycles with a high annealing temperature) and a degenerate primer was used. This protocol has been successful ly used not only on human DNA (Meltzer et al, 1992), but also on Drosophila melanogaster, mouse, cosmid (Telenius et al, 1992b) and pig DNA (Langford et al. 1992).…”

Pure chromosome-specific PCR libraries from single sorted chromosomes.

“…Previous studies have demonstrated that chromosomespecific probes can be generated by PCR amplification of hundreds or thousands of sorted chromosomes (2,4,5,11). This reduces sorting time required but does not alter the requirements for purity of the sorted chromosomes, which often must be assisted by use of somatic-cell hybrids (1,2).…”

“…The development of chromosome-specific libraries to date has been very labor intensive, relying on the existence of appropriate somatic cell hybrids (1,2), the ability to isolate unique chromosomes to relative purity by flow cytometry (1)(2)(3)(4)(5), or the ability to discriminate between and isolate regions of chromosomes by microdissection (6,7). All of these methods have required DNA from hundreds or thousands of chromosomes.…”

Construction of chromosome-specific DNA libraries covering the whole genome of field bean (Vicia faba L.)

“…It is based on multiple locus priming, which is achieved by employing oligonucleotides of partially degenerate sequence as primers and by nonstringent annealing conditions during initial PCR cycles. DOP-PCR using primer 6-MW has been shown to be suitable for amplification of genomic DNA in animal , Langford et al 1992, Breneman et al 1993, Guan et al 1994, Hoebee et al 1994 as well as in plant species (Arumuganathan et al 1994, Pich et al 1994. Amplified fragments can subsequently be cloned via restriction site XhoI, which is present near the 5' end of the primer .…”