Alu polymerase chain reaction: a method for rapid isolation of human-specific sequences from complex DNA sources.

Abstract

Current efforts to map the human genome are focused on individual chromosomes or smaller regions and frequently rely on the use ofsomatic cell hybrids. We report the application of the polymerase chain reaction to direct amplification of human DNA from hybrid cells containing regions of the human genome in rodent cell backgrounds using primers directed to the human Alu repeat element. We demonstrate Alu-directed amplification of a fragment of the human HPRT gene from both hybrid cell and cloned DNA and identify through sequence analysis the Alu repeats involved in this amplification. We also demonstrate the application of this technique to identify the chromosomal locations of large fragments of the human X chromosome cloned in a yeast artificial chromosome and the general applicability of the method to the preparation of DNA probes from cloned human sequences. The technique allows rapid gene mapping and provides a simple method for the isolation and analysis of specific chromosomal regions.

Painting of human chromosomes with probes generated from hybrid cell lines by PCR with Alu and L1 primers

“…The results of this study demonstrate that IRS-PCR with human SINES and LINES directed oligonucleotide primers (Nelson et al 1989;Ledbetter et al 1990) can be used to generate probes from somatic hybrid cell lines that are useful for the painting of specific human chromosomes and chromosomal subregions. This approach provides a new and rapid strategy for the cytogenetic characterization of somatic hybrid cell panels used for human gene mapping studies, and is sensitive enough to detect human chromosomes present in only a minor fraction of the cells in a hybrid cell line.…”

“…Hybrid cell DNA (100ng) was used in a 100lal assay containing l l aM Alu-primer 517 (Nelson et al 1989) or Ll-primer (Ledbetter et al 199(I). 10mM TRIS-HCI (pH 8.4), 50mM KC1, 1.5mM MgCI,, 0.01% gelatin, 2501aM of each of the four dNTPs and 2.5 units of Thermus aquaticus DNA polymerase (Perkin Elmer/ Cetus).…”

“…To increase further the sensitivity of this approach, we have applied IRS-PCR (interspersed repetitive sequence-polymerase chain reaction) (Nelson et al 1989: Ledbetter et al 1990). IRS-PCR allows the specific amplification of human DNA sequences from man-mouse or man-hamster hybrid cell DNA, For this purpose, oligonucleotide primers have been established that under appropriate conditions of stringency hybridize specifically to human Alu repeats or human L1 repeats but not to related sequences present in mouse or Chinese hamster genomic DNA.…”

“…Alu-repeats and Ll-repeats represent short and long interspersed repeat elements (SINES and LINES), respectively, which are present in approximately 10 ~ (Alu) and 104'105 (L1) copies in the human genome. IRS-PCR of hybrid cell DNA with either the Alu primer 517 or the L1 primer recently described by Nelson et al (1989) and Ledbetter et al (1990) results in the amplification of any human DNA sequence located between two inverted blocks of human Alu-or Ll-repeats, provided that these blocks are located within a distance that can be bridged by PCR.…”

Restricted PCR: amplification of an individual sequence flanked by a highly repetitive element from total human DNA

“…Application of the Alu element-mediated polymerase chain reaction (Alu-PCR) became an important method for isolating chromosome-specific human DNA fragments from mixed DNA sources (1). Such fragments are a source of probes hybridizing to chromosome regions of interest and also allow the detection of Alu-related polymorphisms (2,3).…”