Human-specific amplification of radiation hybrid DNA fractionated by pulsed-field gel electrophoresis.
Human-specific amplification of radiation hybrid DNA fractionated by pulsed-field gel electrophoresis.
复制标题
通过脉冲场凝胶电泳分离的辐射杂交 DNA 的人类特异性扩增。
DOI：
10.1093/nar/19.2.401
复制
发表时间：
1991
影响因子：
14.9
通讯作者：
Gorski,JL
中科院分区：
文献类型：
--
作者：
Burright,EN;Reyner,EL;Gorski,JL
Recently, a method named interspersed repetitive sequence polymerase chain reaction (IRS-PCR) was described by which, using primers directed against an IRS, human DNA sequences flanked by the IRS are selectively amplified from complex DNA mixtures such as somatic cell hybrid DNA (1, 2). Hybrids containing human chromosomal fragments, such as radiation hybrids (RH)(3, 4), have been used to further reduce the complexity of the human DNA template and increase the specificity of IRS-PCR amplification (5). Here we describe a method which facilitates the isolation of specific DNA sequences from a subchromosomal region. We have developed a method to amplify human-specific DNA sequences from pulsed-field gel (PFGE) fractionated RH DNA; by amplifying a fraction of digested RH DNA containing an identifiable DNA restriction fragment, this method provides a means to selectively amplify and enrich for DNA sequences contained within a specific DNA restriction fragment.Genomic DNA from RH 128, a RH harboring about 10% of a human X chromosome including region Xpl1. 22-Xpl 1.1 (PN Goodfellow and JL Gorski, unpublished data), was prepared in agarose blocks (6) and digested with SfiI (NEB). DNA blocks were loaded into 1.2% LMP agarose gels (BRL) made with 0.5 x TBE and electrophoresed using a contour-clamped homogeneous electric field (CHEF) gel system (CHEFDRII; BioRad) at 200 V with pulse times of 60s (15 hrs) and 90s (9 hrs). Horizontal 5 mm gel slices were cut; DNA was recovered by electroelution, dialyzed against TE, sequentially extracted with phenol, chloroform, and ether, precipitated using ammonium acetate, ethanol, and carrier yeast tRNA (10 gg/ml), and resuspended in 50 Al of dH2O. PCRs were 100, tl in volume and contained 400 ng of DNA, 50 mM KCl, 10 mM Tris-HCl, 1.6 mM MgCl2, 0.01% gelatin, 250 uM each dNTP, 1 itg RNase A, and 0.5, tM primer; the primer, A-517N (5'AA-GTGCGGCCGCGATCTCGGCTCACTGCAA 3'), was directed against a consensus Alu repetitive sequence (1) modified to contain a 5'NotI restriction endonuclease recognition sequence. PCR reactions were incubated at 37 C for 1 hr, 94 C for 9 min, and after the addition of 5 U AmpliTaq DNA polymerase (Perkin-Elmer/Cetus), 36 cycles of 94 C denaturation (1 min), 55 C annealing (1 min), and 72 C extension (4 min) were performed; amplification products were fractionated on a 1.2% agarose gel and stained with ethidiumbromide. No products resulted from the amplification of hamster DNA indicating that the primer was
