YAC subclone contig assembly by serial interspersed repetitive sequence (IRS)-PCR product hybridizations.
YAC subclone contig assembly by serial interspersed repetitive sequence (IRS)-PCR product hybridizations.
复制标题
通过串行散布重复序列 (IRS)-PCR 产物杂交进行 YAC 亚克隆重叠群组装。
DOI：
10.1093/nar/21.22.5275
复制
发表时间：
1993
影响因子：
14.9
通讯作者：
Gorski,JL
中科院分区：
文献类型：
--
作者：
Pasteris,NG;Bialecki,MD;Gorski,JL
Yeast artificial chromosomes (YACs) are presently the most effective means of cloning large contiguous regions of genomic DNA (1). However, performing a detailed molecular analysis of YAC clone insert DNA is constrained by inherent physical and technical limitations. Typically, an extended analysis of YAC DNA is facilitated by subcloning YAC insert DNA into lambda bacteriophage or cosmid vectors and constructing a contig map (2-4). In the past, a variety of strategies and techniques have been used to construct a contig map of YAC subclones; these have included the use of species-specific repetitive DNA to perform hybridization-based sequence fingerprint analyses (2) and the use of subclone-specific vector-Alu PCR products (3) and RNA end-clone fragments (4) to identify partially overlapping subclones. Each of these techniques, however, requires that individual subclones be isolated prior to analysis. Since, to ensure adequate representation, it is typical to construct a cosmid or bacteriophage library representing 5 to 10 genome equivalents of YAC insert DNA (3, 4), these techniques require the isolation and analysis of numerous individually purified clones. The construction of a contig map would be expedited by delaying the isolation of individual clones until a minimal tile contig is deduced. Here we report on the use of interspersed repetitive sequence (IRS)-PCR products derived from YAC insert DNA to construct a contig map of bacteriophage subclones prior to the isolation of individual purified clones. To simultaneously identify phage clones containing an IRS-PCR product and localize the product to a specific restriction fragment within the YAC insert, each IRS-PCR product was concurrently hybridized both to filters containing phage plaques plated on numbered grids and blots containing digested YAC DNA. High molecular weight yeast DNA was isolated by using the sucrose gradient method (1). DNA was partially digested with Sau3A by serial dilution (5) and fragments ranging from 15 to 20 kb were isolated from a 0.5% agarose gel by electrolution. Ligation products were packaged by using Gigapack II Plus Gold (Stratagene). Phage clones were plated at low density (1000 plaques/150 mm plate) and filters were prepared and hybridized as described (5). To ensure a five-fold representation of YAC DNA, 6,000 plaques were plated. Duplicate filters were screened with total human genomic DNA as probe. The resulting 55 human positive phage were arrayed in a numbered grid and replica-plated for further analysis.To obtain numerous randomly-distributed YAC-specific markers, IRS-PCR amplification of YAC DNA was performed. PCRs were 100 ul in volume and contained 100 ng of YAC DNA, 50 mM KCI, 10 mM Tris-HCI, 1.6 mM MgCl2, 0.01% gelatin, 250 uM each dNTP, and 0.5 uM primer; the primer, TC65, was directed against a consensus 3'Alu repetitive sequence and modified to contain a 5'NotI restriction endonuclease recognition sequence (6). PCR reactions were incubated at 94 C for 10 min and, after the addition of 3 U AmpliTaq DNA polymerase (Perkin-Elmer/Cetus), 35 cycles of 94 C denaturation (1 min), 55 C annealing (1 min), and 72 C extension (4 min) were performed as described (7). A portion of the amplification product was digested with EagI and cloned into a plasmid vector (pBluescript KS). Of 22 recombinants examined, 10 unique amplification products ranging from 0.5 to 3.5 kb in size were identified.
