A rapid and simple method to isolate and characterize highly polymorphic markers from the centromeric regions of the human chromosomes.

Using oligonucleotide primers complementary to the 3' ends of either the Alu or the L1Hs consensus sequences in conjunction with a primer complementary to alpha satellite subsets specific to different human chromosomes, it was possible to detect and characterize polymorphisms originating from the microsatellites which are often present downstream these repetitive elements. The methodology does not require cloning, sequencing or synthesis of specific primers. Centromeric location was confirmed by linkage analysis, in situ hybridization and sequencing. The method is proposed for the generation of polymorphic markers from all centromeric regions.     

Alu-splice PCR: a simple method to isolate exon-containing fragments from cloned human genomic DNA

We have developed a simple, straightforward procedure to isolate exons from cloned human genomic DNA. The method is PCR based and relies upon the conservation of splice-site sequences and the frequency of Alu repeat elements in the genome to capture coding sequences. We designed two different sets of primers: a primer from each end of the Alu element and primers with the 5′ or 3′ splice-site consensus sequences. Putative exons were amplified by PCR using YAC DNA as starting material. We applied Alu-splice PCR to two overlapping YACs, 72H9 and 860G11, from human chromosome 21. Sequence and northern analysis of 37 initial clones resulted in the identification of five novel exons. Received: 17 July 1997 / Accepted: 28 August 1997     

A highly polymorphic region 3'' to the human type II collagen gene.

We have characterised a highly polymorphic region 1.3kb downstream of the human Type II collagen gene. It consists of a highly AT-rich tandem repetitive region (minisatellite) approximately 650bp long. Two alleles had been observed previously, differing in size by approximately 300bp. When this region was cloned from four unrelated individuals carrying the larger allele, DNA sequence data identified three alleles, suggesting far higher polymorphism than was originally supposed. This minisatellite was shown to be present in a single copy in the human genome, and to have arisen after the divergence of Old and New World monkeys.     

A simple and rapid method to isolate purer M13 phage by isoelectric precipitation

M13 virus (phage) has been extensively used in phage display technology and nanomaterial templating. Our research aimed to use M13 phage to template sulfur nanoparticles for making lithium ion batteries. Traditional methods for harvesting M13 phage from Escherichia coli employ polyethylene glycol (PEG)-based precipitation, and the yield is usually measured by plaque counting. With this method, PEG residue is present in the M13 phage pellet and is difficult to eliminate. To resolve this issue, a method based on isoelectric precipitation was introduced and tested. The isoelectric method resulted in the production of purer phage with a higher yield, compared to the traditional PEG-based method. There is no significant variation in infectivity of the phage prepared using isoelectric precipitation, and the dynamic light scattering data indirectly prove that the phage structure is not damaged by pH adjustment. To maximize phage production, a dry-weight yield curve of M13 phage for various culture times was produced. The yield curve is proportional to the growth curve of E. coli. On a 200-mL culture scale, 0.2 g L^?1 M13 phage (dry-weight) was produced by the isoelectric precipitation method.     

Recombinant microsatellite amplification: a rapid method for developing simple sequence repeat markers

Recombinant microsatellite (simple sequence repeat; SSR) amplification is a technique by which DNA sequences flanking microsatellites can be isolated rapidly on a large scale. The approach selectively amplifies microsatellite-containing sequences and recombines the amplicons by redigestion and ligation, in order to increase the yield of microsatellite flanks per clone two-fold and to further increase selectivity of amplification. Since this method does not require prior knowledge of the genomic sequence, it is especially useful for species for which abundant genomic sequences are not available. The feasibility of this approach was demonstrated by developing SSR markers in cultivated oats.     

A simple and rapid method for screening transformant yeast colonies using PCR.

A simple and rapid procedure for screening transformant yeast colonies is described. In this method, a trace amount of plasmid DNA is isolated from a small amount of yeast cell mass; then, the presence of the exogenous DNA in each yeast colony is detected by PCR amplification.     

Hypervariability of simple sequences as a general source for polymorphic DNA markers.

Short simple sequence stretches occur as highly repetitive elements in all eukaryotic genomes and partially also in prokaryotes and eubacteria. They are thought to arise by slippage like events working on randomly occurring internally repetitive sequence stretches. This predicts that they should be generally hypervariable in length. I have used the polymerase chain reaction (PCR) process to show that several randomly chosen simple sequence loci with different nucleotide composition and from different species show extensive length polymorphisms. These simple sequence length polymorphisms (SSLP) may be usefully exploited for identity testing, population studies, linkage analysis and genome mapping.     

Polar body diagnosis for hemophilia a using multiplex PCR for linked polymorphic markers.

Preimplantation genetic diagnosis (PGD) is usually performed on blastomeres. In Germany, the only possibility to perform PGD is by analysis of polar bodies. We performed PGD using polar bodies in a woman who is a carrier of hemophilia A. Multiplex PCR followed by nested fluorescent PCR for five linked polymorphic markers was established. From 11 analyzed polar bodies, only 1 showed alleles linked to the mutation. The corresponding oocyte was transferred and no pregnancy was established. As seen in other investigations, the rate of heterozygous first polar bodies is surprisingly high.     

Isolation of a human chromosome 14-only somatic cell hybrid: analysis using Alu and LINE-based PCR.

Interspecific somatic cell hybrids containing single human chromosomes are valuable reagents for localization of cloned genes and DNA fragments to specific chromosomes, for the development of chromosome-specific libraries, and for generation of hybrid cell lines containing subchromosomal regions. A CHO somatic cell hybrid containing a single, intact human chromosome 14 (MHR14) was developed and confirmed by LINE PCR amplification gel pattern, by Alu-517 PCR product dot blot hybridization, and by cytogenetic analysis. MHR14 will serve as the chromosome source for the development of a radiation map of human chromosome 14.     

SiteFinding-PCR: a simple and efficient PCR method for chromosome walking

In this paper, we present a novel PCR method, termed SiteFinding-PCR, for gene or chromosome walking. The PCR was primed by a SiteFinder at a low temperature, and then the target molecules were amplified exponentially with gene-specific and SiteFinder primers, and screened out by another gene-specific primer and a vector primer. However, non-target molecules could not be amplified exponentially owing to the suppression effect of stem–loop structure and could not be screened out. This simple method proved to be efficient, reliable, inexpensive and time-saving, and may be suitable for the molecules for which gene-specific primers are available. More importantly, large DNA fragments can be obtained easily using this method. To demonstrate the feasibility and efficiency of SiteFinding-PCR, we employed this method to do chromosome walking and obtained 16 positive results from 17 samples.     

Isolation of region-specific and polymorphic markers from chromosome 17 by restricted Alu polymerase chain reaction

We demonstrate that the digestion of template DNAs with restriction endonucleases prior to Alu polymerase chain reaction ("restricted Alu-PCR") reduces the complexity of the Alu-primed amplification patterns of human DNA in somatic cell hybrids and allows a direct informative comparison of these patterns. A comparison of restricted Alu-PCR patterns of a monochromosomal hybrid retaining a human chromosome 17 (MH22-6) and a hybrid retaining a human chromosome 17 deleted for band p11.2 (DH110-D1) revealed four Alu-PCR products that were present in the former but absent in the latter hybrid. Hybridization of these fragments to the total Alu-PCR amplification products of the two hybrids confirmed their absence in DH110-D1 amplification products. Hybridization to a panel of somatic cell hybrids indicated that two of these fragments were deleted in the hybrid DH110-D1 and mapped to 17p11.2, as expected. However, two additional fragments were not deleted in the hybrid DH110-D1 and mapped to other regions of chromosome 17. An insertion-deletion polymorphism was associated with one of the latter fragments, which may be the mechanism for the lack of its amplification in the hybrid DH110-D1. Restricted Alu-PCR should enhance the applications of Alu-PCR and provides a new method for the identification of chromosome-specific polymorphic markers.