Phenol Emulsion Reassociation Technique Research Articles

DNA Reassociation using Oscillating Phenol Emulsions

Reassociating double-stranded DNA from single-stranded components is necessary for many molecular genetics experiments. The choice of a DNA reassociation method is dictated by the complexity of the starting material. Reassociation of simple oligomers needs only slow cooling in an aqueous environment, whereas reannealing the many single-stranded DNAs of complex genomic mixtures requires both a phenol emulsion to accelerate DNA reassociation and dedicated equipment to maintain the emulsion. We present a method that is equally suitable for reassociating either simple or complex DNA mixtures. The Oscillating Phenol Emulsion Reassociation Technique (OsPERT) was primarily developed to prepare heteroduplex DNA from alkali-denatured high molecular weight human genomic DNA samples in which hundreds of thousands of fragments need to be reannealed, but the simplicity of the technique makes it practical for less demanding DNA reassociation applications.

Subtractive hybridization of biotinylated DNA in phenol emulsion

A method of subtractive hybridization using biotinylated DNA and phenol emulsion reassociation technique (PERT) has been proposed. A possibility of combining these techniques has been shown for the first time. The effect of biotinylation degree on the formation of water-insoluble Bio-DNA aggregates was studied. The conditions when Bio-DNA aggregation is actually absent were revealed. A possible use of the above method in hybridization experiments in a wide range of DNA concentrations has been shown. The time of hybridization was 0.5–1 h. The method was approved on a model system, and its possible application for the enrichment of rare mRNA was shown. No less than 300-fold enrichment is achieved for a rare transcript (IL-2) in three cycles of subtractive hybridization.

Molecular relationship between the A and B chromosomes of Crepis capillaris

The molecular structure of the B chromosome of Crepis capillaris has been analysed by DNA comparisons of plants with 2B and 0B chromosomes. No additional band, and no differences in the band intensity were detected in 2B plants after digestion with 18 restriction enzymes. One hundred and five clones were obtained from 2B plants using the phenol-emulsion reassociation technique in formamide (F-PERT). Only one (clone pCc21) gave rise to additional hybridization bands in the DNA from 2B plants, indicating that this sequence occurs both in A and B chromosomes. Clone pCc21 contains a 54-bp microsatellite sequence rich in CA dinucleotides, similar to those found in other eukaryotic genomes. We conclude that the B chromosome of C. capillaris carries no specific highly repetitive sequence, but consists of sequences also present in the A chromosomes. The possibility remains that the heterochromatic pericentromeric region of the B has a different organization with respect to standard heterochromatin.

Sexing of Bovine Embryos with Male-Specific Repetitive DNA by Polymerase Chain Reaction: Cloning and Characterization of Bovine Male-Specific Repetitive DNA.

Bovine male-specific repetitive DNA's were cloned for the sexing of bovine embryos by means of the polymerase chain reaction (PCR). The cloning method used was PERT (phenol emulsion reassociation technique), in which most male DNA's were absorbed by an excess amount of female DNA's to enrich Y-specific DNA's. Among 20 clones examined, one clone was male-specific. The others were gender-neutral; three of these were used as probes for internal controls. The male-specific clone was used as a probe to isolate other male-specific clones from a bovine male genomic DNA library. A clone thus obtained contained at least 3 male-specific EcoRI fragments. The original and the secondarily cloned DNA's were all sequenced. They were repetitive in nature. Oligonucleotides (20 mers) were synthesized on the basis of both the male-specific and gender-neutral DNA sequences as primers for PCR. A combination of 2 pairs of primers, i.e., male-specific and gender-neutral, gave discrete PCR products allowing discrimination between male and female DNA. The detection limit of the template DNA was 10 pg, equivalent to the DNA from 3 cells.

Construction of spinal cord cDNA library and application for subtractive cloning of spinal cord-specific cDNAs

Neurodegenerative diseases are characterized by neuronal degeneration of specific neurons, e.g., degeneration of motoneurons in amyotrophic lateral sclerosis. As an approach to understand molecular mechanisms of neuronal degeneration of human spinal cord motoneurons in various motor neuron diseases, we have constructed a human spinal cord cDNA library and developed a strategy for isolating spinal cord-specific genes by subtractive cloning. We constructed human spinal cord and brain cDNA libraries from postmortem human spinal cord and brain. To isolate human spinal cord-specific cDNAs, a spinal cord-enriched [32P]cDNA probe was generated by the phenol emulsion reassociation technique. Forty-eight cDNA clones out of 10,000 colonies gave strong signals with the subtracted probe, and individual spinal cord cDNA clones were isolated. Northern blotting analysis confirmed that two spinal cord cDNA clones are, in fact, more abundant in spinal cord compared to brain.

Characterization of N-myc amplification in a human neuroblastoma cell line by clones isolated following the phenol emulsion reassociation technique and by hexagonal field gel electrophoresis.

The N-myc amplification of human neuroblastomas was characterized by the amplified DNA cloned from the cell line MC-NB-1 using the phenol emulsion reassociation technique (PERT). A number of PERT clones exhibiting amplification in this cell line were tested for amplification in other neuroblastoma cell lines. In almost all cell lines examined, only a few clones were co-amplified with N-myc and most of the others were exclusively amplified in a subset of the cell lines. The total aggregate size of the Hind III fragment identified by the PERT clones was approximately 350 kb. Most of the PERT clones were mapped to human chromosome (chr) 2p23-2pter, where the N-myc gene is located. Four types of amplicons, the 100, 420, 480 and 520 kb fragments, shown to be Not I fragments, were identified by hexagonal field gel electrophoresis. Three fragments are ordered in a head-to-tail array, and the remaining fragment is either ordered in a tail-to-head array or something else. Despite the extremely unusual construction of the amplified sequences in this cell line as compared with others, there was a low degree of sequence heterogeneity among the amplicons within this cell line. These observations lead to the idea that the complex rearrangements that give rise to the heterogeneous organization of the amplified sequences among the different cell lines precede the amplification of these sequences.

Cloning and physical mapping of DNA sequences encompassing a region in N-myc amplicons of a human neuroblastoma cell line

Cloning and physical mapping of DNA sequences encompassing N-myc amplicons of a human neuroblastoma cell line were done. A number of lambda phage clones within this region were isolated using the probes prepared by the phenol emulsion reassociation technique. Based on the restriction mapping, they were integrated into 8 contigs with sizes of 25-60 kb which, in total, encompassed a 330 kb region. Several amplicons, 100, 420, 480 and 520 kb in size as a Notl fragment, were identified using hexagonal field gel electrophoresis, and the contigs were assigned in these Notl fragments. The region encompassed by the contigs was equivalent to some 60-80% of the amplicons identified as a Notl fragment. In order to compare the amplified regions flanking the N-myc gene among the cell lines, the phage clones to cover the whole contigs were used for hybridization as a probe. The results showed that the portions of the whole contigs ranging 18-45% were also amplified in the cell lines examined. These results allowed us to identified the 'rearranged sites' which were rather evenly distributed, one at every 40 kb, through the contigs. These observations lead to the idea that an amplified DNA domain is constructed after the multiple rearrangements and then increases in number, finally resulting in the formation of subsets of amplicons with sequence homogeneity.

A novel avian W chromosome DNA repeat sequence in the lesser black-backed gull (Larus fuscus)

The phenol emulsion reassociation technique was used to isolate and clone a female specific, repetitive DNA sequence from Larus fuscus. The repeat, designated P2000-17, is restricted to the W chromosome, although related sequences occur elsewhere in the genome of L. fuscus. Similar sequences were detected in the genome of six other bird species from outside the genus Laridae, but the sequence occurs less frequently and to a similar extent in both sexes. The 298 bp DNA sequence of P2000-17 was determined and found to have extensive sequence identity to the rabbit dihydropyridine (DHP) receptor calcium channel. P2000-17 is represented once within a larger 8.6 kb tandem repeat (LfW-1), which has a complex internal DNA sequence. LfW-1 is highly conserved between repeat motifs and may comprise 3% of the female genome. The possible evolutionary origin of LfW-1 is discussed in relation to the repeat types found on the W and Y chromosomes of other species.

Genomic analysis II: isolation of high molecular weight heteroduplex DNA following differential methylase protection and Formamide-PERT hybridization.

Understanding the nature of DNA sequence differences among individuals is important to the understanding of fundamental questions in biology. To analyze such differences in complex genomes new approaches must be developed. We report two new techniques which aid in this effort. First, we have developed a modification of the Phenol Emulsion Reassociation Technique (PERT) that allows hybridization of long (20 kb and longer) single copy heteroduplex DNA fragments from human genomic DNAs. Secondly, by using a differential methylase protection technique we have shown that double methylase resistant heteroduplex DNA molecules can be size fractionated away from reannealed single methylase resistant homoduplex DNA molecules. These methods will be useful in obtaining DNA from chromosomal subregions linked to the inheritance of a specific trait or condition as described in the preceding paper and could also be used to create a map of the chromosomal subregion which includes the gene for the trait.

Factors affecting the kinetics of DNA reassociation in phenol-water emulsion at high DNA concentrations.

AbstractDNA reassociation kinetics using the phenol emulsion reassociation technique (PERT) [Kohne, D. E., Levison, S. A. & Byers, M. J. (1977) Biochemistry 16, 5329–5341] has been investigated at high DNA concentrations using an endonuclease S1 assay of reaction progress. Apparent second‐order rate constants fall on two intersecting straight lines when presented as a function of DNA concentrations on a log–log plot. In the low DNA concentration range, the rate constants drop about 10‐fold when concentration increases 1000‐fold. In the high DNA concentration range, the rate constants drop more than 10‐fold when concentration increases 10‐fold. The slopes of these lines are the same in different solvents and at different temperatures. The intersection between the lines occurs when the available catalytic surface is saturated. At high DNA concentrations, high‐complexity heterologous denatured DNA apparently competes 2–4 times better for the surface than homologous DNA because it does not participate in a reassociation reaction. Native and partially native DNA molecules cannot compete with single‐stranded DNA for a saturated surface. At high DNA concentrations, reactions using PERT become dependent on the single‐strand DNA length. Increasing length lowers reassociation rates.

Molecular Relationship of the DNA and RNA of Intracytoplasmic A Particles to Mouse and Mammary Tumour Virus Genomes

We have directly tested the hypothesis that single-stranded cytoplasmic A particle-associated DNA (ss CAP DNA) is a murine mammary tumour virus (MMTV) proviral intermediate by hybridizing 125I-labelled ss CAP DNA to MMTV RNA or to MMTV complementary DNA (cDNA). 125I-labelled CAP DNA did not form duplexes with either MMTV RNA or MMTV cDNA. In contrast, CAP RNA hybridized readily with MMTV cDNA. CAP RNA contained all the MMTV virus sequences, but at lower concentrations than in MMTV virus particles. Single-stranded CAP DNA hybridized readily with mouse DNA from several sources. A study of the rate of hybridization of CAP DNA to cell DNA at various driver to probe ratios showed that its rate of hybridization is similar to that of tumour cell DNA reassociation. Further, in reassociation studies accelerated by using the phenol emulsion reassociation technique (PERT), CAP DNA originally isolated as single-stranded DNA was shown to reanneal (70%), to protect 125I-cell DNA to the same extent (67%) and to do so with kinetics of reassociation equivalent to that of mouse DNA. Although CAP DNA isolates were slightly enriched for MMTV specific sequences when compared to total cellular DNA, we conclude that the majority of ss CAP-associated DNA is equivalent to a random sample of total tumour cell DNA.

Room temperature method for increasing the rate of DNA reassociation by many thousandfold: the phenol emulsion reassociation technique

Room temperature method for increasing the rate of DNA reassociation by many thousandfold: the phenol emulsion reassociation technique