Double-stranded Deoxyribonucleic Acid Research Articles

3P091走査型トンネル顕微鏡によるCu(111)基板上の二重鎖DNAの構造観察

3P091走査型トンネル顕微鏡によるCu(111)基板上の二重鎖DNAの構造観察

Differential metabolism of arachidonic acid in nasal polyp epithelial cells cultured from aspirin-sensitive and aspirin-tolerant patients.

The mechanism of aspirin (acetylsalicylic acid [ASA]) sensitivity associated with severe asthma and chronic rhinosinusitis with nasal polyps ("aspirin triad") has been attributed to arachidonic metabolism alternations, although the putative biochemical defects have not been elucidated. The aim of this study was assessment of the hypothesis that local production of eicosanoids in the respiratory epithelium in patients with ASA-sensitive asthma/rhinosinusitis (ASARS) differs from that of ASA-tolerant patients with rhinosinusitis (ATRS). Nasal polyps were obtained from 10 patients with ASARS and 15 with ATRS during routine polypectomies, and epithelial cells (ECs) were cultured on bovine collagen type I matrix (Vitrogen 100), in medium supplemented with growth factors. The generation of eicosanoids in supernatants of confluent ECs (6 to 8 d of culture; purity > 98%) was quantified by immunoassays. Unstimulated ECs from ASARS patients generated significantly less prostaglandin E(2)(PGE(2)) compared with ATRS (0.8 +/- 0.3 versus 2. 4 +/- 0.5 ng/microg double-stranded deoxyribonucleic acid [dsDNA], respectively), although a similar relative increase in response to calcium ionophore and inhibition with ASA was observed in both groups. Basal levels of 15-hydroxyeicosatetraenoic acid (15-HETE) were not different between groups, and calcium ionophore enhanced its production to a similar extent. However, cells incubation with 200 microM ASA for 60 min resulted in a significant increase (mean +359%) in 15-HETE generation only in ASARS patients, whereas no effect of ASA on 15-HETE generation in ATRS patients was observed. PGF(2alpha) generation was similar in both groups, and no significant generation of PGD(2) or leukotriene C(4) (LTC(4)) was observed in epithelial cell cultures from either group. Our results indicate that nasal polyps ECs from ASA-sensitive patients have significant abnormality in basal and ASA-induced generation of eicosanoids which may be causally related to the mechanism of ASA sensitivity.

Application of chimeric RNA–DNA oligonucleotides to the detection of pathogenic microorganisms using surface plasmon resonance

Chimeric oligonucleotides consisting of 21 bases of ribonucleic acid (RNA) with six bases of deoxyribonucleic acid (DNA) at the 3′-hydroxyl terminus (chimeric RNA–DNA primer) and the recombinant thermostable DNA polymerase derived from Thermus thermophilus (r Tth DNA polymerase) were utilized to efficiently amplify DNA fragments using the conventional polymerase chain reaction (PCR). The reaction required the use of both DNA polymerase and reverse transcriptase during each thermal cycle to form a double-stranded DNA in which one terminus was an RNA : DNA hybrid. Due to the ability of r Tth DNA polymerase to function as both the DNA polymerase and reverse transcriptase, a chimeric RNA–DNA primer was shown to serve as a primer in the conventional PCR procedure. We further demonstrate the advantages of using these PCR products to identify microorganisms using surface plasmon resonance (SPR) technology. This detection system was able to distinguish Shiga toxin-producing Escherichia coli O157 : H7 strains from other bacteria such as Salmonella typhimurium and S. enteritidis.

Double-stranded deoxyribonucleic acid binds to HLA class II molecules and inhibits HLA class II-mediated antigen presentation.

CD4+ T cells proliferating in response to purified double-stranded deoxyribonucleic acid (dsDNA) have been recently demonstrated in peripheral blood mononuclear cells of patients with systemic lupus erythematosus. Their activation was inhibited by anti-HLA class II (HLA-II) monoclonal antibodies; thus, the existence of a molecular interaction between dsDNA and HLA-II is conceivable. In this report we show that dsDNA specifically bind to HLA-II. After preincubating cells with purified dsDNA or synthetic oligonucleotides, dsDNA was detected on the cell membrane and in the lysates of HLA-II+ but not of isogenic HLA-II- cell lines. We demonstrate that dsDNA binding inhibits that of a specific peptide to HLA-II. Mixed lymphocyte reaction and antigen-specific T cell proliferation were inhibited by the preincubation of stimulator cells or antigen-presenting cells with dsDNA. These results suggest the existence of a novel mechanism of down-modulation of the CD4+ T cell function generated by lack of stimulation due to the HLA-II presenting molecules being "occupied" by dsDNA.

Double-stranded deoxyribonucleic acid binds to HLA class II molecules and inhibits HLA class II-mediated antigen presentation

Double-stranded deoxyribonucleic acid binds to HLA class II molecules and inhibits HLA class II-mediated antigen presentation

Conformations of Long Deoxyribonucleic Acid Molecule on Silicon Surface Observed by Atomic Force Microscopy

By means of atomic force microscopy (AFM), the conformational structures of the circular double-stranded Deoxyribonucleic Acid (DNA) (2.96 kilobase pairs) on step-controlled silicon surface are studied. The DNA molecules observed on the terrace region maintain the supercoiled structure. On the other hand, the DNA molecules observed on the step-bunched region have a modified higher-order structure. Thus, we clarify that the conformations of long DNA are affected by the surface morphology of the substrate.

Gene expression profiling of cells and tissues

The behaviour of individual human cells and of tissues in health and disease has long been a focus for study. Increasingly, we recognize the roles that alterations in the pattern of cellular gene expression play in normal processes, such as human embryonic development, and in abnormal processes, such as tumourigenesis. The Human Genome Project, which aims to identify and sequence all of the approximately 80 000 genes within the human genome, is already at an advanced stage. As this wealth of information becomes available, the focus in research is shifting towards the functional analysis of human genes in health and disease. This requires methods which allow the global pattern of gene expression in cells and/or tissues to be sampled and analysed rapidly. The human genome consists of approximately 3.3 x 10 9 base-pairs of double-stranded deoxyribonucleic acid (DNA) and contains approximately 80000 genes. Within the genome, a gene is that amount of DNA base sequence which codes for a single protein. A gene is said to be expressed when its base sequence is transcribed from DNA into an intermediate form known as messenger RNA (mRNA). Base triplets (codons) within the mRNA determine the incorporation of amino acids into the newly synthesized protein in the order determined by the original DNA strand. Most human genes are expressed in the majority of cell types; however, a minority of genes (15% or less) are expressed only in a specific cell type or at a specific developmental timepoint) These genes confer defined structural and functional properties on the cell. Gene expression and its regulation are processes which determine many aspects

An outcome analysis of 100 women after explantation of silicone gel breast implants.

A prospective outcome analysis was conducted on 100 consecutive women who requested explantation of their silicone gel breast implants from January 6, 1992 (the moratorium), through 1995. Eighteen patients were referred by rheumatologists with a diagnosis of autoimmune or rheumatic disease. Six had autoimmune disease (systemic lupus, 2 patients; rheumatoid arthritis, 2 patients; multiple sclerosis, 1 patient; and Raynaud's disease, 1 patient). Twelve had rheumatic disease (fibromyalgia, 10 patients; inflammatory arthritis, 2 patients). All of these 18 patients had developed symptoms of their disease after they had received implants. All 100 patients were extensively evaluated pre- and postoperatively by interviews, clinical assessment, and by assay of the following laboratory tests: rheumatoid factor, ESR, ANA, and anti-Ro/SSA, -La/SSP, -Sm, -RNP, -double-stranded deoxyribonucleic acid, -Scl-70, -centromere, and -cardiolipin. Patients were also evaluated by a questionnaire that was sent at a mean time of 2.7 years postexplantation (range, 1-5 years), which had a 75% response rate. Reasons for implants were augmentation, 75%; lifting, 11%; reconstruction, 12%; and congenital aplasia, 2%. The mean age at first implant was 28.9 years (range, 13-55 years) and at explantation was 41.5 years (range, 25-65 years). The mean duration of implantation was 12.0 years (range, 1-27 years). Thirty-six percent of the patients had undergone at least one closed capsulotomy and 54% at least one open capsulotomy. The mean reasons for explantation were suspected silicone-related health problems, 76%; suspected rupture, 59%; breast firmness, 36%; breast pain, 36%; and musculoskeletal pain, 23%. Before explantation 75% of the questionnaire respondees had lost some sensitivity in their nipples following their breast augmentation. In 36% of those 75 patients, that loss was almost complete. Loss of sensitivity was related to capsular contracture and to pain (p < 0.05). Following explantation there was significant improvement in nipple sensitivity in 38% of breasts in the 75 respondees. A total of 186 implants were removed. Fifty-seven percent had failed by rupturing or leaking. Only 3.2% demonstrated extravasation extracapsularly. Twenty-five percent of the capsules were calcified, demonstrating visible plaques of calcification on their inner surface. Forty-two percent were colonized by bacteria. The prevalence of class III-IV capsular contracture was 61% and it was related to implant location, duration in situ, and capsular calcification (p < 0.05), but not to capsular colonization or implant integrity (p > 0.05). Only 43 of the 100 patients elected to have saline implants inserted. Of the others, 56% felt that the shell of the saline implant could be associated with medical problems. The others felt that breast size was of minor importance to them at this time. There were few complications from the explantation procedure. Two "masses" were discovered-one was an occult carcinoma, the other a galactocele. There was one wound infection, which responded to antibiotics. Three patients developed decreased sensitivity and 3 developed increased breast pain. From the patient questionnaires, in those women who did not have saline implants inserted, 15% felt that their breast appearance was improved after explantation, 36% were "pleased," 33% were disappointed, and 13% felt "mutilated". In women who did have saline implants inserted, 18% felt that their breast appearance was now improved, 60% were "pleased," and 14% were disappointed, mainly because of wrinkling. At a mean time of 2.7 years (range, 1-5 years) after explantation, 45% of the 75 questionnaire respondees felt that their implants had caused permanent health problems and 56% felt that they had not been given adequate informed consent by their original surgeon (particularly regarding implant rupture and a possible relationship to medical disease). (ABSTRACT TRUNCATED)

Direct determination of diene conjugation and deoxyribonucleic acid (DNA) oxidative damage can explain the role of DTPA-Fe(2+)-O2 complex in a linoleic acid-DNA system.

Exposure of linoleic acid to diethylenetriminepentaacetic acid (DTPA)-Fe(2+) complexes resulted in fast diene conjugation and peroxidized products which could further react with deoxyribonucleic acid (DNA) to cause DNA oxidative damage. In this paper, we have detected diene conjugation and DNA oxidative damage in a linoleic acid-DNA model system driven by DTPA-Fe(2+) and found that: 1. in air or oxygen-saturated reaction systems, addition of hydrogen peroxide resulted in a decrease in diene conjugation and double-stranded DNA content, but had no obvious effects on the formation of DNA fluorescent products; 2. in anoxic conditions, addition of hydrogen peroxide had no effect on the formation of diene conjugation and fluorescent products, but resulted in a decrease of double-stranded DNA content; 3. in the presence of DTPA, Fe(3+) did not stimulate the formation of diene conjugation; 4. the formation of diene conjugation and fluorescent products was not inhibited by superoxide dismutase, catalase, sodium benzoate, sodium azide and mannitol. However, these 'scavengers' increased the percentage of double-strand DNA to different degrees. α-tocopherol, but not reduced glutathione (GSH), inhibited the formation of diene conjugates. α-tocopherol and GSH both could reduce the amounts of fluorescent products and DNA strand breaks. Taken together, these data further indicate that chelator-Fe(2+)-O2 complex, a perferryl-type oxidant, is probably an important initiator of lipid peroxidation in the linoleic acid-DNA system.

Selective acoustic plate mode DNA sensor

Acoustic wave sensors have been proposed for (bio)sensing applications for the past 20 years. One of the most attractive acoustic wave sensor geometries for these applications is the acoustic plate mode (APM) delay line. However, the use of APM delay lines has been hampered by the relative immaturity of the associated design techniques. The principle issue in the design of APM delay lines is to excite and detect electrically a single acoustic mode within the plate with distortion from intermode interference or multiple waveguide reflections. The use of single-phase unidirectional transducers (SPUDT) enables the excitation and detection of a single acoustic mode, reducing the distortions that occur in conventional transducer designs. The current work examines the sensing properties of the resulting APM device for the selective detection of chemically denatured, double-stranded deoxyribonucleic acid (ds-DNA) obtained through polymerase chain reaction (PCR) amplification of genomic viral DNA from cytomegalovirus (CMV).

EPR Spin-trapping studies of radical damage to DNA

EPR spectroscopy has been employed, in conjunction with spin-trapping [using 2-methyl-2-nitrosopropane dimer (MNP)] to study the attack of ˙OH (generated by the Fe2+–EDTA/H2O2 couple) on deoxyribonucleic acid (DNA) and its components. The results confirm that for pyrimidine deoxyribonucleosides and deoxyribonucleotides the alkenic C5–C6 double bond of the base moiety is the predominant site of attack (as with the pyrimidine bases, nucleosides and nucleotides) though weak signals from sugar–derived radicals are also observed. Both sugar and base radicals have also been detected for purines (both ribose and deoxyribose derivatives), providing evidence that rapid transfer occurs from the initially formed base radicals to the sugarphosphate backbone.The spectra of the adducts from double-stranded (ds) and single-stranded (ss) DNA are found to vary with pH and ˙OH flux. With dsDNA at pH 7, an anisotropic spectrum due to a slowly tumbling adduct of a high molecular weight polymer radical-adduct is accompanied by a more isotropic signal due to a mobile, low molecular weight radical-adduct, the relative yield of which increases as the pH is lowered. Close similarities are observed between the spectra of dsDNA at pH 2 and ssDNA at pH 7, with a greater occurrence of strand-breakage under these conditions. Treatment of the partially-immobilised DNA adducts formed at pH 6–7 either with the enzyme DNase or with acid releases more mobile adducts enabling information to be obtained about sites of initial attack (at pyrimidine bases) and mechanisms of transfer of damage.

17 beta-estradiol, but not 5 alpha-dihydrotestosterone, augments antibodies to double-stranded deoxyribonucleic acid in nonautoimmune C57BL/6J mice.

The influence of sex hormones on the immune response to foreign antigens as well as self-antigens is now recognized. In this study, we investigated the influence of gender and sex hormones on the development of antibodies to double-stranded DNA in nonautoimmune C57BL/6J mice. Immunoglobulin G (IgG) anti-dsDNA antibodies are commonly present in lupus patients and several autoimmune disease-prone murine strains. We found that C57BL/6J mice have detectable antibodies (IgM and IgG, but not IgA) to dsDNA. Interestingly, the incidence and level of IgG anti-dsDNA antibodies were lower in male than in female mice. Orchidectomy or administration of 5 alpha-dihydrotestosterone to orchidectomized male mice had minimal effects on these antibodies. In contrast, administration of 17 beta-estradiol to orchidectomized or intact males significantly increased both the incidence and levels of anti-dsDNA antibodies. In female mice, ovariectomy decreased whereas administration of estrogen augmented the incidence and levels of anti-dsDNA antibodies in ovariectomized as well as intact female mice. Kinetic studies revealed that estrogen treatment of male and female mice induced earlier and sustained expression of IgG anti-dsDNA antibodies compared to controls. IgG subisotype analysis showed IgG2b to be predominant. In summary, our findings suggest that estrogen, but not dihydrotestosterone, promotes anti-dsDNA antibodies in normal mice.

Molecular Weight Distribution and Intrinsic Viscosity of Sonicated and Successively Fractionated Double-Stranded Deoxyribonucleic Acid and Polyribonucleotides

The ultrasonic scission was studied for deoxyribonucleic acid (DNA) and five polyribonucleotide duplexes; three antiparallel-stranded helices (Poly(G)·Poly(C), Poly(A)·Poly(U), Poly(I)·Poly(C)) and two parallel-stranded helices (Poly(C)·Poly(C+) and Poly(A)·Poly(A+)). The sonication was carried out in 0.1—0.2 M NaCl solutions at 0°C and 200 watts for totally 20 minutes under helium gas atmosphere. The sonicated sample was fractionated by successive precipitation with acetone as the precipitant. For each duplex, several fractions were obtained in large quantities. The weight-average molecular weight Mw, the degree of polydispersity Mw/Mn, and the molecular weight distribution were determined for unsonicated, sonicated, and fractionated samples by the gel-permeation-chromatography/low-angle-laser-light-scattering method. The Mw values were reduced to a limit of 105 and the Mw/Mn values were narrowed to a 2—1.5 range by sonication. These values were further refined by fractionation. The molecular weight distribution profiles of well-defined fractions could be fitted by the logarithmic-normal function. The relationship between intrinsic viscosity and Mw was evaluated for each duplex. The result showed that the rigidity or flexibility of polymer chain depends on the molecular weight of fractionated samples and the kind of duplex examined.

Atomic force microscopy of single- and double-stranded deoxyribonucleic acid

The atomic force microscope (AFM) was used to image double-stranded DNA and single-stranded DNA adsorbed onto mica modified by Ba (II). Images of double-stranded DNA show an open circular shape without drastic contortions, and contour lengths that measure within 7% of the calculated length. Images of single-stranded DNA show two distinct forms; compact open circles with ‘‘nodes’’ or lumps almost uniformly distributed are the most frequently observed while less abundant are highly elongated circles with a few nodes. Heating the single-stranded DNA sample and altering the solvent conditions prior to adsorption was found to increase the number of elongated circular molecules observed in the AFM images.

Determination of dissolved nucleic acids in seawater by the fluorescence dye, ethidium bromide

A new method for the determination of dissolved double-stranded deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in seawater was developed, evaluated and used to study the fates of these nucleic acids in marine ecosystems. These nucleic acids, which were pre-concentrated on a hydroxyapatite column, were determined fluorometrically by the use of ethidium bromide dye, which binds specifically to the double-stranded polynucleotide. No dissolved organic matter coexisting in the pre-concentrated sample solution interfered in the analysis of DNA and RNA. Column recoveries of DNA and RNA in a sample volume of up to 11 were 93% and 97%, respectively, and 90% of both at 51. The detection limits of DNA and RNA concentrated from a 51 sample by this fluorometric method were 0.6 and 1.1 μg l −1, respectively. The concentration of dissolved nucleic acids in the waters from Tokyo Bay and Sagami Bay showed great variation in space and time. DNA ranged from 1 to 32 μg l −1, and RNA from below the detection limit to 34 μg l −1. The total amount of phosphorus in nucleic acids was an important fraction (12.9 ± 8.2%) of the dissolved organic phosphorus (DOP) and showed a good correlation with DOP.

Organotin compounds and deoxyribonucleic acid

The interaction of organotin(IV) moieties SnIVR2 and SnIVR3(R = Me, Et, Bun, C8H17 or Ph) with calf thymus deoxyribonucleic acid (DNA) has been studied. The experimental conditions have been determined for the formation of condensates SnIVR2– and SnIVR3–DNA by the reaction of ethanolic organotins [SnR2Cl2(EtOH)2 and SnR3Cl(EtOH)], as well as aqueous SnIVMe3 species, with aqueous DNA. The nature of the condensates has been investigated by 119Sn Mossbauer spectroscopy. The species SnEt2(DNA phosphate)2 and SnR3(DNA phosphate)(R = Me or Et) have been assumed to occur, where the environment of the tin atoms would be trans octahedral and trigonal bipyramidal respectively. In the reaction with aqueous DNA of SnR2Cl2(EtOH)2 and SnR3Cl(EtOH)(R = Bun, C8H17 or Ph), stannoxanes (SnR2Cl)2O and hydroxides SnR3(OH) would form too, being possibly the main reaction products for the moieties SnIV(C8H17)2, SnIV(C8H17)3 and SnIVPh3. Possible bonding situations have been advanced for SnIVRn–DNA condensates, involving e.g. two vicinal phosphodiester groups (from the DNA double strand)cis-co-ordinated giving an octahedral geometry, as well as a phosphodiester residue axially binding SnIVR3 to give a trigonal-bipyramidal structure.

Characterization of a polyreactive monoclonal antibody to dsDNA, F × 1A, and heparan sulfate generated from [formula omitted] mice immunized with rat renal homogenates

Monoclonal antibodies (MoAb) were prepared by fusing spleen cells from BALB c mice immunized with rat renal cortical homogenates to the mouse myeloma cell. One of them, designated MoAb26-3, revealed a positive antinuclear activity by screening an indirect immunofluorescence test on kidney cryostat sections. The reactivity of MoAb26-3 with double-stranded deoxyribonucleic acid (dsDNA) was confirmed by the Crithidia luciliae assay. The isotype of MoAb26-3 was determined to be IgM-κ. To test the nephritogenity of MoAb26-3, the hybridomas were grafted intraperitoneally into BALB c mice. A deposition of IgM was observed along the base of the epithelial foot processes and on the luminal surface of the endothelium by immunoelectron microscopy. By direct enzyme-linked immunosorbent assay (ELISA), MoAb26-3 was shown to react not only with dsDNA but also with fraction 1A of renal cortical supernatant (F × 1A) and heparan sulfate. On the basis of inhibition ELISA, the dsDNA inhibited F × 1A and heparan sulfate binding of MoAb26-3 and F × 1A blocked the reactivity of Mo26-3 with dsDNA and heparan sulfate, while heparan sulfate showed a less inhibition on the binding of MoAb26-3 with F × 1A, dsDNA, and even with heparan sulfate. Using immunoprecipitation with radiolabeled F × 1A, MoAb26-3 was shown to react with MW 330,000, 440,000 and 700,000 bands which were the same with those which polyclonal Heymann nephritis serum could react. An intravenous injection of MoAb26-3 to rats resulted in the deposition of IgM along the glomerular capillary wall, but resulted in an only transient appearance of proteinuria.

Genetic contributions to lupus-like disease in NZB/NZW mice

In contrast to parental New Zealand Black (NZB) or New Zealand White (NZW) mice, (NZB × NZW)F 1 mice exhibit a lupus-like disease characterized by high levels of immunoglobulin G (IgG) antinuclear antibodies in their serum and a fatal immune-complex glomerulonephritis. At least three gene loci have been identified in NZW mice that could potentially contribute to a T cell-dependent autoimmune disease, including the T cell receptor alpha- and beta-chain gene complexes and the major histocompatibility complex (MHC). The NZW T cell receptor beta-chain, complex appeared to be particularly unusual in that the C-beta-1, D-beta-2, and J-beta-2 gene segments have been deleted. Approximately one half of (NZB × NZW)F 1 × NZB backcross mice developed severe renal disease and elevated levels of IgG antibodies to double-stranded deoxyribonucleic acid and histone, suggesting that only one dominant gene or closely linked group of genes accounts for the NZW genetic contribution to F 1 disease. Despite the extremely unusual nature of the NZW T cell receptor beta-chain gene complex, we found no association of disease expression with the presence of this allele in the backcross mice. There was also no correlation of disease incidence with the presence of the NZW T cell receptor alpha-chain allele. In contrast, nearly 90 percent of the backcross mice with the NZW MHC expressed severe autoimmune disease compared with 12 percent of the mice that did not carry this haplotype. Thus, the NZW MHC or gene(s) linked to this locus appears to be the only dominant NZW genetic contribution to F 1 disease. Recent preliminary studies mapping genes that are located centromeric and telomeric to the NZW MHC suggest that the disease-associated gene(s) lies within the MHC.

Evidence for absence of histones in the Crithidia luciliae kinetoplast: a study with anti-H2A and monoclonal anti-H3 antibodies.

Recently, the specificity of the Crithidia luciliae immunofluorescent assay for the detection of antibodies to double-stranded deoxyribonucleic acid (dsDNA) has been questioned. It has been proposed that the kinetoplast of this trypanosome-like organism contains antigenic determinants other than dsDNA, presumably histones. Using anti-H2A and monoclonal anti-H3 antihistone antibodies, we have proved that there are no histones present in the Crithidia luciliae kinetoplast. False-positive results from the assay may be due to binding of antibodies to unidentified kinetoplast acid-extractable antigens other than histones.

16] A new and simple rapid method for sequencing DNA

Publisher Summary This chapter describes a current and simple rapid method for sequencing long fragments of deoxyribonucleic acid (DNA) by combining the advantages of the two systems: (1) a systematic shortening of double-stranded DNA and (2) subcloning. In this method, a fragment of DNA carrying the origin of replication from a single-stranded phage, F1 or M13, is inserted into a plasmid to make a single-stranded:double-stranded bifunctional plasmid of the shortened fragments into sequencing vectors. The new family of bifunctional plasmid constructed is of pAA-PZ1, pAA-PZ2, pAA-PZ3, and pAA-PZ4. The chapter presents the methods in a simplified way for selecting the transposon-generated deletion plasmids of different sizes and for isolating the single-stranded plasmid DNA for DNA sequence analysis. The chapter discusses solutions, buffers, media, and sources of enzymes for the method. The chapter also discusses construction and properties of pAA-PZ plasmids, construction and property of E. coli strain AA102F', cloning of foreign DNA into pAA-PZ plasmids, and several related concepts and procedures.