A bacteriophage M13 based sandwich hybridization assay for the detection of hepatitis B virus in human blood.

A two step hybridization procedure was developed to detect the presence of hepatitis B virus in blood samples using bacteriophage M13 radiolabelled DNA as probe. During the first step of hybridization, single-stranded bacteriophage M13 tg 130 DNA, with 3.2 kb HBV DNA cloned into it, was hybridized to target HBV DNA immobilized on nitrocellulose membrane filter. In the second step of hybridization, M13 DNA annealed to HBV target is detected with the help of double stranded form of M13 DNA. The assay offers minimum 4- to 6-fold higher sensitivity in comparison to single-step conventional hybridization assays. Additionally M13 DNA offers itself as universal probe.     

Synthesis of a fixed-length single-stranded DNA probe by blocking primer extension in bacteriophage M13

A simple and efficient technique has been developed for preparing radiolabeled single-stranded (ss) probes of determined length and high specific radioactivity. The human beta-globin gene intervening segment II (IVSII) fragment (0.9-kb) was inserted between the EcoRI and BamHI sites of M13mp11 and used as a template for ss probe synthesis. The M13 hybridization probe primer (M13 Hpp) was annealed to the recombinant M13mp11-beta IVSII template DNA. This M13 Hpp was next blocked by the enzymatic addition of a dideoxy adenosine monophosphate (ddAMP) residue to the 3' OH group of the primer. The M13 universal sequencing primer was then annealed and used to prepare an ss copy of the beta-IVSII fragment. Synthesis of the ss fragment was terminated by the presence of the dd-blocked M13 Hpp yielding a specific 0.9-kb ss beta-IVSII probe.     

M13 phage DNA as a universal marker for DNA fingerprinting of animals, plants and microorganisms

Hypervariable polymorphic patterns were detected with M13 phage DNA as a probe in genomic DNA of organisms belonging to different taxonomic groups including animals (vertebrates and invertebrates), plants and microorganisms. Individual-specific restriction pattern analysis (DNA fingerprinting) with this probe proved to be useful for individual identification, analysis of somatic stability and paternity testing in man. The nuclear type of inheritance indicates that the hypervariable DNA regions in question are located in the chromosomes, not in the mitochondrial DNA. The data obtained also demonstrate a potential range of M13 DNA applications as a probe for DNA fingerprinting of animals, plants and microorganisms, particularly for the determination of inbred lines, identification of bacterial strains and establishing stock, variety and strain distinctions.     

Reverse Southern hybridization.

A DNA oligomer 25 nucleotides long which contained an HMT (4'-hydroxymethyl-4,5', 8-trimethylpsoralen) furan side monoadduct to thymidine at a 5'-TpA-3' site was used as a probe for the polylinker sequence present in single-stranded M13 mp19 DNA and in double-stranded pUC 19 DNA. Hybridization and photofixation were carried out simultaneously in solution under conditions approximating the melting temperature of the probe-target hybrid. Use of probe concentrations greater than 10(-8) M permitted hybridization times of a few minutes. Irradiation with near ultraviolet light converted the HMT monoadduct present in hybrid complexes into an interstrand crosslink. Efficient photofixation removed hybrid from the equilibrium distribution and resulted in the formation of additional probe-target complex. After removal of excess probe by centrifugation through a semi-permeable membrane (Centricon-30), samples were electrophoresed through an alkaline agarose gel which was analyzed by autoradiography. When using an HMT-modified 25-mer probe end-labeled with 3,000 Ci/mmole 32P, 0.015 ng (3.8 X 10(6) copies) of M13 DNA could be detected. With this same probe 10 micrograms of denatured human DNA (corresponding to 3.0 X 10(6) copies) did not give a signal.     

Screening recombinant phage M13 plaques with RNA probes; a one-step procedure which identifies clones containing either of the complementary DNA strands

We describe a method for detecting specific DNA sequences cloned in M13 phage vectors, based on the procedure of Woo (in Wu, R., Methods in Enzymology, Vol. 68, Academic Press, New York, 1979, pp. 389-395). M13 plaques are adsorbed to a nitrocellulose filter that has been pre-saturated with bacteria. The filter is incubated on an agar plate to amplify the phage; the DNA is alkali-denatured and then hybridized with a radioactive RNA probe. Unlike standard procedures, this method detects and distinguishes M13 plaques containing phage particles which harbor either the coding or non-coding (RNA-like) DNA strand, when single-stranded RNA is used as probe. We have optimized this procedure with M13 clones containing mouse histidine tRNA gene sequences and have used it to determine the sequence of both strands of a mouse glycine tRNA gene.     

Two regions of M13 phage genome hybridizing with human DNA are similar to several keratin genes.

DNA from two regions of the phage M13 genome hybridizes with DNA restriction fragments from genomes of various species including man [15, 20]. As the pattern of hybridization is individual-specific, this phage M13 probe can be used for DNA fingerprinting. We demonstrate here that the regions of many keratin genes coding for glycine-rich parts of C and N end domains are very similar to the phage M13 probe, and this similarity may be responsible for hybridization.     

A DNA probe for specific detection of Mycoplasma pulmonis

Mycoplasma pulmonis was specifically detected by using a 2.3 kilobase pair (kbp) cloned DNA fragment derived from M. pulmonis m 53 as a probe. This probe recognized 2.3-kbp DNA fragments of three M. pulmonis strains in Southern hybridization, while it did not hybridize with the DNA of M. arthritidis or M. neurolyticum. Determination of the sensitivity of the probe by dot hybridization revealed that 10 ng of M. pulmonis DNA was detected by a biotinylated probe and 1 ng of M. pulmonis DNA was detected by a radioactive probe.     

Genetic analysis of fingerprints in Mérinos d''Arles X Booroola Merino crossbred sheep

The M13.13 minisatellite probe, consisting of a polymer of the M13 VNTR consensus sequence, cross-hybridized to ovine DNA and allowed detection of several polymorphic loci. Individual specific patterns were obtained in sheep using this probe. Pedigree analysis showed that individuals were heterozygous for most of the DNA fragments detected (88%). By studying the segregation of male's variable DNA fragments, a minimum of 10 loci were defined. The ovine DNA 'fingerprint' obtained with M13.13 is polymorphic enough to be used efficiently in animal identification, paternity testing, and possibly as a source of genetic markers for linkage analysis.     

Genomic variations in mosquitocidal strains of Bacillus sphaericus detected by M13 DNA fingerprinting.

The genomic variation of Bacillus sphaericus reference and local strains belonging to different serotypes was examined by DNA fingerprinting. A phage M13 DNA probe detected a number of variable fragments in the restriction digests of total strain DNAs. The patterns of band distribution showed a certain homology among mosquitocidal strains, expressed by similarity index D and might be a reliable criterion for assessing the level of genomic similarity between closely related strains. An important advantage of DNA fingerprinting is the differentiation of one bacterial strain from another, both expressing common phenotype and possessing highly similar genomic portions. The strain variation revealed by the M13 probe will be useful for characterization of individual strains within a serotype. It could help as well to solve some uncertain cases based on the results obtained by other methods of identification.     

间接核酸杂交方法的建立

建立了一种新的核酸杂交手段一间接核酸杂交方法,其突出的优点是用一种共同的核酸标记物就可检查不同的基因组或不同的基因。我们重组乙型肝炎病毒或EB病毒的核酸片段于噬菌体M_(13)mp8载体,以此重组的单链DNA为第一夹心层,用~(32)P标记的双链噬菌体DNA作为共同探针,检查乙型肝炎病毒和EB病毒的核酸,获得满意的结果。应用该法进行细胞内的原位杂交,检查细胞内存在的EB病毒基因效果亦佳。     

The making of strand-specific M13 probes

A novel approach has been developed for the preparation of highly radioactive, strand-specific M13 probes. A universal primer, complementary to the region 5' to the multiple cloning sites of M13mp7, was used to initiate the DNA synthesis of the complementary strand of the M13 sequence downstream from the inserted sequence. The synthesis of the (?) strand, which was labeled with a radioactively labeled precursor, did not proceed to completion so that the inserted sequence was kept single-stranded. Thus, a partially double-stranded probe that had the specificity of this inserted sequence was obtained. As an example for the application of single-stranded specific hybridization probes, an M13mp7 subclone of a zein cDNA clone of maize (A30) was labeled and used in a dot hybridization test to select from the hundreds of M13mp7 subclones of the zein genomic clone, 24, the sequences complementary to the probe. The specificity of the probe was confirmed by dideoxy chain terminator sequencing experiments.     

A new and versatile reagent for incorporating multiple primary aliphatic amines into synthetic oligonucleotides.

A novel and versatile phosphoramidite, N-Fmoc-O1-DMT-O2-cyanoethoxydiisopropylamino-phosphinyl-3-am ino-1,2-propanediol (1, Fig. 1), has been synthesized and used to incorporate primary aliphatic amines into synthetic oligonucleotides. Its convenient preparation and use in solid phase oligonucleotide synthesis is described. Using phosphoramidite 1, an amino-modified oligonucleotide probe complementary to M13mp18 DNA was constructed with five primary amines attached to the 5'-terminus. The amino-modified oligonucleotide was subsequently labeled with biotin and employed in a dot-blot hybridization assay. As little as 0.5 ng of M13mp18 target DNA was colorimetrically detected.     

Genetic variation between strains of the Mediterranean fruit fly, Ceratitis capitata, detected by DNA fingerprinting.

DNA fingerprinting has been used to detect genetic variation in the Mediterranean fruit fly, Ceratitis capitata. Three different probes have been identified that can be used to detect DNA restriction fragment length polymorphisms between strains of this species. The strains used in this study differ only in terms of their geographic origin or genetic background. One of the probes used is the bacteriophage vector M13, and the other two are repetitive sequences derived from the medfly genome based on a weak homology to M13. Within a strain, each probe produces a consistent restriction fragment profile that is not affected by the method or timing of DNA extraction. Between strains, when M13 is used as a probe, an average of 10% of the observable bands are polymorphic. Use of the medfly genomic sequences as a probe increases the proportion of polymorphic bands between strains up to 30%. The fact that genetic differences between even such closely related strains can be reliably detected by this method holds great promise for studies of insect pests including the ability to monitor the movements of pest species, determining the extent of genetic variation in pest populations, and in making identifications from otherwise unidentifiable material.     

Genomic fingerprinting of microorganisms: its use as a hybridization probe of phage M13 DNA

Hypervariable nucleotide sequences detected by hybridization with the phage M13 DNA probe were found in the chromosomal DNAs of certain pathogenic microbial species. DNA fingerprinting, based on hybridization of M13-probe with hypervariable chromosomal DNA sequences, opens new approaches to epidemiological analysis, epidemiological prognosis, taxonomy, and other theoretical and applied fields of bacteriology.     

Molecular cloning of human satellite DNA sequences and their use in detecting DNA polymorphism

A approximately 400 bp HaeIII human genomic satellite DNA band was cloned into pUC18 to construct a partial library. A fragment of bacteriophage M13 containing a sequence homologous to the human minisatellite core was cloned in pUC18 and was used as a probe to isolate a approximately 350 bp human satellite clone (pTRF5.6) from the partial library. Other clones from this library showed a wide variation in terms of size and hybridization to the pTRF5.6 clone. Human DNA from different individuals was digested with restriction enzymes, Southern transferred and probed with TRF5.6. Individual-specific complex pattern of DNA bands was produced. TRF5.6, therefore, could be useful as a probe for detecting genetic polymorphism.     

cDNA cloning and mapping of the human creatine kinase M gene to 19q13

We describe the first isolation of a human creatine kinase M cDNA clone and its mapping of the gene to human chromosome 19. A human creatine kinase M cDNA clone, pJN2CK-M, harboring a 1,160-bp insert, was isolated by colony hybridization with a previously sequenced chicken creatine kinase M cDNA probe. The human cDNA was used as a probe in Southern transfers of TaqI-digested genomic DNA from mouse/human somatic-cell hybrids to localize the human creatine kinase-M gene to chromosome 19. In situ hybridization of the tritiated cDNA probe to metaphase chromosomes of peripheral blood lymphocytes from normal males revealed significant labeling to chromosome 19. These two independent methodologies assign the human creatine kinase-M gene to chromosome 19. Since greater than 69% of the grains of chromosome 19 label band q13, the human creatine kinase-M gene has been mapped to 19q13. On the basis of high-resolution G-banding, the predominant labeling site was 19q13.2-q13.3.     

Efficient cDNA cloning method using solid phase DNA probe.

We are now developing a novel and efficient method using solid phase DNA probe to isolate a particular recombinant cDNA from single stranded cDNA library. Target clone coding metapyrocatechase (MPC) and cDNA library constructed from mRNA of U-937 (human lymphoma cell line) were converted to single stranded form by superinfection of helper phage (M13KO7). Probe DNA (25 mer) composed of a portion of the target cDNA was synthesized, attached to an HPLC gel and used as a solid phase DNA probe. Hybridization between probe DNA and target clone was performed in an Eppendorf tube within a few hours. Competent cell (JM109) was transformed with about one-twentieth of hybridized and eluted fraction by Hanahan's method. From the mixture of 1 ng of MPC vector and 5 micrograms of cDNA library, we obtained 50 colonies containing MPC gene out of 63 transformed colonies.     

A Lactobacillus helveticus plasmid detects restriction fragment length polymorphism in different bacterial species

A small cryptic Lactobacillus helveticus plasmid, pLBL4, was able to reveal restriction fragment length polymorphism in different bacterial species including Lactobacillus species, Bacillus species, and Escherichia coli when used as a DNA probe. The observed polymorphism was a result of the combined hybridization of several microsatellite sequences. The 6-bp sequence (TTGTTT) was repeated 12 times, seven of which were concentrated within the region between 1791 and 1997 bp of the plasmid sequence. The polymorphic patterns generated with pLBL4 differed from those obtained with M13 DNA in the larger number of bands observed. The results presented here open the possibility of using pLBL4 as a new broad-spectrum polymorphic DNA probe for fingerprint analysis.     

Genomic fingerprints of organisms from different taxonomic groups: the use of phage M13 DNA as a hybridization probe

Hypervariable polymorphic patterns were detected using wild-type M13 DNA as a probe in genomic DNAs of very different organisms ranging from procaryotes and lower eucaryotes to upper plants and animals, including human beings. Due to somatic stability of highly polymorphic patterns and their discrete inheritance, individual-specific restriction pattern analysis ("DNA fingerprinting") with this test probe was found to be useful in applied human genetics, in particular, for identifying paternity and maternity, and mapping of human genomes. The data obtained also demonstrate some possibilities of the DNA fingerprinting technology in genetics and selection of agricultural plants and animals, such as variety analysis, classification and registration of individual inbred lines and strains, as well as identification of bacterial strains.