Partial digestion with restriction enzymes of ultraviolet-irradiated human genomic DNA: a method for identifying restriction site polymorphisms

A method for partial digestion of total human DNA with restriction enzymes has been developed on the basis of a principle already utilized by P.A. Whittaker and E. Southern (1986, Gene 41: 129-134) for the analysis of phage lambda recombinants. Total human DNA irradiated with uv light of 254 nm is partially digested by restriction enzymes that recognize sequences containing adjacent thymidines because of TT dimer formation. The products resulting from partial digestion of specific genomic regions are detected in Southern blots by genomic-unique DNA probes with high reproducibility. This procedure is rapid and simple to perform because the same conditions of uv irradiation are used for different enzymes and probes. It is shown that restriction site polymorphisms occurring in the genomic regions analyzed are recognized by the "allelic" partial digest patterns they determine.     

A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP.

Partial digestion of DNA fragments is a standard procedure for subcloning analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-methyl-dCTP into the PCR product to protect most of the restriction sites. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reaction done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditional methods. Furthermore, using fluorescein-labeled primers in the reaction, we were able to detect the fluorescein-labeled end fragments resulting from the enzyme digestion using a fluorimager or anti-fluorescein-AP antibody and thus determine the restriction maps.     

Generation of deletion subclones for sequencing by partial digestion with restriction endonucleases

A method for creating a group of deletion subclones for DNA sequencing by partial digestion of M13 bacteriophage constructions is outlined. The M13 construct is linearized at a unique site and then subjected to partial digestion with a frequent-cutting restriction endonuclease. The insert is truncated at different locations. The vector DNA is also partially digested. The products of a single partial digestion are repaired, recircularized by ligation, and used for bacterial transfection to generate subclones with a spectrum of deletions in the insert; most deletions in the vector DNA will disrupt vital viral genes and will thus disappear in the transfection. The subclones are sorted by size by gel electrophoresis of single-stranded viral DNA. This method is simpler and thus may be more reliable than established subcloning schemes.     

λ噬菌体和Cosmid重组DNA克隆的快速限制性内切酶图谱分析方法

cosmld克隆的线性化用λcos末端酶来完成,线性的cosmid或λDNA经部份限制性内切酶酶解后,分别与已标记的cos顺序探针杂交(探针为分别与λ的左端或右端的cos顺序互补的12核苷酸单链片段),杂交后的部份酶解片段经电泳分离和自显影后,酶切点位置可直接在X-底片上读出。在本实验室条件下,可一次完成二个克隆包括5—6种限制性内切酶的图谱分析,分析和作图可通过计算机或手工进行。     

Rapid restriction mapping of DNA cloned in lambda phage vectors

A protocol for the rapid restriction mapping of phage λ clones has been developed. Partial digestion products are selectively labelled at the right or left cohesive λ DNA termini by hybridisation with [³²P]oligonucleotides complementary to the single-stranded cos ends. After gel electrophoresis and autoradiography, the restriction map can be directly determined from the “ladder” of partial digestion products.     

Tn5cos: a transposon for restriction mapping of large plasmids using phage lambda terminase.

A method for the rapid restriction mapping of large plasmids has been developed. A 400-bp fragment of phage lambda DNA containing the cos region has been inserted into Tn5. After in vivo transposition of this Tn5cos element into the plasmid of choice, the plasmid is isolated and linearized at its cos site with phage lambda terminase (Ter). Such Ter linearization was about 70% efficient. After partial digestion of the linear molecules with the appropriate restriction enzyme, the products are selectively labelled at the right or left cohesive phage lambda DNA termini by hybridization with digoxygenin (DIG)-11-dUTP-labelled (using terminal transferase) oligodeoxyribonucleotides complementary to the single-stranded cos ends. After pulsed field gel electrophoresis, the labelled fragments are visualized in the dried gel using a DIG-detection kit. The restriction map can be directly determined from the 'ladder' of partial digestion products.     

A computer program package for restriction map analysis and manipulation.

Programs for the calculation, storage and analysis of restriction maps derived from the analysis of partial digestion products from end labelled DNA (1,2,3) and their correlation with digestion - and hybridisation patterns in total digestions and Southern blot experiments are described. These programs allow direct input of gel patterns from partial or complete digestion experiments using a digitizer tablet, calculation of molecular weights and restriction maps, plotting of maps and actual or predicted fragment patterns and automated identification of overlapping cosmids from partial restriction mapping results. Programs are written in PASCAL and have been implemented on a VAX/VMS system, with a HP-7221T plotter and a digitizing tablet.     

A molecular approach to the identification of S-alleles in Brassica oleracea

A molecular technique for the identification of S-alleles involved in self-incompatibility has been used to analyse the S-allele reference collection of Brassica oleracea. The reference collection contains nearly 50 different lines each with a different S-allele present in the homozygous state. The technique consists of amplifying by the polymerase chain reaction (PCR) sequences belonging to the S multigene sequence family using a single pair of conserved primers. PCR products are then analysed further by digestion with six restriction enzymes followed by gel electrophoresis of the digestion products. A simple method of estimating the band sizes of the digestion products is described. The S-locus-related sequences can be distinguished from S-locus glycoprotein and S-receptor kinase genes by the restriction patterns. Furthermore, with any one restriction enzyme, several alleles showed the same restriction pattern. Alleles could therefore be grouped together. With two exceptions, each member of the S-allele reference collection showed a unique set of restriction patterns. Investigation of the exceptions using pollen tube growth tests showed that these accessions represented duplications within the collection. This technique therefore provides a simple and useful method for identifying different S-alleles.     

Restriction mapping of a chlorobenzoate degradative plasmid and molecular cloning of the degradative genes

The genes for the degradation of 3-chlorobenzoic acid ( 3Cba ) are present in a 110-kb plasmid pAC27 . A circular map is established using the restriction endonucleases EcoRI, HindIII and Bg/II. The map is derived from the results obtained by partial restriction digestion, complete single and double restriction digestion and finally confirmed with hybridization of the digested fragments using different purified fragments as probes. The 3Cba degradative genes are found to be clustered in one region of the map (EcoRI fragment A) as judged by molecular cloning with a broad host range vector pLAFRI . A portion of the 3Cba degradative gene cluster appears to undergo ready recombination with the chromosome, even in a recA host, suggesting the probable transposable nature of such gene cluster.     

Restriction endonuclease map of Euglena gracilis chloroplast DNA.

A physical map of the Euglena gracilis chloroplast genome has been constructed, based on cleavage sites of Euglena gracilis chloroplast DNA treated with bacterial restriction endonucleases. Covalently close, circular chloroplast DNA is cleaved by restriction endonuclease SalI into three fragments and by restriction endonuclease BamHI into six fragments. These nine cleavage sites have been ordered by fragment molecular weight analysis, double digestions, partial digestions, and by digestion studies of isolated DNA fragments. A fragment pattern of the products of EcoRI restriction endonuclease digestion of Euglena chloroplast DNA is also described. One of these fragments has been located on the cleavage site map.     

Mapping genes within a YAC by computer-assisted interpretation of partial restriction digestions.

Partial restriction digestion is used to map restriction sites and the location of genes within yeast artificial chromosomes (YACs). Locus-specific probes are hybridised to the partially digested YAC DNA and the fragments to which they hybridise are compared with the pattern of partial digestion products that include each map region. A least squares criterion is presented which allows for error in fragment length determination. This rapidly defines the most likely location of a marker within the restriction map and permits the combination of results from digestions with different restriction enzymes. Approximate confidence intervals may be assigned to gene locations, and tests of goodness-of-fit of the data may be performed. Since the number of erroneously matched fragments increases in proportion to the square of the number of sites, denser maps are not necessarily more informative. Simulations indicate that the optimal number of internal restriction sites given typical experimental error (1% of YAC length) is about five sites; the associated broad support interval (on average one third of YAC length) may be reduced by combining results from different enzyme digestions. Application of a computer implementation of this model to experimental data showed that the model fitted well, and estimates of location were found to be consistent with other evidence.     

Utility of internal markers to improve the accuracy of cystic fibrosis genotype analysis.

DNA diagnostic tests often utilize restriction endonuclease digestion of PCR-amplified portions of genes under analysis. When partial digestion occurs, the resulting patterns may lead to error in diagnosis. To overcome such potential errors in cystic fibrosis testing, we have developed internal markers that can increase the precision and reliability of genotype assignments.     

Cloning of two type II methylase genes that recognise asymmetric nucleotide sequences: FokI and HgaI

Summary The modification genes of Flavobacterium okeanokoites and Haemophilus galinarum have been cloned into the vector pBR322 and expressed in Escherichia coli cells. FokI methylase gene is contained on a 3.80 kb piece of F. okeanokoites DNA. Plasmid constructs carrying this fragment of DNA are resistant to digestion by FokI restriction endonuclease but are sensitive to cleavage by HindIII, EcoRI and PstI. Unmodified DNA molecules, exposed in vitro to cell extracts prepared from cells habouring this plasmid, became resistant to digestion by FokI.The smallest HgaI methylase clone carries the pBR322 plasmid containing a 3.50 kb piece of H. galinarum DNA. This plasmid is resistant to digestion by HgaI.Neither the FokI nor the HgaI restriction endonuclease was detected in either clone. This is the first report of cloning modification genes whose protein products recognise asymmetric nucleotide sequences.     

Dictyostelium 17S, 25S, and 5S rDNAs lie within a 38,000 base pair repeated unit.

Mapping with the restriction enzymes Sal 1 and R1 has generated a picture of the organization of Dictyostelium ribosomal DNA. The DNA which codes for 17S and 25S ribosomal RNAs is located within a stretch of repetitive DNA at least 38,000 base pairs long. This repeated unit includes 5S DNA, linked to 25S DNA. Two techniques were especially useful in the mapping: "cloning" 14S + 25S DNA on the plasmid pMB9 to amplify individual R1 fragments, and digesting DNA with R1 in the presence of the antibiotic distamycin A to produce specific partial digestion products.     

Rapid and reliable identification of rice genomes by RFLP analysis of PCR-amplified Adh genes.

The rice genus (Oryza L.) consists of 24 species with 10 recognized genome types. With the realization of many useful genes in species of wild rice, continuous efforts have been made to understand their genomic composition and relationships. However, the identification of rice genomes has often been difficult owing to complex morphological variation and formation of allotetraploids. Here we propose a rapid and reliable method for identifying rice genomes based on the restriction sites of PCR-amplified Adh genes. The experimental procedure was as follows: (i) amplify a portion of Adh1 and Adh2 genes with the locus-specific PCR primers; (ii) digest PCR products with restriction enzymes that distinguish different genomes; and (iii) run the digested products on 1.4% agarose gel, and photograph. Using various combinations of restriction digestion of the two Adh genes, all of the rice genomes can be identified.     

Partial physical map of human chromosome 21 from fibroblast and lymphocyte DNA

A partial physical map of the human chromosome 21 including 26 genes and anonymous sequences was established by pulsed-field gel electrophoresis analysis of restriction fragments obtained from lymphocyte and fibroblast DNAs. The sizes of the restriction fragments obtained by total digestion with eight different enzymes were compared in these two tissues. Differences resulting from the variations in the methylation state of the restriction sites were frequently observed. These differences and partial digestions were used to estimate the order and the distances between genes and sequences. Six linkage groups were defined: D21S13-D21S16, D21S1-D21S11, D21S65-D21S17, (D21S55,ERG)-ETS2, BCEI-D21S19-D21S42-D21S113-CBS-CRYA1, and COL6A2-S100B. For six intergenic distances the resolution of previous maps was significantly increased.     

HindIII and Sst I restriction sites mapped on rabbit poxvirus and vaccinia virus DNA.

The DNAs of two closely related orthopoxviruses, rabbit poxvirus (RPV) and vaccinia virus (VV), were mapped by overlapping-fragment analysis using restriction endonucleases HindIII and Sst I. The exact arrangement of these fragments was accomplished by total digestion of isolated partial restriction products and by end-fragment determination. RPV and VV DNAs showed identical restriction patterns in an internal region comprising approximately 60% of the genome. The size, by electrophoretical analysis of the RPV DNA, was 118 X 10(6) daltons, some 6 X 10(6) daltons less than VV DNA. The two opposite terminal restriction fragments of RPV DNA cross-hybridized to each other.     

Long range restriction analysis of the bovine casein genes.

Pulsed field gel electrophoresis (PFGE) was used to analyse the organization of the bovine alpha s1, alpha s2, beta and kappa casein genes. High molecular weight DNA was prepared from fibroblasts and lymphocytes embedded in agarose and was digested with the restriction endonucleases Clal, Sall, Smal, Xhol. The digestion products were separated by PFGE, transfered to nitrocellulose filters and hybridized to probes corresponding to the cDNAs of the four bovine casein genes. The casein genes were demonstrated to be physically linked within a region of 300 kb, represented by two adjacent Xhol fragments in fibroblasts and by a single fragment in lymphocytes. A restriction map of the casein locus was derived and the order of the genes was shown to be kappa, alpha s2, beta, alpha s1.